THE COVER

The Nobel Prize in Physiology and Medicine for 1969 was awarded on December .10, 1969 to Dr. Max Delbrilck, Dr. Alfred Hershey and llr. Salvador Luria for "their discoveries regarding the mechanism of reproduction of viruses and their genetic structure."

Professor Delbrilck is shown receiving his Prize from King Gustav VI.

Professor Murray Ge/I.Mann of our Division of Physics (in the background) received the 1969 Prize in Physics at the same occasion.

We are proud.

A Report for the Year 1969-70

on the Research and other Activities

of the

Division of Biology

at the

California Institute of Technology

Pasadena, California

Tf

STAFF OF BIOLOGY 1970

Fannie L. Warren: Compilation and typing.

Elizabeth T. Hanson: Copy editing.

The helpful assistance of Gene Bollt, Geraldine Cranmer, Polly Eiker, Miriam Eisberg, Eleanor Kempees, and Isabella Lubomirski is greatly appreciated.

RESEARCH REPORTS

Much of the research work summarized here has not y-e_ b<:c11 n?ported in print, in many instances because it is not yet con1p/e'-e. i ,)r that reason this report is not intended as a publication and shoLlcJ ·101 be cited as such. Individual projects should be referred to only if spc· c1fic permission to do so is obtained from the investigator respuis1 Jic for the material. References are made here to published papers beaw1g on the projects reported. Publications by members of the Division, C·>v ering the period July 1969.June 1970, are listed separately, at tile end of the research reports of each group.

T A B l E 0 F C 0 N T E N T S

In trod 1.--~ i ~).-1

Staff :•f ,·,;1ruction and Research I I

· lll'JCllL\ :c;•1;y lill:ILJNOGENETICS, BIOPHYSICS

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:;c)quonco studios on chromosomal RNA wnd studios on chroma-· somal RNA binding ;irotcin 21

:ti romosoma I RNA u posit i vo agent In contra I of gone E1ct iv i ty 21 Interaction of chromosomal RNA with tho repetitive soquoncos

of tho genome 22 3-:-udios of the size and distribution of tho ropoui·od

soq uoncos of ch romosoma I DNA 23 Tho bi ogonos is of ch romosoma I RNA 23 l:;olation of high molecular weight RNA by gol filtrntion 011

So;iharoso 26 24 Ionic strength effect on tho molting of nucloohistono 25 Ronaturation of nucloohistone 25 Model of two molting bands by histono binding in nucloo-

h i stone 26 T'io suporcoi led structure of nucloohistono 26 Limited hetorogonoity of tho major nonhistono chrwosomal

;iroteins 27 Di~A-binding proteins in rat I Ivor nucleoplasm 27 Characterization of mammalian RNA ;iolymoraso 28 Hormona I induction of now gone ex;iressi on by ostro::ions 29 Art! facts of tho RNA polymerase sys tom 29 Tho role of a factor in tho initiation of RNA synthesis by

E. coli RNA polymerase on various templates 30 Initiation of transcription by E. coli RNA polymorasG on

T7 DNA 30 Studies on enzyme differentiation in the cellular ~;limo

mold Dictyostol ium discoidoum: Glycogen phos;ihorylnso 31 /\mi no acid-catabolizing and am;:>hibolic onzymos during slimo

mold development 32 f;ogulatlon of amino peptidase activity in sl imo meld dovolop-

mont 33 C:hnractorlzation of tho s I imo mold gonomo 33 c;ono regulation in rcgonoratlng I Ivor 34 '.;tudios of brain chromatin 34 Studios of tho chromatin of Drosophila molanogasfor 35 ~Jtudios on tho Bursa of Fubricius in pigeons and lts contri ..

butions to tho immune response 38 11 study of the effect of i rrad i at ion on tho pi goor' karyoty ;io 39 l~ultiplo effects of anti-lym;ihocytic scrum 39 1\n attom;it to dovolop a toct)niquo for the idontificution of

individual eel Is producing anti-bactoriophngo <rntibody 40 l,1aintcnance of polymorphism for trunsforrins in tho ;iigco11 40 ilohavloral biology of rotifcrs 41

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:>I. Tho uso of antibodies directed against cell surfocos, io interfere with normal development of the pigeon '"'"i' 42

32. P,utomati on of Phycomyces export ments 44 33. Long wuvclongth phototropic action spectra of Phyconr;ccc; 45 34. Dlchroism and orientation of Phycomycos visual p\,,rno,,t 'i'J 35, Munipulation of a cl rcadian rhythm: A new offoc'r O'' true

Ii ght on Phycomycos 46 36, Ufccts of cold periods on the stlmulus-rosponso sys •'om

in Phycomycos 47 37. Phycomyces phototrop Ism: Loca I I zati on of adapta n 0.1 :me

the role of eel I rotatI on 47 38. The modi atl on of tho avoidance response of Phyc01nycec, 4U 39. Effect of changes in relative humidity on tho gr-ov11n rul"

of Phycanyces sporanglophores 48 40. Mononucleate spores of Phycomyces 49 41. Cnaracterlzation of the mad mutants of Phycomyces 49 42. Biochemical investlgatioOS-ot photomutant Phycomycos 50 43. Genetic curing of blindness In Phycomyces 51 44. Processes affect Ing the growth of Phycomyces sponn~; opiw.,·s •,I 45. Ultrastructure of Phycomyces sporanglophores froz:in :n sc:;L;r-·

fluid helium 52 46. Electrical conduction in lipid bi layers 53

EllOP\\YSICS, PhYSIOLOGICAL PSYCHOLOGY, CELL BIOLOGY

4 7. Tho size and order of bactcrl ophagc iilX 17~ genes 48. Multiple length DNA molecules of bacteriophage (llXI'/" 49. Tho go net i c map of bacteri ophagc iilX 174 50. Cenetic recanbination of bacteriophage iilX174 51. i1Xl74-·specitic proteins: Sedimentation analysis 52. Opal <UGAl nonsense codon mutants of \<!XI 74 53. Studios with 0Xl74 mutants blocked In progeny sir.g' )-·e>TJcuocl

llNA synthesis 54. Characterization of a spoci tic membrane fragment <i:;·;oc c ic:d

with ropl icatlng DNA 55. f:tfoct of maomycin Con lysis of iilXl74 infected ho;t ·:ol 1;; 56, IJacteriophage \<!Xl74 DNA synthesis in vitro 57. Nature of the comp lomentary strand synthos i zed i 11 v i'r'u , rom

ultraviolet-irradiated circular single-stra11d0:: IJ:'1/. 58. Fff 11 DNA; resistance to DNA polymerase-assoclukcl ,;xora.: .• oo-

lytic activity 59. The in vivo iilX messenger RNA 60. lhe ln vitro \<!Xl74 messenger RNA 61. 1°1ethy lat ion of l:IX174 DNA 62. \IXl74 antigonlcity: A comparison of l<!X174 am3 a·1d

chemically altorod particle lacking tho."spike" co1

pononts 6 3. A rad i oi nmuno assay for iilX anti gens 64. Rep I !cation of bacteriophage PM2 DNA: A closoc: c1 'cu'''

double-stranded molecule 65. I mp roved puri ti cation for Qfl virus 66, Rep I ication of Sindbls-speci tic RNAs In tissue cui·rurc ·;: 11• 67. Sindbis viru~-induced host eel I plasma morrbranc co1·:ve:cs <.:.-il 68. Charucteriza·rion of an infcctivlty assay for Sinclt·r·; :•l J';

69. Nourophyslological correlates of Pavlovian concllth:iiic·

55 56 56 58 58 59

59

60 61 6i

66 6b

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71. Cholinorgic and adronergic effects on instrumont;cl behcvior with brain shock, on neural activity in subcortical regions, and on brain norop i neph r i no 79

7i, Evoked potentials elicited by oloctricul stimulwtion in ttio medial forebrain bundle 82

;.>, Neuronal activity during self-stimulation 82 '\. Tho action of chlorpromazino on conditioned unit activity b3 !1. Unit activity and conditioned behavior 84 75. Tho effect of cortisol on oxtruhypothalamic neurons 85 h. Ono trial loaming with electrical brain stimulation as

roi nforcomont 86 n, Dove I opmenta I and hormona I aspects of tho septa I·· raga

syndrome 87 7:1. Inhibition of hippocampal unit activity with motor movement 88 n. Estimation of genetic determination of tho readiness to

respond to electrical stimulntion of tho hy;oothelarnu<; as a reinforcing agent 88

in, Hypothalamic unit activity and feeding bohavio1- 89 b. Hypothalamic unit activity and drinking in rats 89 n::. Unit responses to thirst induced by hyportonic s2lino in

chronic rats 90 i<\. Operant conditioning of hippocampal neurons in r;it 90 (V,. Bonding moments in f I ago 11 a 94 f\'i, Un coup Ii ng tho ATPaso of sea urchin sperm f I ago Ii a 95 "" Chomotactic behavior of Tubularia spermatozoa 95

GUit·r ·c;, :1\CT!:RIOPHAGE GENETICS, CELL BIOLOGY, BIO-ORGANIC CHEl'iSTRY, nroc,,;:- '' ,'' llEHAVIORAL GENETICS

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Indications of genetic hotorogonoity in singlo··uscosporo colonies of Nourospora crassu

A character in Nourospora showing both Mondo I ii.in crnd matorna I i nhori tanco

Tho subunit structure of bactoriophago T4 tail fiber precursors

Tho genetic control of early stops in T4 tni I fihor assomb ly

Tho role of tho head in T4 tai I fiber attachrrcnt AcJsor;ition mutants of T4 I sol ati on of a phago-codod amber suppressor Tllo lysis mechanism of phage T4 Com;:>os it ion and propcrtios of Nourospora tyros! nusos Growth of Nourospora at low water activities· Studios of tho role of cyclic AMP in tho doroprnssion

of tyros i naso in Nourospora What is tho function of chloroplast glycol ipids? Investigations of mitosis Chloral hydrate inhibits protein synthesis Tho effect of ch lora I hydrate on tho induction of g I utamGfo

dohydrogenaso activity Canavan i no k i II i ng of Ch I arriydomonas ro i nhard i Tomporature-sonsitivo mutants dofoctivo in tho incorporntion

of arginine into protein Tlw effects of penicillin on dovolopmont in col lulcir slirco

molds

98

99

10 I

102 103 104 105 106 107 108

109 11 I 112 113

I 13 114

114

115

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I 0:.1. 106. I 0-/. IO fl. I OSI • 11 o. 11 i. 112.

113.

114. I I 'J. I lG.

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119.

120. 121. 122.

17). !24. IL5. 126,

127.

128. 129.

uo. 1:51 • 132. 133.

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I .56. I .:SI. 153.

I 19. 140. 14 ! • 142. 145" 14•1.

Conservation and air pollution Ery-rhropolotln and erythropoiesls Phenol oxidaso function in Drosophila Somo properties of dysin Biochemistry of phenol oxldase precursor Ai in Drosophi 1,, Chliinaso activity during Drosophila dovolopment Studios on Drosophila phenylalanine hydroxylaso Protein granules of Drosophila larval fat body: Or·isii n

of the prate in Protein granules of Drosophl la larval tat body: lnducticn

in vitro Eledrnphoretic heterogeneity in human lnsul In The molecular organization of tho synaptinomal complo> Isolation and genetic mapping of behavioral mutants 1r ..

lJrosoph i la Neurnphysiological analysls of visual mutants of Drosop!i; lo Gonu1-ic surgery of tho visual system of Drosophila b;• tw•

use of mos a I cs Electron microscopy of visually defective mutants of

ll rosoph i I a lsolarion of rotinula cells from Drosophila U I trastructure of the dove I op Ing visual system of Dr\"<o,oili i .c Two-·dirrensional countercurrent method for Isolation o·.'

optomotor-dof iclont mutants "Ecsi ly shocked" - A now mutant phenotype In Drosophi :a Phototaxls of Drosophila larvae Ci r·cad I an rhythm mutants In Drosoph i I a Circadian activity rhythms in single individuals of

Drosophila rre lanogaster Loca 11 zat ion of tho s i to of act! on of a rhythm mu tat i ·)n

within the fly Production of mosaics for an autosomal mutation 8-c.lanine rretabolism in wi Id type Drosophila and tw0 cnu·: '"',

ob any and b I ack Catecholamine levels in adult Drosophila Ca"locholamlne rretabol !sm in Drosophila Ccmmunicai"ion of direction in the honey bee lsolntion of fundamental polypeptide subunits of biolo,1 e<ci

membranes Pndial characterization of tho fundarrental protein Sllb: ,,,.;

of co 11 u I ar rremb ranes A pm posed mode I for tho genera ti on of graded potonT i u ! :

in rod outer segments: Ionic gradients and rn"io1\_liun of dark adaptation by Na-K-Mg-ATPase

F'rotolns derived from the surfaces of embryonic col is Studies on membrane proteins of E. coli Studios on membrane proteins of wi Id typo (74-Al and J'.''."'L

(mi-I) Neurospora mitochondria 11 no11 system for identifying proteins \for·k at JPL supported by the President's Fund Mc,ss spectrometry of protein amino terminal soquonc•l'' Chomical ionization mass spoctranetry of peptide doc. variv,,;s S·ITucturo and function of tropoelasti n fl,o development of adult menbrano markers on the red b ku;i

col ls

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141 148 \ L~ 9 ! ::o I: I I '.·2

B Lli.'.JG I Cf,L !;ys TEMS ANALYSIS, NEUROPHYSIOLOGY, PSYCHOBIOLOGY, DROSOPH I LA GPKT I CS

I 4 _5, '.46. . 47. 148. f 49. 150.

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lhe human oculomotor system Visual evoked potentials In man Stereopsls as a function of color and luminance The function of S-potentlal In the visual process Analysis of S-potentlals from the channel catfish retina Concomitants of spreading depression and asphyxiation of

central nervous tissue Effect of glutamate on cortical tissue Glutamate release during glutaraldehyde fixation lhe effect of monocular deprivation on axosomatic contacts

of the visual cortex Tho effects of visual deprivation on synaptic contacts of

the retina Input to motoneurons during spastic states Transmission of high frequency trains of impulses Electron microscopy of Insect ganglia The effect of calcium on the impedance of central nervous

tissue Changes in intracellular electrolytes of chicken retine

during spreading depression Binocular interaction In visual responses of the crayfish Visual Integration of lobsters Comp I-ex ly integrating un I ts of the opt! c nerve in the rock

lobster Units in the optic nerve of a South American rock lobster Visual responses from tho optic nerve of a crab The Influence of state of excitement of tho animnl on

discharges in the optomotor fibers of a crnb Unldlrectlonal optomotor fibers In Carcinus Optomotor fibers in the rock lobster Coordination and activation of swimmoret movements in the

crayfish Discharge pattern of joint movement f I bors In tho crab Observations on the crayfish sinus gland In vitro entrainment of the circadian rhythm of optic nerve

Impulses from eyes of Aplysla The effect of actinomycin Don the circadian rhythm of the

eye of Aplys!a californica Molecular weight distribution of proteins synthesized in

single, identified neurons of Aplysia ATPase activity In Aplysia nervous tissue Seasonal reproduction In Aplysla: Bag eel I perfusion Habituation and dish ab i tuati on in the absence of a centra I

nervous sys tern Intracellular calcium injection causes incroasod potassium

conductance In Aplysla nerve cells Synaptic Influences on k<jentlfled neurons in an aberrant

parleto-vlsceral gangl Ion of Aplysia Right hemispheric dominance for tho perception of part-wholo

relationships In human commlssurotomy patients Lateralization of perceptual unification In commissurotomy

patients

Page

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161 162 163

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164 165 166 167

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168 171 172

172 173 174

174 175 175

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176 177 177

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182 183 184

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186

188

189

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181. 182. 183.

184. 185.

186. 187.

188.

189. 190.

191. 192.

193. 194,

195. 196, 197. 198.

199.

200. 20 I.

202.

203.

204.

205. 206. 207.

The periodicity of earliest eye movements Coord I natl on of eye and hand>.! n human infants Detection and discrimination of visual stimuli on both

s •dos by subjects with disconnected hemispheres Eye movomonts in commissurotomy subjects Finger-tapping test for distribution of attention in bruin­

bisocted patients Completion of visual percepts by disconnected hemls?horos Perception of chimeras by commissurotomy patients: Facoo,

nonsense shapes, objects and patterns Recognition of auditory and visual Images by commlssuro·romy

poti ants Brain mechanisms for perception and naming of colors Hemispheric mechanisms for perception of spatial orientatio:1

and motion Pred i ctab i Ii ty of a cross-moda Ii ty test Effect of right- or left-handedness on perceptual and

reasoning abilities 1970 Revision of spatial abi llty test Impairment of singing after unilateral injection of sod;um

a<!1yta I Musical abi I it I es of the cerebral hemispheres Dovelopmont of visual behavior in kittens Development of compensatory visual functions In kittens Retlnotectal connections fol lowing removal of half-toctum

in the goldfish Specificity and plasticlty in neural roconnections botwoon

tho eye and the brain of adult goldfish Ylsual responses of optic tectal units in the goldfish The processing of visual informat!On associated with learning

in chicks Absenco of amnesic effect with current administered to chici\s

reexposed to the training situation Rotrogrado amnesia gradients by subconvulsivo and high

convu Is i ve transcran la I currents in ch I cks Two soparab le phases of behavloral ly demonstrab lo memory

for one-trial learning in chicks Analysis of regulatory genes in Drosophila Gone functions tn a smal I region of a Drosophl la chrornosc,rne A new cnso of comp I ementary I etha Ii ty in Drosoph i I a

BIOPHYSICAL CHEM ISTFN, CELL BIOLOGY

208. 209. 210. 211.

212. 213.

214. 215. 216.

Complex mltochondrial DNA In loukomlc lymphocytes Circular dimer mitochondrial DNA in mallgnant melanocyfos Complox mitochondrial DNA In eel Is in culture Effeci"o; of tho intercalating dye ethidium bromide on tho

mitochondrial DNA of eel Is in culture Microlnjoctlon of mitochondria into the ferti 1 lzed moLJse ousi lllstrillution of closed circular mitochondrial DNA in thu

animal kingdom Rolatednoss among mitochondrlal lJNAs in the animal kingdom Tho mode of repl!cat!on of mltochondrial DNA in HoLa coil,; A buoyant method for the determination of tho suporhol ix

clonslty of closed circular DNA

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/'17. Tho sedimentation velocity behavior of closed circular SV40 DNA as a function of suporholix density, ionic strongth, temperature, and countcrlon 220

/18. Tho effect ot bound basic proteins on tho su;iorholix donsity of closed circular SV40 DNA 221

2.19. Investigations of intercalative dye-DNA interactions 221 l20. Viscometric dye tltrations of closed circular DNA 221 n1. Sedimentation properties of purified catenated mitochondrial

DNA from Hela eel Is 222 ?22. Alkali-Induced strand breakage of circular mitochondrinl DNA 223 223. Intracellular closed circular forms of SV40 viral DNA in

African Green Monkey eel ls in culture 223 ?/'4. Replicative intermediates of the si ngle-strandod DNA of the

mi nuto virus of tho mcuso 224 ;>;>5. Rapidly labeled heterogeneous RNA and discrete RNA com;ionents

from the mitochondrial fraction of Hela col Is 225 226. Evidence for complete transcription of mitochondrial DNA in

Holacells 226 227. Electron microscopic visualization of mitochondrial RNA-DNA

hybrids 227 228. Titration of mitochondrial genes for 165, 12S and 4S RNA in

He la co 11 s 228 729. Mitochondrial mlnirlbosomes in Hola cells 228 230. Tho relationship of mitochondrial RNA synthesis fo the col I

cycle in Hela eel Is 229 231. Studies on tho Isolation and subfractionation of mitochondria

of Hela eel Is 230 232. Studies on RNA synthesis by rat brain synaptosomos 230 233. In vitro protein synthesis by Hela eel I merrorane fractions 231 234. Studies on membrane structural protein from Hela col ls 232 735. Studios on the stability of messenger RNA in Hola cells 232 236., Fractionation of Hola col I metaphase chromosomos on the basis

of their electrical charge 233 237.. Nuclear magnetic resonance studios of protein conformation

and al lostoric mechanisms 235

Vi ;;i ·ring Lcctu re rs 236

Grnduetos 239

~-Honors, Awar·ds, Appel ntments 241

FinanclDI St1pport 242

l\uthor I ndox lBy pa go numbo r) 245

Financiul Support Index (By page number) 247

NEW FACULTY

On July I, 1970 Professor Leroy Hood joined our faculty. He wi 11 strengthen our program In biochemistry, particularly In the fields of lmmunochem­istry and protein structure. Dr. Hood wl 11 also serve as faculty advisor to pre­medical students.

In addition the faculty wl II be joined during the next year by Professor Richard Russel I and Professor Eric Davidson.

Professor Russel I, originally trained In bacteriophage genetics, has joined those viho see great lntel lectual adventure in neurobiology. He is partic­ularly interested in neurogenetlcs, speclflcal ly In the genetic and biochemical characterization of behavioral mutants In nematodes and In mice.

Professor Davidson Is a developmental biologist with wide interests in the overal I structure and composition of the DNAs of higher organisms and their role In the complex control mechanisms associated with differentiation.

Professor Ler~y Hood

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THE FACULTY

The entire Division was thrlLled to learn on the morning of October 16 of the award of the Nobel Prize to Professor Max DelbrUck who thus became the thl rd Nobel Prize winner In our history (fol lowing Thomas Hunt Morgan and George Wei ls Bead I e) • A Rockefe 11 er Found at I on Research Fe 11 ow In BI ol ogy at Ca I tech In the years 1937-39 and Professor of Biology since 1947, Max has awed, cajoled, encour­aged and inspired successive generations of graduate students and postdoctoral fellows, often to accomplishments of significance well beyond their Initial con­cepts. After hav Ing "fathered" the modern schoo I of bacterl ophage research with Its major role In the.development of molecular biology, Max has turned to the broad problem of sensory transduction as exemplified in the mold Phycomyces. And again a whole nascent "school" Is emergl ng.

To the Biology Division the award of the Nobel Prize to Max DelbrUck was a long overdue recognition of a most extraordinary talent and person.

After the announcement there to 11 owed an I nevi tab le and in I ml tab I e press conference, numerous faculty dinners and other honors, and a climactic musical extravaganza by the Bio-dramatics Seminar (Professor Wood and Sandra Winicur, Chief Instigators) entitled "I am Curious (Max)."

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Dr. Geoffrey Keighley

Professor Alfred H. Sturtevant

1891-1970

5

TERMINATION

Dr. Geoffrey Keighley, Research Associate and long-time collaborator with Professor Erner! tus Henry Borsook, retl red th Is year after 40 years at the Institute. Dr. Ke I gh I ey w 111 cont I nue h Is Interest In eryth ropol etl n th rough research at the .I ackson Laboratory at Bar Harbor.

Our loss Is Santa Cruz' gain. Professor Robert S. Edgar, our distin­guished viral geneticist, has left the Institute to become Provost of the new (embryonic) College Six at the University of California at Santa Cruz. Professor Edgar felt the cal I of the opportunity for major educational Innovation to be stronger than that of the continuance of his elegant scientific research. We wish him we I I •

The death of Professor Emeritus Alfred Henry Sturtevant on Aprl I 5, 1970 marked the end of an era, Professor Sturtevant was assoc I ated w I th the DI vis I on of Biology since Its founding by Thomas Hunt Morgan In 1928, He was the last survivor of the group who with Morgan In the "fly room" at Columbia University made Drosoph-1 la the most fruitful object of genetic research for several decades. One of the most distinguished geneticists of the century, recipient of numercus awards, Professor Sturtevant was a major contributor to the eminence of this Division In genetics.

Professor E. B. Lewis, Dr. Sturtevant 1s successor as Thomas Hunt Morgan Prcfessor of Genetics, paid a moving and Inspiring tribute to Professor Sturtevant at a memorial service _held at the Institute in May:

"Alfred Henry Sturtevant, born on November 2, 1891, at Jacksonvl lie, Illinois, was the youngest of six children of Alfred Henry and Harriet Evelyn (Morse) Sturtevant. His grandfather, Julian Sturtevant, was a Yale graduate, a Congregational minister, and one of the founders and later president of I I llnols College. Sturtevant's father taught mathematics for a while at llllnols College, but for the most part was a farmer, f I rst In I I 11 no! s and later in southern Alabama where the faml ly moved when Sturtevant was seven years old. Sturtevant went to a one-room country school, taught by his future sister-In-law, and went to a pub 11 c h i gh sch oo I I n Mob I I e.

"At the age of 17 he entered Columbia University. That crucial choice came about because his brother Edgar, who was 20 years older, was at that time teaching at Barnard College. Edgar and his wife took the young Sturtevant Into their tami ly, and he lived with them whl le attending Columbia University. Edgar was a scholar who later became a professor of linguistics at Yale and an authority on Hittite languages. Sturtevant said that he learned the.alms and the standards of scholarship and research from him. One can imagine the great source of pleasure it must have been for Sturtevant when he and Edgar were awarded honorary degrees at the same Yale conmencement many years later. Also present was Sturtevant's nephew Jul I an (the son of Edgar) who was at that time and stll I is a prcfessor of organic chemistry at Yale. Jullan Sturtevant's son Bradford, another Yale man, ls on the faculty at Caltech In aeronautics, and A. H. Sturtevant's own children are In the same tradition: One son Is In anthropology, the other is In engineering, and his daughter Is a practicing lawyer speclallzlng In the legal aspects of the use of atomic energy. A student of behavioral genetics might well begin to wonder whether the re may not be some Inf I uence of the genes as we I I as of the env I rcnment on the academic and scientific performances of this line of Sturtevants.

"How Sturtevant became interested in genetl cs and how he came to work w·lth Thomas Hunt Morgan are especially revealing anecdotes. Sturt, as he was

I I I

6

known to his col leagues, has sat d that he ti rst became Interested in genetl cs by tabulating the pedigrees of his father's horses. He continued this Interest whl le an undergraduate at Columbia, and he al~o collected considerable data on his own pedigree. Sturt said his brother Edgar suggested that he go to the library and read some books on heredity to learn more about the meaning of pedigrees. Thus it was that Sturtevant read a textbook on Mendel Ism by the English geneticist Punnett.

"Sturtevant saw at once that Mendellsm could explain some of the rather complex patterns of inheritance of coat colors In horses which he and others before him nad observed. Edgar encouraged Sturtevant to write an account of his findings and take it to T. H. Morgan, who at that time was a professor at Columbia and from whom Sturtevant had already taken a course In zoology during his freshman year. Morgan encouraged Sturtevant to pub 11 sh the paper, and It was sub ml tted to the Biolo9;cal Bulletin In June 1910, at the end of his sophomore year. The paper ·appeared that same year.

"The other result of Sturtevant's Interest In the pedigrees of horses was that he "as given a desk In the famous fly room at Columbia University where only three months before Morgan had found the first white-eyed fly, a discovery that was to revo1 utl on I ze genetl cs. Much of the rest of the story about those ear I y days at Co1umbia, when modern genetics was In a very real sense born, Is a matter of record in the w ri tings of Sturtevant and others.

"Sturtevant had a keen Interest In the h I story of science; his book, The History of Genetics, which was published In 1965, bears witness to this. This -­book contains a typical example of the workings of Sturtevant's mind. In it he comp! led an appendix that contained a series of 11 lntelledual 11 pedigrees of many of the men prominent In genetics or cytology In the early days. Sturtevant, of course, was a direct descendant of T. H. Morgan and of E. B. WI Ison, another emi­nent biologist who was a contemporary and friend of Morgan's at Columbia and who was at that time the authority In this country on the cytological behavior of chro­mosomes and the cell Itself. Morgan and WI Ison were, In turn, direct descendants of Martin and Brooks, two men who were at Johns Hopkl ns Un Ivers I ty where Morgan had obtained his doctorate; Martin was descended from T. H. Huxley, and Brooks from Louis Agassiz, and so It went.

"From Morgan, Sturtevant must have fl rst learned - or at I east seen in operation - the experimental approach. Sturtevant once wrote that he knew of no one else at the time who was so thoroughly committed to the experl~ental approach to biological problems as was Morgan. It was Morgan's aim to produce a mechanis­tic, as opposed to a purposeful, Interpretation of biological phenomena; a great aeal of this approach clearly rubbed off on Sturtevant, for the simplistic ele­gance ot Sturtevant 's expert ments In genetl cs are I egendary.

"Sturtevant had a remarkable memory of, I suspect, a special sort. It was as if his memory were composed of an Infinity of matrices waiting to be fl lied with any data that lent Itself to classlflcatlon Into discrete categories. The data might be In the form of numbers and kinds of bristles missing In a mutant fly; numbers of snai Is with a right-handed col I versus a left-handed coi I - the genetics of which Sturtevant was the first to explain; the relation ben,een In­version sequences In different species; or the host of other char·acterlstlcs he investigated not only In Drosophl la, but In Iris, evening primroses, snal Is, moths, and many other creatures, Incl udl ng human bet ngs. Whatever form the data took, the observations fel I Jn the appropriate matrix In his memory, from which they were read I ly retrlevab le to a degree that was truly phenomenal.

7

"The Caltech period was a time of col laboratlon especially with Sterl Ing Emerson, Theodosius Dobzhansky, George Beadle, Jack Schultz, Edward Novitski, and others. It was Sturtevant's style, at least after he came to Caltech In 1928 with Morgan and Bridges, to spend his mornings doing experiments. Afternoons were re­served for perusing the literature - and there were few journals In any phase of biology that he did not at least dip Into - and there were the wide-ranging discus­sions at the afternoon tea sessions.

"Sturtevant taught the undergraduate course In genet I cs at Ca I tech for many years. From time to time he also gave a course for undergraduates In ento­mology, complete with a field laboratory session. His lectures on topics In advanced genetics were scholarly reviews of speclal lzed areas of genetics - often dealing with organisms with a bizarre genetics, such as the protozoa, for example. These lectures were especially valuable to graduate students since they were In areas of research not directly going on at Caltech, and they served to broaden their genetic outlook, The elementary course In genetics that Sturtevant taught was based on a textbook which he and George Beadle wrote In 1939. It was not as widely used throughout the U.S. as It perhaps should have been, probably because It was considered too difficult for the average student. The trouble was that It was tal lored for Caltech students, and the problems especially were a real chal­lenge, even for Caltech undergraduates.

"Sturtevant and Beadle planned to revise the textbook, but the pressure of other work and the rapidity of developments that fol lowed the discovery of DNA prevented that revision. I would Ilka to mention one episode In this regard. There Is a subtle difference In the way geneticists use the word gene. Sturtevant discovered that he and Beadle had In fact used the word dlfferently when they wrote the book, and he always facetiously blained their I nab I llty to get out a second edition on this disagreement. Cheracterlstlcal ly, when he became aware of this amb I gu I ty In the usage of gene, he wou Id ask every genetl cl st he met hew he used the term, and he then promptly cataloged his colleagues according to whether they thought of the gene the way he did or the way Beadle did. The person asked did not, of course, need to worry about his answer because he would be sure to be In good company In either case.

"Sturtevant read widely and was extremely well Informed on every topic of current interest, especially politics. He would, for example, read the Sunday ~ York TI mes and the Manchester Guard I an Weekly from cover to cover. He was especially happy If he could do the crossword puzzle In the Guardian at one sit­ting. Those who know those puzz I es w 111 know that only a very spec I a I breed of person attempts them, let alone solves them In one sitting. Sturtevant was fasci­nated with puzzles of al I kinds - especially puzzles Involving three-dimensional objects. When Anne Roe made a study some years ago of what makes scientists tick (The Making of~ Scientist), she wisely chose Sturtevant as one of her subjects. He was not only flattered, but overjoyed at the opportunity to take the tests, which he viewed as simply a new set of puzzles to work out.

"Sturtevant would develop a topic logically and succinctly, whether he was pub Ii sh Ing a paper or giving a formal lecture. In private conversation, how­ever, he always seemed to assume that the 11 stener was at I east as we 11 versed In the subject matter as he was, so he'd leave out the prellmlnarles and get right to the point. This could be mystifying to some; for others It was a challenge to be­come versed enough to profit by I lstenlng to his Ideas or by tapping the tremendous store of Information always at his fingertips on almost any topic of substance. His papers were so wel I written that one would assume that he had labored over each word. I have seen his panel lied manuscripts; they rarely contained more than a few minor word changes inserted Into the original draft, which was always done in

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longhand. I once asked him hCM he did this; he told me that he usually spent many oays mulling the paper over In his mind until all the words fell Into place, and then a I I he had To do was w rl te It down from memory.

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"Sturtevant's col leagues and students and friends at Caltech wl 11 always remember the warmth of his personallty. His love for people and for al I I ivlng things was expressed in many ways. For example, In 1954 he gave the presldentlal adoress before the Pacific Division of the American Association for the Advancement of Science, and he dealt with some of the social Implications of human genetics. In this address he warned of the hazards to human beings of the tal lout from the atmospheric testing pt atomic bombs. What had provoked Sturtevant was a strong statement issued by the executive branch of the government that the fallout levels from testing we re tar be I ow any that cou Id cause damage to human be! ngs.

"Although many assumed that the only purpose of Sturtevant's remarks was to halt bomb-testing, he was completely objective about the whole problem. He felt there might be a need for bomb-test! ng but that the pub It c shou Id be gl ven the best estimate thet scientists could make about the nature of the danger ot tal lout levels of radiation to the unborn. To use the language of today (some 16 years later), Sturtevant was decrying the credlbl I tty gap that was developing in the government's handling of Information on environmental pol lutlon with radiation.

"For Sturtevant, 11 te must have been an excl ting, reward Ing, and perhaps sometimes heartbreaking journey Into the unknown. It was fortunately a long 'jour­ney, which Involved many detours to many realms, and I am convinced that he savored every minute of It. His explorations In genetics will make the journey into the unknown a 11 tt I e eas I er tor the human race."

TEACH I NG

The introductions of a tutorial program and of a Biology "Scholars Pro­gram" for Biology juniors and seniors provide additional experimental approaches toward the objective of less structured curricula for students of adequate maturi­ty and self-direction. The Scholars Program permits the formulation of individual academic programs combining course work, independent study and research adapted to each student's Interests and requirements. Such programs are In principle com­pletely free in scope, subject only to the general requl rements of the Institute. Each program must be acceptable to and ts supervised by a faculty committee; work is undertaken and evaluated on the basts of a written "contract" between the student and his committee and Instructors.

These Initiatives exploit and explore the concept of the Institute - its faculty, Its laboratories, Its I ibrarles, Its courses and Its research programs -as a rl ch resource for the educatl on of the se I f-motl vated student. They seek to take advantage of the extraordinary qua I lty of our students and of our unique iy high ratio of faculty to students.

CONFERENCES

During this year the Division organized and hosted two symposia. In March a three-day conference was held on the "Biological Bases of Human Behavior." Seventeen speakers discussed evidence derived from anthropology, primate studies, genetics, physiology and biochemistry relative to the origins and variability of human behavior. Several of the more wel I-developed papers were published in the Apri I 1970 issue of Engineering and Science.

This conference was the first in a series of four to be held at the Institute in 1970 under the general title of "The Nature of Man."

In May a one-and-one-ha If-day conference was he Id on "Current Topi cs in Neurochomi stry." Nine speakers described recent progress in such fie Ids as the biochemistry of neurogonic amines, axonal transport, and neuroendocrine relations.

Symposium Participants

9

Left to right: R. Price Peterson, Univers~!Y of Pennsylvania; David L. Barker, Harvard Medical School; William O. McClure, University of Illinois; R. Wayne Albers, National Institute of Neurological Diseases and Stroke; Bruce S. McEwen, The Rocketol ler University; Donald W. Pfaff, The Rockefeller University; Roger A. Gorski, University of California, Los Angeles; Samuel H. Barondes, University of California, San Diego; Julius Axelrod, National Institute of Mental Health (not shown).

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LECTURESHIPS

The fl rst Jean Welgle M~morlal Lectureship was presented on Feoruary 12, .:~ 1970 by Professor Herman Kalckar of Harvard Medlcal School. His lecture subject ! was "High Affinity Perlplasmatlc Galactose Binders in E. coll Mutants: Correlations'! with Transport, Scavenging, Chemotaxls."

The first Albert Tyler Memorial Lecture was presented on May 26, 1970 by Dr. Thaddeus Mann, Marshal I-Walton Professor of Physiology of Reproduction at the University of Cambridge. Dr. Mann's subject was "Research Problems in Animal Re­production." Dr, Mann also presented a seminar on "Spermatozoa and Spermatophores" and led a group discussion on "Present Trends In Reproductive Biology" during his visit here.

Dr. Horman Kalckar

Dr. Thaddeus ~ann

11

STAFF OF INSTRUCTION AND RESEARCH

DIVISION OF BIOLOGY

Robert L. Slnshelmer, Chairman

Ernest G. Anderson, Ph.D. Henry Borsook, M.D., Ph.D. George E. MacGinltle, M.A. * Alfred H. Sturtevant, Ph.D.

Giuseppe Attardl, M.D. Seyroc>ur Benzer, Ph.D. James Bonner, Ph.D. Charles J. Brokaw, Ph.D. Max Delbrilck, Ph.D. WI JI lam J. Dreyer, Ph.D. Rebert S. Edgar, Ph.D. Sterl Ing Emerson, Ph.D. Derek H. Fender, Ph.D. Arie J. Haagen-Smlt, Ph.D. A I an ,I. Hodge, Ph.D. t Nerman H. Ho row I tz, Ph .D. Edward B. Lewis, Ph.D. Herschel K. Mltchell, Ph.D. James 0 I ds, M. D., Ph.D. Ray D. Owen, Ph.D., D.Sc. Robert L. Slnsheimer, Ph.D. Roger W. Sperry, Ph.D. Fe 11 x Strumwasser, Ph.D.

Professors Emert ti

Genetics Biology Biology

Thomas Hunt Morgan Professor of Biology

Professors

Biology Biology Biology Biology Bl elegy Biology Biology

Genetics Biology and Applied Science

Biology Biology Bl ology

Anthonie Van Harreveid, Ph.D., M.D. Jerome Vinograd, Ph.D.

Thanas Hunt Morgan Professor of Biology Biology

Behavioral Biology Biology

Biophys I cs Hixon Professor of Psychobiology

Biology Physiology

Chemistry and Biology Bio logy Corne I is A. G. Wiersma, Ph.D.

Geoffrey L. Keighley, Ph.D. Ken-lchl Naka, Ph.D. WI I I i am S. Stew art, Ph.D.

* Deceased Apr! I 5, 1970 t Not on campus

Research Associates

Biology Biology and Appl led Science

Biology

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Harbans L. Arora, Ph.D. John W. Kauffman, Ph.D.

William B. Wood, Ph.D.

Visiting Associates

Associate Professor

Senior Research Fei lows

David S. Dennison, Ph.D. Romi I io T. Espejo, M.S. Eva Fifkova, M.D., Ph.D. WI II i am R. Gray, Ph.D. Evelyn Lee-Teng, Ph.D. Peter H. Lowy, Doctorandum Robert W. Meech, Ph.D. Marianne E. Olds, Ph.D. John A. Petruska, Ph.D. Lajos Pik6, D.V.M. Colwyn B. Trevarthen, Ph.D.

Daniel McMahon, Ph.D. James H. Strauss, Jr., Ph.D.

Assistant Professors

Gosney Research Fe I lows

Ruxton H. VJ llet, Ph.D. Mogens Westergaard, Ph.D.

Yosef Alon I, Ph.D. I Stephen Arch, Ph.D.2 Kimiko Asano, Ph.D. I rvlng H. Brown, Ph .o.3 Gisela Charlang, Ph.D. David A. Clayton, Ph.D. Natalie S. Cohen, Ph.D. Robert J. Cohen, Ph.D.2 Nachum Dafny, Ph.D. Thomas A. De VI i eger, Ph .D .4 Lawrence B. Dumas, Ph.D.2 David B. Dusenbery, Ph.D.2 Robert Eason, Ph.D. James M. England, Ph.u.2 Arnold Eskin, Ph.D. I Linda Fagan, Ph.D.

Research Fe I lows

Akio Fukuda, Ph.D. El lls E. Golub, Ph.D.2 Gerhard G!lpe I, Ph.D. 5 Richard L. Hallberg, Ph.D.2 Thomas E. Hanson, Ph.D.6 Stephen E. Harris, Ph.D.2 Robert T. Heath, Ph.D.2 Ross B. Hodgetts, Ph.D.7 Yoshiki Hotta, Ph.D. Muneyuki Ito, M.D. Stanley Jaffee, Ph.D. Harumi Kasamatsu, Ph.u.8 Santosh Kumar, Ph.D. Marion T. Laico, Ph.D. Muriel Lederman, Ph.D.2

Biology Biology

Biology

Biology Biology Biology Biology Biology Biology Biology Biology Biology Biology Biology

Biology Biology

B lo logy Biology

Hsuoh-Jei LI, Ph.D. Is rae I Lieb I i ch, Ph .D. Ronald B. Luftlg, Ph.D.6 Livia Pica Mattoccla, Ph.D. John E. Mayfield, Ph.D.2 Robert F, Mayol, Ph .D.2 John R. Merriam, Ph.D.2 Ma0; B. Mitchell, M.A. Carl R. Morgan, Ph.D.2 Leelavatl D. Murthy, Ph.D.9 David S. Papennaster, M.D. Margarete Petzuch, Ph.D.2 Anthony G. Ph i I I I ps, Ph .D. MI ch ae I I . Ph I I I I ps , Ph • D • John S. Pierce, Ph.D. I I Ralllf"lndra K. Poddar, Ph.D. Aharon Razin, Ph.D.2 Bernard M. J, Revet, Ph.D. Erkkl I. Ruos Jahtl, M.D.2

I I nternatlona I Agency for Research

on Cancer Fe 1 IOll 2National Institutes of Health,

Pub Ii c Hea I th Service Fe I low 3Natlonal Cancer Institute of

Canada Fe I I ow 4

Free Un 1 vers I ty, Amsterdam 5North Atlantic Treaty Organization

Fellow '6

National Science Foundation FellOll 7Jane Coffin Chi Ids Memorial Fund

for Medical Research Fel '°"

Mark G. Rush, Ph.D. Gernot Sander, Ph.D. Dennis G. Searcy, Ph.D.2 Yurly M. Sivolap, Ph.D.12 John E. Smart, Ph.D.6 Ellen G. Strauss, Ph.D. David T. Sul JI van, Ph.D.2 Henry T. Tai' Ph.D. 13 Keh-Ping Ting, Ph.D. Janett Trubatch, Ph.D. Alex J. Van der Eb, Ph.D.14 Sharon T. Vaughan, Ph.D. Mary Avis Weir, Ph.D.2 J. Mi liar Whalley, Ph.D. David L. Wi Ison, Ph.o,8 Jung-Rung Wu, Ph.D. II Keljl Yanaglsawa, Ph.D. Myonggeun Yoon, Ph.D.2

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8 Helen Hay Whitney Foundation Fellow

91nternatlonal Atomic Energy Agency

Fellow 10 American Cancer Society Fellow 11

Damon Runyon Memorl a I Fund Fe I low 12

1nternatlonal Research and Exchanges Board Fe 11 ow

13unlverslty of Southern Cal I fornia

14 Nether I ands Organ I zatl on for the

Advancement of Pure Research Fe I low

Graduate Fellows and Assistants

Barbara F. Attard I, B.S, I Steven K. Beckendorf, A.B.2,3 Robert M. Benbow, B.S.4 Larry I. Benowltz, B.E. I Kostia Bergman, B.A.I Charles R. Bl rdwel I, B.s. I Steven A. Bissel I, M.S.1 Alan 8. Blumenthal, A.B.1 Wesley M. Brown, M.A.3 Patricia N. Burke, B.A.I E. Denise Campbell, B.A.* Ming-Ta Chong, M.D.5 J. Lee Compton, A.B. I Sydney P. Craig, B.S.6 Jane H. Cramer, B.S.I John W. Cross, Jr., B.A. I

Stephen G. Oennls, S.B.i** Tom C. Douglas, A.B.3 W. Jack Driskell, B.s.I Moises G. Eisenberg7 . 2 Sarah C. R. Elgin, B.A. EI I en J • E I I i ott, B, A , 2 Richard A. Flrtel, A.B.2 P, John Flory, B.A.I Kenneth w. Foster, B.s.5 Jeffrey A. Frelinger, B.A.I Stanley C, Froehner, B.S. I John E. Geltosky, B.S.3 E. WI II i am Goode II , B. S. I Harold W. Gordon, B.S. I David E. Hiatt, M.A.I Wiiiiam A. Hill, B.A.I

14

David S. Ho.mes, B.A.5 Wray H. Huestis, B.A.2 John B. Jamieson, B.A.6 Alglrdas J, Jesaltis, B.S.3 Richard D. K. Joss I in, S.B.I Michael B. Klayman, B.S.I Ronald J. Konopka, B.S.2 Carol L. Kornb I Ith, M.A. I Lee-Ming Kow, M.S.5 Pau I T. Langstroth, B.A. ·i· Jane E. Latta, A.B.I . Jerre Levy, M.S. I Mary Ann Linseman, B.A.5 Cary Lu, A.B.9 David J. Mcconnel I, B .A. 10 Paul S. l'.e\tzer, A.B.2 ';,usan \.. Vlel'<\n, '2..1\,\ Ronald L. Meyer, B.A.3 Mark J. Ml I \er, B.1\.6 WI 111 am I. Murphy, B.S.2 Robert D. Nebes, B.S.I John E. Newbold, B.Sc.5 Charles E. Novitski, B.A.6

1National Institutes of Health Graduate

Research Training Grant 2Natlonal Science Foundation Fellow

3Natlonal Defense Education Act Fellow 4

Earle C. Anthony Fel ION 5

11-rthur McCal \um Fellow 6~ationa\ Science Foundation Graduate

\raining Grant

* On leave of absence

** On leave of absence third term

-r Deceased December 12, 1969

John S. Parkinson, A.B.5 Jeffrey L. Ram, A.B.2 Donald L. Robberson, B.s.6 Robert G. Rohwer, B.S. I Barry S. Rothman, B.S. I Gary Scheidt, B.s.2 John w. Sadat, B.A.I WI I I lam D. Seybold, B.Sc.5 Danie\ T. Slnvnons, B.s.I c. Allen Smith, s.B.I Lloyd H. Smith, B.s.3 Brian Storrle, B.S.2 David Tang, A.B.I Wiiiiam A. Thomasson, M.A.9,1 Clark J.B. Tibbetts, B.A.I Lois A. Toevs, B.A.I L.c I-\; f>n I\ • Tt!>\<.(>s , 12. • S • I Jessica Tuchman, B.1\.1 S~ue\ Ward, l\.B.6 John H. WI Ison, A.B.4 Sandra Wlnlcur, M.s.I Anthony J. Zuccarelli, M.s.2

7 Rockefe 11 er Foundatl on Fe 11 °"

8Beschorman Fe I I ow 9 •

National Institutes of Health, Public Health Service Fel\C4'1

10Lucy Mason Clark Fellow

Administrative Assistant

Lib rarl an

Dana L. Roth

Visiting Biologist

Solomon A. Kaplan

Eugene T. Bol It

Consu I tants

WI II lam M. Blackmore, D.V.M. Joseph E. Bogen, M.D. Ronald E. Saul, M.D.

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Research Engineer

Floyd Schlechte, B.S.

Engineering Test Assistant

James J. Gilliam

Computing Analyst

Jordan Rosenberg, M.A.

Laboratory Special lsts

WI I 11 am J • A II an David M. Schembe rger, B .S. Ronald W. Siemens, B.S.

Associate Biologists

Eliana C. Espejo, M.S. Inge S. Frahm Patrl ck F. Koen Robert M. Watson, A.A. Laverne T. Wenzel, 11.B.

Electron I c Spec I a I I sts

Comellus M. Dekker Kenneth L. Ho I I is Peter Jonkhoft

Research Assistants

Renate Alvarez, M.T.A. Bruce R. Benedict, M.S. lngelore S. Bonner Kathleen R. Burroughs, B.A. Denise I. David, B.S. Gloria C. Davis, A.A. Ursino E. Del Val le, B.S. Ruth J. Dusenbery, Ph.D. Jean Edens Ruth E. Estey W II 11 am G. Fry, M .S. Kay D. Grelsen, B.S. George Horn Sarah M. Ingersol I, M.A. Ruth T. Johnson Bertha E. Jones Nancy C. Joyce, B.A. Benne ta Kee I ey May G. Kelgh ley, B.A. Sandra J. Kiddy, M.S. Li I a Mendenh a I I Kort Charles D. Kreuter, M.A. 1 lga Lielausis David E. Lindsay, S.S.

Suzy Bower

Elizabeth P. Blankenhorn, A.B. Eve Ii ne El chenberger

Harriett L. Lyle, B.A. Lois E. MacBI rd J. Geoffrey Magnus, M.S. Margaret T. Novitski, B.A. Deanna K. Ojala, B.S. Josephine Pagano Cornell C. Phil lips, B.S. Margaret M. Ph I I 1 l ps Ml ldred A. Quisenberry, M.S. John Raes, B.S. Umavathl Ramaswamy, B.S. Patricia M. Reau, B.S. Joan L. Roach, B.A. Marla Rouklove Loverlza A. Sarmiento, B.S. Karen B. Searcy, M.A. Alma J, Shafer, B.A. Amel la M. Smit Marlyn R. Tep I itz, M.S. Ursula M. Tracy Sandra M. Webb, M.S. Lydia C. Yuan, B.S.

Laboratory Ass I stant

Research A I des

Susan F. Foster, B .A. Ee f Goedemans

15

Josie E. Jaraml I lo, S.S. Rosina K. T. Kinzel Buda J. Martonyl Walter K. McClelland, B.S. Jeanette Navest Wanda L. Owens She I I y A • Hempe I

Ludi a Br01Vn Shirley M. 13r0\'ln Christine U. Edmonds Robert Eibergen Chery I A. Everton Sue Fl tch Melissa A. Honack

Marla Rivera, A.S.S. Denz I 11 e T. Stephens Barbara Van Gelder Maude E. Ve nnaes George. Ync I an Dahlia Zaidel, B.A.

Laboratory Al des

Jeannette Johnstone Lynda A, Macleod Judith E. Rush Kitty L. Van Bueren Jul le Van den Kolk Caro 11 ne Vermaes Irene E. von Hartmann

California Foundation for Biochemical Research Trainees

David I. Lewin H. Peter Zassenhaus

National Science Foundation Undergraduate Summer Research Trainees

Charles V. Barber James R. Cooper, Jr. Stephen F. Cooper David E. Fast Al fen G. HI rsh Jeffrey B. Hurn

Michael J. Abrams Robert W. Brackenbury Peter C. Brown Paul T. Christensen Dennis L. Di Bartolomeo J 111 D. Fabri cant Michael B. Farber Robert S. Fi sher Steven Flanagan Yee-WI ng Fong James L. Gou Id Michael A. Henerey Jeffrey 8. Hurn

Marci a J. Lev1 ne Douglas A. Lockwood Ann L. Matthews Kevin R. O'Hara Jeffrey W. Salzer Jeanne M. Wal lace

Student Assistants

Doy le Kn I ght Victor Lee Robert K. Lew Is Michael C. Macleod Isaac Majerovlcz James H. Richards Charles J. Romeo, Jr. Cameron J. Sch I ehuber Jeffrey M. Schredder Robert W. Se I I ent Rand Wa I tzman

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Senior Instrument Specialist

Frank L. Ostrander

Maintenance Electrlclan

Machinist-Mechanics

Richard J. Broderick Dale L. LI nder

Super I ntendent

17

Ben F. Fisher Kerckhoff Marl ne Laboratory: Joe R. Deem

Division Secretary

Geraldine S. Cranmer

Secretarl es

Fannie L. Warren, Office Supervisor

Mary W. Elker Mi rl am H. El sberg Elizabeth T. Hanson

El I zabeth Koster Bern I ta K. Larsh Marijo Valene I ana

Jo Ann Ch I kah I ro Ruth M. Erickson

Accounting Assistants

Eleanora Kempees, Supervisor

Stockroom

Isabel la Lubomirskl Margaret B. W rl ght

Jane C. Keasberry, Acting Supervisor

WI I I I am F. Lease Edward C. Lohr

Kare I Burm Ml lton D. Grooms Eml le Lemmens

Amos Ch rl sty Benny De Rul ter Harvey Ma I one

Aleksandar Petrovich

Animal Room Staff

E. K. Mayfield, Supervisor

Building Custodians

Ernest Radney Hendrl k S. Ro rye Daniel R. Shaw

Marius Weintre, Supervisor

George San Diego Jimmy Walker John Warnar, Sr.

18

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BIOCHEMISTRY :1 ~ '

IMMUNOGENETICS

BIOPHYSICS :;

:;' Kerckhoff - Church \i ,, 1:: (First Basement)

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Professor: ,/ ames Bonner

Research rel lows: Mayfield, Carl R. Yuriy M. Sivolap,

Stephen E. Harris, H. Gunnar Jansson, Hsueh-Jel Li, John E. Morgan, Leelavatl D. Murthy, Gernot Sander, Dennis G. Searcy, Keh-Pl ng Tl ng, Jung-Rung Wu

Gradu~te Students: Ming-Ta Chong, Sarah C.R. Elgin, Richard A. Flrtel, Stanley C. Froehner, Davids. Holmes, David J. McConnel I

19

Research Assistants: lngelore S. Bonner, Denise I. David, Nancy C. Joyce, Cornell C. Phillips, John Raes, Anll Sadgopal

Laboratory Aides: Ludla Brown, Sue M. Fitch

The work of our group concerns the mechanisms by which the genes of the ch romosanes of h I gher organ Isms are caused to be repressed, turned oft, or de re­pressed, turned on. Understanding of these matters is of interest because we now know that development and differentiation are due to the orderly sequential turn­ing off and on of the appropriate genes at the right times in the right places. We ask, how does this work? We study the question with Isolated lnterphase chromo­sanes, ch rornatl n.

On the level of repression Itself we know that the portion of the genomal DNA of higher organisms which Is complexed by histone is turned off, that is, not available for transcription by RNA polymarase. Our earlier work has sho.;n that there are 7 major hlstone species, that their primary structures have been amazing­ly conserved during evolution (2 conservative amino acid replacements among the 102 residues of hlstone IV as between pea plant and cowl, and that binding of histone to DNA, altl1ough it prevents transcription, does not prevent replication. The na­ture of the hf stone-DNA complex Is examined in detal I below.

Chromatin contains proteins which are not hlstones. We have devised methods for their separation and study. There are a limited nuniler, ca. 12, of major nonhistone chromosomal proteins and they are quite slmi lar as between chro­matin of d I fferent organs of the same creature and even as between different crea­tures. We suspect they represent enzymes of chromosome metabolism: in any case, none have been found to have specific repressor activity. The same is true of the DNA-binding proteins of the nucleoplasm. Since a principal enzyme of chromosome me­tabolism Is RNA polymerase we are purifying this enzyme from higher creature nuclei so that we can compare its properties with those of the wel I known E. coli RNA po I yme rase.

It has been known for some time that histones do not read base sequences, but cornblne with DNA of any kind if left to their own devices. We ask the question, how are histones deposited upon the correct genes, those which are to be repressed? The answer to this question appears to lie in a further constituent of the chromo­sane, chromosomal RNA (cRNAl. cRNA molecules constitute a distinctive class of RNA, characterized by short length (40-200 nucleotides in different creatures), high con­tent of dlhydropyrimldlne (ca. 10 mole%>, high sequence diversity, abl I lty to hy­bridize to a large proportion of the genome (in general 2-5% of denatured DNAl, and ability to form complexes with native daub le-stranded DNA. In the chromosome, cRNA is covalently bound to protein which, as we show below, Is of a single species, at least In the rat. We have ear II er shown that the part I cl pati on of cRNA Is requl rad for sequence-specific interaction of histones with DNA. We now ask the question, does cRNA guide hlstones to the appropriate sites or does it flag and mark those sequences, genes, which are not to have hlstones deposited upon them. The latter

20

is correct. We show this by dissociating chromatin of a given organ into Its rro­lecular components, adding to the mixture separately purified cRNA (with protein attached) prepared from chromatin of a different organ of the same creature, and reconstituting the chromatin. We show below ·That the template activity of the hy­brid reconstituted chromatin is equal to the sum of the template activities of the two different chromatins tested separately, and by DNA-RNA hybridization, that In the hybrid chromatin the genes active In the chromatin which served as donor of the heterologous cRNA are active, as well as those active In the original receptor chromatl n.

Clearly, then, cRNA together with Its binding protein somehow causes his­tone not to be deposited along a length of DNA. How It does so we do not yet know.

cRl>lA is then a central element in control of gene action In higher orga­nisms. We have therefore concentrated on Its properties. We have found that cRNA interacts and binds to the repetitive sequences which characterize the genomes of higher organisms. By the same token that cRNA Is a control element, so also are the repetitive sequences whose function has been hitherto unknown. Since cRNA mole­cules are short one would expect the repetitive sequences to be short also. We have devised a method for visualizing them by electron microscopy and have found that the repetl t Ive sequences are indeed only ca. 150 base pa I rs In I ength and that they are interspersed among the unique DNA sequences which we presume represent the· structura I genes.

cRNA becomes labeled only slowly In pulse-labeled eel Is. We have there­fore looked for molecules which bear a precursor relationship to cRNA. It has been known for several years that in pulse-labeled eel Is, a particular class of nuclear RNA becomes rapidly labeled. This Is the so-called rapidly-labeled, high molecular weight, rapidly-turning over, nuclear RNA. This RNA, of peak sedimentation coeffi­cient ca. 705, Is again cleaved without leaving the nucleus, hybridizes to the repet­itive portion of the genome and hybridizes with some 3-8% of denatured DNA. We have found by hybridization-competition that substantially al I sequences found In cRNA are found In rapidly-labeled nuclear RNA. We presume such RNA to be the precursor of cRNA. The long tapes of rapidly-labeled nuclear RNA, each containing many dif­ferent sequences, are, we presume, specifically cut to cRNA length, bases then modi­fied to introduce the required dlhydropyrlmldine, and each resulting cRNA molecule attached to a molecule of binding protein.

We have sought to study the sequence of events which accompany the turn­ing on of previously repressed genes in a variety of systems Inducible by hormones, In all of the cases we have studied - estrogen/uterus, cortisone/liver, insulin/liv­er, plant hormones/plant tissue - genes are turned on In large groups and template activity for RNA synthesis of chromatin Isolated after administration of hormone In vivo is Increased by 30-80% in the several cases. The system which has, however, yielded the most Insight Into this matter Is that of the regenerating rat liver. Within a few minutes after removal ot 2 of the I Iver lobes, new species of rapidly­labeled nuclear RNA begin to be produced and total number of rapidly-labeled se­quences present doubles within an hour. With a lag of ca. I hour, new sequences of cRNA appear and these then double within ca. 4 hours. Template activity of I Iver chromatin for RNA synthesis Increases with a lag of ca. 2 hours, and reaches Its maximum, an increase of 60%, in ca. 6 hours. We Imagine that the act of excision of I Iver lobes results In a chemical signal which derepresses sensor genes which then produce the relevant rapidly-labeled nuclear RNAs. These are processed Into cRNA molecules which then bind to the relevant repetitive sequences, each adjacent to a repressed structural gene. Such binding would appear to be the signal for hlstone removal, a process which we are currently studying.

l !

I

21

We are sti I I far from total understanding of the regulatlon of gene ac­tion. We have however made significant progress toward an overal I view of the prlnclples which underly this central process.

'

I. SEQUENCE STUDIES ON CHROl-OSDMAL RNA AND STUDIES ON CHROl-OSOMAL RNA BI ND I NG PROTEIN

I nvestl gator: James Bonner

Support: Natlonal Institutes of Health, Publlc Health Service

Ch romosoma I RNA ( cRNAl Is known as a nat Ive component of pea bud chroma­t In, chick embryo chromatin, Novikoff ascltes chromatin, and calf thymus chromatin. Such cRNAs represent a new class of RNA characterized by their high (ca. 10 mole%> content of dlhydropyrlmldlne, short chain length (40 to 200 nucleotldes depending on source), hybridization properties which are unllke those of m-, t-, or rRNA, and by the fact that they are covalently I Inked to a protein which we here designate chromosomal RNA binding protein. We have further studied the nature and nucleotlde sequences of cRNA and the nature of Its binding protein.

We have found that the ollgonucleotldes produced by hydrolysls of cRNA with Tl and/or pancreatic rlbonuclease digests contain a semi-Infinite number of ollgonucleotldes. This fact supports the Inference that cRNA is hlghly sequence heterogeneous. We have looked for evidence as to whether cRNA contains any uni­versal sequences as have been found In tRNA. None were found with the exception of the 5 1 termlnal pCP and the 31 termlnal pyrimidine pCOH· The finding that several of the Tl RNase-produced ol lgonucleotldes contained dihyaroT (dfhydrorlbothymldlnel Ind I cates that these res I dues are not a II c I ustered at one pos I ti on In the molecule.

The behavior of the cRNA binding protein on acrylamlde gel electrophore­sis, gel filtration, and In the ultracentrlfuge provides strong evidence that the protein Is sequence homogeneous. The siml lar distributions of amino acids around different RNA protein attachment sites also supports this view. In any case, the cRNA binding protein of rat ascltes tumor eel Is would appear to be a single, homo­geneous mater I a I •

This work was done In col laboratlon with Dr. Ralph A. Jacobson, Depart­ment of Chemistry, University of Oklahoma, Norman, Oklahoma.

2. CHROl-OSOMAL RNA A POSIT I VE AGENT IN CONTfnL OF GENE ACTIVITY

I nvestl gators: James Bonner, Nancy C. Joyce

Sup port: National Institutes of Health, Public Health Service

We have shown earl fer (Bekhor, Kung and Bonner, 1969) that participation of chromosomal RNA CcRNAl Is required tor sequence-specific interaction of chromo­somal proteins with chromsomal RNA. Thus chromatin Is dissociated Into Its molec­ularly dispersed components by the presence of 2 M NaCl-5 M urea. The chromatin Is now reconstituted by removal of the salt by gradient dialysis fol lowed by re­moval of the urea. RNA transcribed from such chromatin Is ldentlcal to that tran­scribed from native chromatin, as judged by hybridization-competition. If, however, cRNA Is removed from the dissociated chromosomal mixture and reconstitution then performed, the repressor proteins deposit randomly on the DNA template and RNA

22

transcribed from It is ineffective in competing with RNA transcribed from native chromatin. We na; ask, does cRNA guide the repressor hlstones to the genes to be repressed, or does it protect those genes which are to be active from hlstone de­posltlon7 We have found that the latter ls true. To dissociated pea cotyledon chromatin was added cRNA (with Its protein attached) prepared from pea bud chromatin. After reconstitution the template activity for RNA synthesis of the hybrid chroma­tin was tested and found to be equal to the sum of the template activities of pea cotyledon and pea bud chromatins. The RNA thus formed was found to be effective In competing with RNA transcribed from pea bud chromatin (which RNA transcribed from native pea cotyledon chromatin is not). Similar results are found when cRNA from pea cotyledon chromatin is added to dissociated pea bud chromatin and the mixture then reconstituted. It appears therefore that the presence of particular cRNA molecules protects particular DNA sequences from hlstone deposition. cRNA is a positive agent which results in gene activation. We have In our hands a tool tor the derepression of particular and specific classes of genes.

Reference:

Bekhor, I., Kung, G., and Bonner, J. (1969) J. Mel. Blol. 39: 351.

3. INTERACTION OF CHROt>IOSOMAL RNA WITH THE REPETITIVE SEQUENCES OF THE GENOME

Invest I gators: Yurly M. Slvolap, James Bonner

Support: International Research and Exchanges Board National Institutes ot Health, Publlc Health Service

It is known that participation of a species of chromosomal ly-assoclated RNA (chromosomal RNA, cRNAl Is essential to sequence-specific Interaction of chro­mosomal proteins with DNA. In this function cRNA Interacts and binds to DNA. The DNA of higher organism; Is composed ot sequences each repeated a single time In the genome, the unique sequences, and ot sequences repeated a few to many times per ge­nome, the repetitive sequences. We ask the question, with which of these classes ot DNA does cRNA lnteract7 We have found that cRNA Interacts with the repetitive sequences. Since cRNA is establlshed as a control element of the chromosomes of higher organism;, it tel lows that the repetitive sequences of the DNA are llkewlse, in part at least, also control elements.

Whole chromosomal DNA of pea plants was separated Into 3 classes on the basis ot degree of repetitiveness. Class I consists of sequences each repeated an average ot 10,000 times; class I I consists of sequences each repeated an average 240 times, while class 111 consists of sequences repeated an average of I time. cRNA from homologous chromatin hybridizes extensively to repetitive DNA of class I, less extensively to repetitive DNA ot class I I, and barely to DNA of class I II. In one sense It Is already known that cRNA Is complementary to and interacts with t~e ia?et\t\~e sequences of the genome. Thus, In earlier studies of Bonner and Widhalm< 1967) hybridization to saturation ct pea cRNll w\t'n aenatll>ea ?ea 01-111 'rias obtained at a time and RNA concentration value of Cot 10 <moles per liter x sec­onds). Significant hybridization ot RNA to unique DNA sequences does not occur over this time period. Values slml lar to those reported by Bonner and Widhalm have been reported by Dahmus and McConne 11 ( 1969) and Huang and Huang ( 1969) tor hybrid-1 zatl on of cRNA of rat ascltes tumor and of chick embryos to their respective DNAs. The ti ndl ngs of our present study underscore the earl ler ones, and by a di tterent experlrrental approach emphasize the Interaction of cRNA with the repetitive se­quences of the genome.

Re fe re nces :

Bonner, J. and Widhalm, J. (1967) Proc. Nat. Acad. Sci. 57: 1379.

Dahmus, M. and McConnel I, D. (1969) Biochemistry 8: 1524.

Huang, R. c. c. and Huang, P. C. ( 1969) J. Mol. Biol. 39: 365.

4. STUDIES OF THE SIZE AND DISTRIBUTION OF THE REPEATED SEQUENCES OF CHRJM:JSOMAL DNA

* I n vest I gators: Jung-Rung Wu, Jeffrey Hurn James Bonner

Support: Damon Runyon Memorial Fund National Institutes of Health, Public Health Service

23

The chrcxnosomal DNA of higher (eukaryotJc) organisms consists In part of sequences each repeated but once and believed to be the structural genes, and In part of sequences repeated many times, the repetitive or redundant sequences. We have studied the length and distribution of the repetitive sequences. It ls known that about one-half of manrnal Ian DNA consists of repetitive sequences. The genomes of simpler creatures are characterized by a lesser proportion of repetitive se­quences. We have used DNA of Tetrahymena (genome size BX that of E.coli and 8% repetitive) and of Drosophl la (genome size 50X that of E. coll and 24% repetitive). The DNA was sheared to an average molecular weight of 7 x J05 ( IOOO base pairs), denatured by heating to J00°C, and then reannealed (25° below Tml for periods such that only the repetitive sequences reannealed. The reannea-led samples were exam­ined by two variations of the standard basic protein (Klelnschmldtl technique for electron microscopy. By the standard aqueous technique only double-stranded DNA is visualized and single-stranded DNA collapses Into bushes because of random base pairing Interactions. Addition of fonnamlde to the mounting medium permits vlsual­lzatlon of single- as well as double-stranded DNA.

We have found by the fl rst method that the repetl ti ve segments of both Tetrahymena and Drosophl la DNA are 150-180 nucleotides In length. We have shown by the second technique that In Tetrahymena the repetitive sequences are distributed according to a very uniform pattern, namely, singly and at spacings of 1000-1500 bases. Thus the repetitive segments are separated by stretches of unique DNA se­quences of approx I mate I y gene I ength. In Drosoph I I a DNA, one to three d I f fe rent repetitive segments are clustered and slml larly separated by gene length stretches of unique DNA.

5. THE BI OGENES IS OF CHROM:JSOMAL RNA

I nvestl gator: Leelavatl D. Murthy

Support: International Atomic Energy Authority National Institutes of Health, Public Health Service

It Is known that when the eel Is of higher organisms are pulse-labeled for a short period, 1-30 minutes, with precursors of RNA, a major portion of the incor­poration is Into a traction now known as rapidly-labeled, high molecular weight,

* Undergraduate, Callfornla Institute of Technology.

24

nuclear RNA. Among the are largely orentlrely within the nucleus.

properties of such molecules are (I) the relevant sequences confined to the nucleus, and (2) they turn over entirely

It is also known that nuclei contain a class of low molecular weight RNA, chromosanal RNA (cRNAl, associated with chromosomes. These molecules are character­ized by their small size, high content of dlhydropyrlmldlne, and covalent attachment to a particular cRNA-blndlng protein. It is known that the participation of cRNA ls essential to DNA sequence-specific binding of chromosomal proteins to cRNA (Bekhor et al., 1969; Huang and Huang, 1969). Rapidly-labeled, high molecular weight, nuclear RNA and cRNA hybridize to denatured DNA with slml lar characteris­tics. Thus, both RNA classes hybridize with large fractions of the genome, 4% of whole DNA in the case of RNAs from Ehrlich ascttes tumor cells. Both also hybrid­ize principally to the repetitive segments of the genome, as is evidenced tran the rapidity of their hybridization reactions. We have Isolated rapidly-labeled, high molecular weight (sedimentation coefficient of 70S and over>, nuclear RNA from ascltes tumor cells. We have also Isolated cRNA from such cells following the pro­cedures of Dahmus and McConnell (1969). We have found by hybridization canpetitlon that cRNA ts a highly effective competitor to the hybridization of rapidly-labeled, high molecular weight, nuclear RNA to denatured DNA. Apparently al I sequences con­tained in cRNA of ascltes tumor eel Is are also found in high molecular weight, rapidly-labeled, nuclear RNA. The latter does, however, contain some 10 to 12% of sequences not found In cRNA.

The kinetics of labeling of rapidly-labeled, high molecular weight, nu­clear RNA and of cRNA are also in agreement with the supposition that rapidly­labeled, high rrolecular weight, nuclear RNA Is the precursor of cRNA. It would appear, therefore, that cRNA is generated in the form of long tapes, each contain­ing many different sequences of cRNA. After the production of these transcripts, the long tapes are apparently cleaved sequence-specif I cal iy by appropriate enzymes, the dihydropyrimidlne introduced Into the cRNA molecules at the appropriate places, and the thus altered molecules covalently fastened to cRNA-btndlng protein.

Re te re nces :

Bekhor, I., Kung, G., and Bonner, J. (1969) J. Mol. Biol. 39: 351.

Huang, R. C. C. and Huang, P. C. (1969) J. Mol. Biol. 39: 365.

Dahmus, M. and McConnel I, D. ( 1969) Biochemistry 8: 1524.

6. ISOLATION OF HIGH MOLECULAR WEIGHT RNA BY GEL FILTRATION ON SEPHAROSE 2B

Investigator: David S. Holmes

Support: McCal !um Fund National Institutes of Health, Public Health Service

Whole nuclear RNA Isolated from rat ascltes cells by a method modi tied from Attard! et al. (1966) can be resolved into 3 different size tractions on the resin 5epharose 2B. The sample is applied in 0.002 Na phosphate, pH 6, and 0.001 M MgClz and washed from the gel with the same butter. The first peak, corresponding to the excluded volume, contains mainly RNA of sedimentation coefficient greater than 505 as determined by sucrose density gradient centrifugation. The second and third peaks contain RNAs of sedimentation coefficients 185 to 455, and 45 to i8S

25

respectively.

An advantage of the new method over the classical one of sucrose density gradient sedimentation is that large quantities of high molecular weight RNA can be freed from 45S, 28S, t8S and low molecular weight RNA In less than 3 hours.

Reference:

Attardi, G., Parnas, H., Hwang, M.-1. H., and Attardl, B. (1966) J. Mol. Biol. 20: 145-182.

7. IONIC STRENGTH EFFECT ON THE MOLTING OF NUCLEOHISTONE

I nvestl gator: Hsueh-Jel LI

Support: National Institutes of Health, Public Health Service

Native and partially dehlstonlzed nucleohlstones In 2.5 x 10-4 M EDTA, pH B.O butter show three melting bands, at 42 (f), 66 (If!) and 81°C CIV) respec­tively, with a shoulder near 52°C ( 11). Increasing the ionic strength of the s61u­tlon in which melting occurs reveals that ionic strength effect on Tm Is largest for melting band I, intermediate for melting band 111 and smal test for melting b'and IV. This Indicates that electrostatic shielding is least for those DNA segments of melting band I, Intermediate for those of melting band I II and largest for those of melting band IV.

Experiments with the removal of hlstones show thaf melting bands I II and IV are both due to complexing of hlstones to DNA. We estimate that the effective DNA charges on segments of melting band Ill are twice those available on segments of melting band IV.

8. RENATURATION OF NUCLEOHISTONE

I nvestl gator: Hsueh-Jet LI

Support: National Institutes of Health, Public Health Service

We have shown that hlstone binding to DNA in nucleohlstone results in two different extents of stab I lizatlon. We now ask the question, are these two kinds of DNA segments complexed by histones adjacent or close to one another? The method of study is to denature the lower-melting histone-DNA segments and to determine whether the higher-melting segnent, by virtue of its Intactness, keeps the DNA In register, and thus permits rapid reanneat Ing. We have found that the amount of the lower-melting histone-DNA segments reannealed Is Inversely proportional to the amount of the higher-melting histone-DNA segments denatured. Thus Intactness of the double helix of the higher-melting hlstone-DNA tends to enable restoration of the lower-melting segments to double helix form, even when the latter have been pre-denatured. These results support the view that the lower- and higher-melting hi stone-DNA segments are physically connected.

26

9. MODEL OF TWO MELTING BANDS BY HISTONE BINDING IN NUCLEOHISTONE

Investigator: Hsueh-Jei Li

Support: National Institutes of Health, Public Health Service

We have shown that histone binding to DNA in nucleohistone stab! llzes the latter to two different extents. In addition, it is known that molecules of his­tone IV, I I and I contain unequal distributions of basic amino acids along their lengths. Al I of these hlstones exhibit clustering of more basic amino acids in one end and a lesser concentration of such amino acids In the other end. We ask the question, does the more basic end of the molecule stab! lize DNA against melting more than does the less basic end of the molecule? Histone I lb2 has methionine residues in the middle of the molecule and these can be speciflcal ly cleaved by cyanogen bromide. The histone llb2-N terminal half and histone I lb2-C terminal half were purified by Sephadex G-75 and Amberlite CG-50 column chromatography. Their Identities were confirmed by both disc electrophoresis on polyacrylamide gel and by their amino acid compositions.

Reconstituted histone Ii-DNA complexes show two melting bands at 66 and 80°C. These are essentially the same as in native nucleohlstone. Melting spectra of hf stone llb2-N-DNA complexes show one melting band at 70°C while hlstone I lb2-c stabilizes DNA to 57°C. Thus histone I lb2-N with 0.32 basic amino acids per resi­due stabilizes DNA to 70°C which Is 13° higher than the stab! lization of DNA by hlstone I lb2-C with 0.20 basic amino acids per residue (57°Cl. It Is close to the difference between the two stab I llzations of DNA caused by native hi stone I I (80°-660C = 14°>.

These results, together with those of the previous reports concerning Ionic strength and renaturation effects, support a model in which the higher melt­ing hlstone-DNA segment is due to the binding of the more basic end of histone to DNA, while the lower melting hi stone-DNA segment is due to binding of the less basic end.

10. THE SUPERCOILED STRUCTURE OF NUCLEOHISTONE

Investigator: Hsueh-Jei Li

Support: National Institutes of Health, Public Health Service

Native nucleohlstone Is supercoiied with a pitch of ca. 110 ~. We in­quire into the origin of such supercol ling. One condition required for supercoil­ing is occurrence in the polymer of a structural unit which Is bent into a three­dlmenslonai arc and which occurs repeatedly along the molecule. The questions to be raised are whether nucleohistone contains such a structural unit and if so whether It appears periodically or at least quasi-periodically along the nucleohls­tone.

According to our model, the binding of a histone molecule to DNA results in two different kinds of segments, those to which the more basic end is bound and those to which the less basic end is bound. This is true for all hlstones even though there are differences among them as to amino acid composition, amino acid sequence, and length. A DNA segment bound by one histone molecule therefore con­stitutes a structural unit of nucleohistone. The next question then is whether such a histone-bound DNA segment is bent and therefore not a simple Watson-Crick helix.

•

\

\

27

Neither end of a hlstone molecule possesses strict regularity of distri­bution of basic amino acids and these do not completely neutral lze al I phosphates of the ONA to which the hlstone Is bound. Their Interaction with phosphates there­fore very probably occurs unevenly along the DNA segment. This is equivalent to applying an uneven force on the DNA segment, and this would tend to bend It. The two ends of the hlstone molecule have different proportions of basic amino acids and thus may form differently bent structural units. in nucleohlstone such hlstone­bound segments occur continuously along the molecule. Binding of histone to DNA therefore satisfies the conditions requl red for supercol I ing of nucleohistone.

I I . LIMITED HETEROGENEITY OF THE MAJOR NONH I STONE CHROM)SOMAL PROTEINS

Investigator: Sarah C. R. Elgin

Support: National Science Foundation National Institutes of Health, Public Health Service

There has been much discussion but little detal led work on the chemistry and biology of nonhlstone chromosomal protein CNHC protein). The principal h"ln­drances In their study have been the tendency of the proteins to aggregate and the difficulty in dissociating them from histone and DNA. We have Isolated the N~C proteins In soluble form In sodium dodecyl sulfate (SDS> and have analyzed them by SDS ge I electrophoresl s Cmolecu Jar we I ght s levi ng). Purl fled ch romatl n was used as the starting material. The hlstones were extracted with 0.4 N HzS04, and the remaining ONA and protein were then solubl II zed In 1% SDS, 0.05 M Tris, pH 8. DNA was next removed by ultracentrifugation; the NHC protelns'ln supernatant could then be examined by SDS gel electrophoresis. Our preparations of rat I Iver NHC protein include 13 major polypeptide bands of molecular weight ca. 5,000 to ca. 100,000. Homologous peptides are found In chicken liver NHC protein, whl le an additional high molecular weight band Is found In preparations from chicken erythrocyte. Rat kidney NHC protein Jacks 2 and possesses I additional band relative to rat I Iver NHC protein fractions. Pea bud NHC proteins Include half of these same bands. The striking siml larlty of NHC proteins of different organs and creatures suggests that some of them are common enzymes, such as those of nucleic acid metabolism, and/or convnon structural proteins <analogous to histones). Some of the apparent differ­ences may be organ and species specific.

Re fe ranees :

Elgin, S. C.R. and Bonner, J. (1970) Biochemistry {Jn press).

Elgin. S. C.R., Froehner, S. C., Smart, J.E., and Bonner, J. (1970) In: E. J. DuPraw Ced.), Advances In Cel I and Molecular Biology, Vol. I. Academic Press, New York (In press).

12. DNA-BINDING PROTEINS IN RAT LIVER NUCLEOPLASM

Investigators:

Support:

Keh-Ping Ting, Gernot Sander

National Research Councl I of Canada Deutsche Forschungsgeme I nschaft National Institutes of Health, Public Health Service

We ask the question, do the nuclei of the eel Is of higher organisms

28

contain proteins which bind nucleotide sequence-speclflcal ly to DNA and If so what are their funct1ons1 We have studied this matter with the soluble proteins which remain after the chromatin ls pelleted from lysed, highly purified, rat I Iver nu­clel. Such proteins Include a highly active protease. Study of the protein mix­ture requires that the protease be first Inactivated. This we accompllsh by treat­ment with dllsopropylfluorphosphate (DFPl. The nuclear protein mixture Is then applied to a DNA-cellulose column, prepared with homologous Cratl DNA, and eluted with a gradient of NaCl. The bulk of the nuclear protein binds at low C<O.I Ml 1 on le strength and e I utes at an NaCl concentratl on of 0. 3 M NaC I • Over 80% of the protein which binds to homologous DNA Is not retained on a DNA cellulose column of heterologous (E. coll) DNA •. The binding of this fraction Is therefore sequence­specific.

SOS acrylamlde gel electrophoresls of the speclflcal ly-bound fraction re­veals that It includes ca. 12 major and many minor components. These have been separated Into 5 tractions by chromatography on Sephadex G-100. 3 fractions exhib­it the property of Increasing rate of transcription of homologous DNA (but not of heterologous) by E. col I RNA polymerase. This effect ls not observed 1 f rat I Iver ch romat 1 n 1 s used as temp I ate and we presume such ch romat In conta I ns the transcrl p­t I on factors as Integral components. One fraction exhibits the property of inhib­iting transcription of DNA by E. coll RNA polymerase. Since this effect is found both with homologous and heterologous DNA It Is not a sequence-specific one.

We conclude that llver nuclel do contain sequence-specific DNA-binding proteins, that these include specific promoters of transcription, but that they do not Include sequence-specific Inhibitors of transcription.

13. CHARACTERIZATION OF MAMMALIAN RNA POLYMERASE

I nvestl gator: Stanley C. Froehner

Support: Natlonal Institutes of Health, Publlc Health Service

It ls known that eukaryotlc organisms contain multlple RNA polymerase ac­tivities (Roeder .and Rutter, 1969; Kedlnger et al., 1970l. Rat I Iver and calf thy­mus contain polymerase I (nucleolarl and polymerase II (nucleoplasmlcl whl le sea urchin embryos and rat embryos contain these as wel I as polymerase 111 (nucleoplas­mlcl. I am attempting to purify these 3 enzymes from Novikoff ascltes tumor eel Is, which contain large amounts of al I 3, to compare their subunit structures, and to determine whether they control gene expression as Is the case with E. col I RNA polymerase. Ear Iler Isolation procedures have been Improved, thus facl lltatlng the extraction of large quantities of tissue. The crude extract ls then purified on DNA eel lulose which binds only ca. 5% of the total nuclear protein. Further puri­fication and separation of the multlple forms ls accomplished by DEAE-Sephadex chromatography •

Comparison of the subunit structures of marrmallan and E. coll RNA polymerase may yleld insight Into the problems Involved In transcription of mamma­l Ian DNA and chromatin with exogenous bacterial polymerase (Blology 1969, No. 58),

Re fe ranees:

Roeder, R. G. and Rutter, W. J, (1969) Nature 224: 234.

Kedlnger, C., Gnlazdowskl, M., Mandel, J. L., Jr., Glsslnger, F., and Chambon, P. (1970) Blochem. Blophys. Res. Commun. 38: 165.

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14. HORl"ONAL INDUCTION OF NEW GENE EXPRESSION BY ESTROGENS

I nvestl gators:

Support:

Dennis G. Searcy•,. Ml ng-Ta Chong

National Institutes of Health, Public Health Service McCal !um Fund

29

The response of the uterus of ovarlectomlzed animals to estrogen is known to be the rap Id express I on of genes p rev I ous ly repressed. We have fo 110<1ed severa I I Ines of study of this response.

I. We have attempted to Induce, In vitro, Increased template activity of Isolated uterine chromatin or of Isolated uterine nuclei of both Immature and ovarlectomlzed rats. Estrogen-binding protein was extracted from the uterine cyto­plasm of both ovarlectomized and Immature rats, and from the nuclei of mature rats. The addition of the estrogen + binding protein complex to chromatin decreased the template ac~lvlty of the chromatin, rather than Increasing It, as occurs in vivo. Purification of the binding proteins did not alter this result. We showed, with 3t-i-estradiol, that the estradlol +binding protein complex saturates chromatin at a weight ratio of chromatin to estradlol of 108:1. Unfortunately, pea plant chro­matin binds 75% as much estrogen + binding protein, with the same saturation 'value. The binding Is therefore not at a specific receptor site for the hormone.

2. Ovarlectomlzed rabbit uterus chromatin increases several-fold In tem­plate activity only 10 minutes after estrogen administration In vivo. In attempts to duplicate this effect In vitro we added a small amount of estrogen to whole ho­mogenates of uteri from ovarlectomlzed rabbits, and measured subsequent rate of RNA synthesis. Even with RNase Inhibitors present, a minute amount of estrogen caused depression of RNA synthesis In the whole homogenate. After Incubation of such an In vitro system tor 15 to 40 minutes, In some cases with additional ATP, the chro­matin was Isolated, and the template activity of the purl fled chromatin assayed with added E. coll RNA polymerase. Chromatin that had been incubated with estrogen In vitro exhibited decreased template activity, as well as a higher ratio of pro­tein to DNA than chromatin that had been Incubated without estrogen. It seems clear that a specific response to estrogen occurs In the uterine homogenate, but that the in vitro system lacks some component required for the derepression of the chromatin.

15. ARTIFACTS OF THE RNA POLYMERASE SYSTEM

Investigator: David J. Mcconnel I

Support: Lucy Mason Clark Fund National Institutes of Health, Public Health Service

It had been observed that highly purified RNA polymerase catalyzed the Incorporation of Y 32po4 from ATP Into acid-insoluble material in the absence of primer. This was Investigated thoroughly as evidence of formation of a phospho­enzyme. It has been clearly shown that this activity is due to the presence of a contaminating enzyme, polyphosphate kinase, which generates polyphosphate from the Y phosphate of ATP. The activity did not cosediment with RNA polymerase on glyc­erol gradients, nor did It chromatograph with It on phosphocellulose. As with the purified polyphosphate kinase It was strongly Inhibited by ADP and slightly by pyro­phosphate. The p reduct was h I gh mo lecu I ar we I ght, pol yd I sparse on e I ectrophores is, and did not electrophorese with any of the subunits of RNA polymerase. It did not incorporate Y P04 from GTP. This activity represents a serious artifact. It can

30

be inhibited completely by a KCI concentration of 0.008 M, or by smal I amounts of Al1'. It Is also inhibited by the presence of DNA.

A second artifact In the system resu'lts from the presence of polynucleo­tlde phosphorylase which converts XDP into poly X +Pi. This enzyme can be con­veniently removed by chromatography on phosphocellulose, and probably equally well on DNA-cellulose. It is completely Inhibited by 0.8 rrt"1 K2HP04 and this has no effect on the rate of RNA synthes Is. It must be contra 11 ed s I nee commercl a I prepa­ratl ons of nucleoslde triphosphates commonly contain 10% nucleotide dlphosphate.

16. THE R'.lLE OF o FACTOR IN THE INITIATION OF RNA SYNTHESIS BYE. COLI RNA POLYMERASE ON VARIOUS TEt<PLATES

Investigator: David J. Mcconnel I

Support: Lucy Mason Clark Fund National Institutes of Health, Public Health Service

Polymerase was separated 1 nto core enzyme [S2a2w] and o factor by chroma­tography on phosphocellulose. Core enzyme has very low specific activity, <100 units per mg on all DNAs tested: T7, rat liver, rat ascltes tumor, and pea bud. The addition of o factor raises the specific activity by 800% In the case of T7 DNA, but only slightly, around 50%, for the other templates. Since o has been clearly shown to promote accurate Initiation In the case of T4 and T7, these results imply that the frequency of the o-speclflc sites on heterologous DNA ls very low. When It is considered that T7 may have only one such Initiation site per 23 x 106 molecular weight the efficiency with which the heterologous DNA binds polymerase must be very low Indeed. However, high values >30% have been obtained for hybrid­ization of RNA synthesized in vitro on heterologous DNA templates. The most ratio­nal explanation Is that Initiation on such DNA Is a low frequency event, occurring at random sites, perhaps at nicks or ends, to which the enzyme Is known to bind.

Chromatin has also been tested as template. The presence of o makes only a small difference to the efficiency of transcription. If chromatin contains o­llke factors, they are not capable of stimulating E. coll core enzyme.

17. INITIATION OF TRANSCRIPTION BYE. COLI RNA POLYMERASE ON T7 DNA

I nvestl gator: David J. McConnel I

Support: Lucy Mason Cl ark Fund National Institutes of Health, Public Health Service

The transcription of the late genes of T7 depends on the activity of gene I. It appears that gene I can be transcribed by the E. col 1 RNA polymerase, and that it codes for a polypeptide which facl lltates the transcription of at least sane of the late genes. In vitro, E. coll RNA polymerase only transcribes the R strand and then only to make early mRNA. However, If the enzyme ls purified from T7-lnfected eel Is late mRNA is also made. These results suggest that there may only be one or at least a small number of Initiation sites on T7 DNA for E. coli RNA polymerase.

T7 ONA and E. coll polymerase react In form a salt-stab le complex. It may be recovered

++ the presence of GTP and Mg to after a sedimentation through a

l l

~ -

31

glycerol gradient In 0.4 M CNH4 >2S04, separated from free enzyme and free nucleo­tide. If the GTP ls trltlated there is a peak of 3H associated with the DNA and the amount Increases proportionally as the.,enzyme:DNA ratio Is Increased. Pre I I mi nary results are that the amount saturates at one G per DNA. No G cosedlments with the DNA If rlfamplcin Is present. These results suggest that there Is one binding site on the DNA at which a DNA-enzyme-G complex Is formed.

18, STUDIES ON ENZYME DIFFERENTIATION IN THE CELLULAR SLIME MOLD DICTYOSTELIUM DISCOIDEUM: GLYCOGEN PHOSPHORYLASE

I nvestl gator: Richard A. Flrtel

Support: National Science Foundation National Institutes of Health, Public Health Service

The cellular slime mold, Dlctyostellum dlscoldeum, Is an Ideal system for the study of differentiation. The organism gra-is exponentially under conditions of favorable food supply. Upon removal or exhaustion of the food source, the Indi­vidual vegetative eel Is aggregate Into colonies of approximately 105 cells and,dlf­ferentlate into 2 different cell types: spores and stalk cells. Development is synchronous and spores are produced In approximately 24 hours.

As previously described (Biology 1969, No. 69), glycogen phosphorylase is developmentally controlled during slime mold development. Activity Is first detectable at 13 hours, reaches a maximum at 22.5 hours (culmination), and then decreases. Between 14 and 22.5 hours, phosphory lase act I vi TY Increases approxi­mately 40-fold. Experiments with actlnomycln D and cyclohexlmlde Indicate that prior RNA and concomitant protein syntheses are necessary.

Further experiments.with RNA and protein synthesis Inhibitors Indicate that at 8 hours after initiation of development, Dlctyostellum starts to synthesize mRNA for phosphorylase. The phosphorylase mRNA synthesis continues for 10 hours. Experiments suggest that the rate of mRNA synthesis Is constant and that there ls a specific and constant number of protein molecules translated/mRNA. The decrease In phosphorylase activity also appears to require prior RNA and concomitant protein syntheses. The period of RNA synthesis necessary for the decrease is between 14 and 24 hours.

The enzyme has also been examined in several morphologically aberrant and temporally deranged CFR17 and GN3l mutants. Mutants which do not aggregate do not accumulate any activity; a stalkless mutant, KYl9, produces a normal amount of enzyme; and a mutant which normally stops at the pseudoplasmodlum stage <KY3l accu­mulates 1/2 normal activity and the activity does not decay. FR17 produces spores in 16 hours and GN3 In 60-70 hours. Both accumulate phosphorylase at the same stage morphologically with respect to spore formation in wl Id type.

Results indicate synthesis of glycogen phosphorylase Is tightly I Inked to development and that the time of expression with respect to cyto-morphologlcal development Is stringently controlled. The appearance of phosphorylase coincides with the period of soluble glycogen degradation and Its maximum activity occurs within 2 hours of maximum activity of other major developmentally controlled en­zymes believed to be associated with cell wal I synthesis. It is possible that It Is responsible tor the degradation of soluble glycogen and that the glucose-I-phos­phate produced Is the bul I ding block of eel I wal I polysaccharide.

32

References:

Roth, R., Ashworth, J., and Sussman,, J. (1968) Proc. Nat. Acad. Sci. 59: 1235.

Cleland, S. V. and Coe, E. L. (1968) Biochlm. Biophys. Acta 156: 44.

19. AMINO ACID-CATABOLIZING AND AMPHIBOLIC ENZYMES DURING SLIME MOLD DEVELOPM::NT

Investigators:

Suppon:

* Richard A. Flrtel, Robert W. Brackenbury

National Science Foundation National Institutes of Health, Public Health Service

During the vegetative period of growth {Biology 1969, No. 69) Dlctyo­stelium accumulates a large amount of soluble glycogen C5% of eel I dry weight). This accumulation continues during the early period of slime mold development up to the end of aggregation. Subsequently, the glycogen Is degraded and a storage disaccharide Ctrehalose) and eel I wal I material are produced.

The total amount of carbohydrate present during development has been shown to remain approximately constant. During this period, however, there is a 50% loss of cell protein. It has been hypothesized that slime molds use the stored glycogen for cell wal I and trehalose synthesis and the protein as a food source during differentiation. It ls already known that tyrosine tran~amlnase and threo­nine dehydrase are developmentally controlled {W. F. Loomis, personal communication).

In order to obtain a better understanding of protein catabollsm, several amino acld-catabolizing and amphlbollc enzymes were examined. The activities of aspartate transaminase, alanine transaminase, glutamlc dehydrogenase, alanine de­hydrogenase, homoserlne dehydrase, and histldase were examined. Of these, only alanine transaminase was shown to be developmentally controlled. The specific ac­tivity of the other enzymes remained constant or decreased slightly during develop­ment.

Alanine transaminase Increases 2.5-fold during the first 4.5 hours of de­velopment after the food source Is removed. Experiments with cyclohexlmlde and actinomycin D indicate that protein and RNA syntheses are necessary tor the in­crease. The enzyme was also examined in morphologically aberrant mutants <Biology 1970, No. 18). KY3 and KYl9 showed normal enzyme activity whl le an aggregation less mutant CAGG 206) shows no increase. FRl7 shows the same kinetics as wl Id type while GN3 only shows a slight Increase.

Of 4 of the main amino acld-catabollzing enzymes directly I Inked to the Krebs cycle Cglutamic dehydrogenase, aspartate transaminase, alanine transaminase, alanine dehydrogenase) only alanine transaminase is developmentally control led. Unlike tyrosine transaminase, threonine dehydrase, and amino peptidase {Biology 1970, No. 20) which reach maximum activity just prior to culmination, alanine transaminase increases very early in development.

References:

Cleland, S. V. and Coe, E. L. (1968) Biochim. Biophys. Acta 156: 44.

* Undergraduate, California Institute of Technology.

33

Cleland, s. v. and Coe, E. L. (1969) Blochlm. Blophys. Acta 192: 446.

White, G. J. and Sussman, M,~ (1961) Blochlm. Blophys. Acta 53: 285.

20. REGULATICN OF AMll\O PEPTIDASE ACTIVITY IN SLIME MOLD DEVELOPMENT

I nvestl gators:

Support:

* Richard A. Fl rte I, Kenneth D. Plschel

National Science Foundation National Institutes of Health, Public Health Service

We are attempting to find out If, during development, Dlctyostellum ge­netlcal ly regulates enzymes which are responsible for the catabol Ism of eel lular proteins. The enzyme amlnopeptldase was Investigated. It has been found to be developmentally control led and to Increase 2.5-3 times over the fl rst 15-16 hours of development. The Increase Is linear and requires RNA and protein synthesis.

Studies with mutants Indicate that the Increase In spec! fie activity occurs only In those mutants which undergo differentiation. In mutants FRl7 and GN3, the enzyme reaches maximum activity at 9 and 24 hours respectively. •

Although the Increase In specific activity Is not large and occurs grad­ually over 16 hours, It Is under genetic regulation. The enzyme may be responslb le for the hydrolysis of smal I peptides resulting from protein degradation into amino acid.

21. CHARACTERIZATION OF THE SLIME MOLD GENOME

I nvestl gators:

Support:

" Richard A. Flrtel, Ratchford C. Higgins

National Science Foundation

Recent studies have shown that one can obtain an understanding of the mo­lecular composition of genomes by using DNA/DNA and RNA/DNA hybridization. Studies of Britten and Kohne showed that the genome of higher organisms contain different classes of ONA as determined by their rates of renaturation. These can be divided bas I cal ly Into redundant DNA (ONA whose sequence or very slml lar sequence Is pres­ent many times In the genome> and unique DNA (sequence present only once per haploid genome). An analysis Is being made of the genome of Dlctyostelium.

DNA from whole eel Is, nuclei, purified mitochondria, and partially puri­fied nucleol I was Isolated. Total eel I DNA and DNA from organelles were obtained by lyslng the material In sodium lauryl sarcoslne and running lsopycnlc centrifuga­tion of the r.iaterlal directly In CsCI In the presence of saturating ethldlum bro­mide. This al lows one to obtain a good yield of DNA and al lows one to remove most of the protein, RNA, and polysaccharlde In one step.

Total cell DNA purified by this method shows a main peak at p =I .676 CM. I ysode I kt lcus taken at I • 725), a major shou Ider at I .682, and a very sma I I shou Ider at p = 1.686. The large satellite comprises approximately 30% of total cell DNA. A Tm of 79 .5°C In 0.12 M phosphate buffer as wel I as a density of 1.676 glee agrees

* Undergraduate, California Institute of Technology.

34

with a 22% G-C content of the main component. When DNA is Isolated from purified nuclei or from whole cells treated with ethldium bromide, most of the main satel­lite Is lost but the small satellite is still present. The large satellite appears to be cytoplasmic and probably represents mitochondrial DNA. A pure DNA fraction from purified mitochondria has the same buoyant density as the main sate I lite com­ponent. The smal I satellite comprises approximately 10% of the nuclear DNA.

Hybridization of purified ribosomal RNA <rRNAl to total cell DNA shOfls that the genes tor I 7 and 265 rRNA make up about I .6% ot the DNA wh I I e rRNA hyb rl d-1 zes to approximately 2.2% ot nuclear DNA. This ls consistent with the amount ot DNA present in the small satellite If non-conservation of rRNA precursor and rRNA gene spacer regions are considered. DNA trom partially purl tied nucleol I shOt1s the same density as the small satellite. The hybridization results yield approximately 300 rRNA genes/haploid genome.

When DNA/DNA hybridization of total eel I DNA Is fol lowed by decrease In absorbance at 260 nm and Is plotted% <renatured) vs. log (DNA concentration x time, a Cot plotl, one sees three main rates ot reassociation: a very fast component of approximately 10-15% of the DNA, a medium fast component of approximately 30%, and a slow component whose rate Is in agreement with a unique DNA haploid nuclear genome size ot 0.1 µµg.

Reference:

Britten, R. J. and Kohne, D. E. ( 1968) Science 161: 529.

22. GENE REGULATION IN REGENERATING LIVER

I nvestlgator: John E. Mayfield

Support: National Institutes of Health, Pub Ile Health Service

Chromosomal RNA (cRNAl has been Isolated from the I Iver of rats before and after partial hepatectomy. It has been found that the regenerating I Iver pro­duces both Increased amounts and new sequences of cRNA after such hepatectomy. The template activity ot the chromatin ot regenerating rat liver Increases after hepa­tectomy. It has been found that the increase in cRNA sequences precedes the In­crease in template activity of chromatin but lags behind the appearance ot new RNA sequences in rapidly-labeled, high molecular weight, nuclear RNA, as studied by Church and McCarthy (1967). These findings are In accordance with the concept that rapidly-labeled nuclear RNA is the precursor of cRNA and that the role ot this RNA Is that ot a gene activator.

Reference:

Church, R. B. and McCarthy, B. J. ( 1967) J. Mol. Biol. 23: 459.

23. STUDIES OF BRAIN CHROMATIN

I nvest i gator: Stephen Harris

Support: National Institutes of Health, Public Health Service

Chromatin from whole rat brain has been Isolated by the standard methods. ~h chromatin exhibits a typical two-step melting protl le with Tm of 66° and 83°

35

respectively. Its chemical composition ls similar to that of chromatin isolated from other mammalian tissues. Thus Its hlstone, non-hlstone, and RNA to DNA ratios are 1.01, 1.10, .104, respectively. Approximately 40% of the RNA associated with such chromatin is recovered In the protein-associated RNA fraction and is thus apparently chromosomal RNA. Brain chromatin exhibits a template activity relative to deprotelnlzed DNA of 5%.

24. STUDIES OF THE CHROMATIN OF DROSOPHILA MELANOGASTER

Investigator: Stephen Harris

Support: National Institutes of Health, Public Health Service

Chromatin has been Isolated from 0 to 12-hour-old embryos of Drosoph I la melanogaster by the standard methods. The chemical composition of such chromatin is as follows: hlstone, non-hlstone, and RNA to DNA ratios are 0.95, 1.2, 0.10 re­spect! vely. Drosophila chromatin exhibits a template activity of 20-25% of that of deproteinized Drosophl la DNA. The melting prof I le of Drosophl la chromatin Is monophaslc and typical of the chromatins of many other creatures and organs.

PUBLICATIONS

Bekhor, I., Bonner, J., and Dahmus, G. K. ( 1969) Hybrid I zatlon of chromosomal RNA to native DNA. Proc. Nat. Acad. Sci. 62: 271-277.

Bekhor, I., Kung, G. M., and Bonner, J. (1969) Sequence specific interaction of DNA and chromosomal prote In. J. Mol • Bl ol • 39: 351-364.

Brutlag, D., Schlehuber, C., and Bonner, J. (1969) Properties of formaldehyde­treated nucleohlstone. Biochemistry 8: 3214-3218.

Dahmus, M. and Bonner, J. C 1970) Nucleoprotelns In regulation of gene function. Fed. Proc. 29: 1255.

Delange, R. J., Fambrough, D. M., Smith. E. L., and Bonner, J. (1969) Calf and pea hlstone IV. I II. Complete amino acid sequence of pea seedling hi stone IV; comparison with the homologous calf thymus hlstone. J. Biol. Chem. 244: 5669-5679.

Elgin, S. c. R., Froehner, S. c., Smart, J. E., and Bonner, J. (1970) The biology and chemistry of chromosomal proteins, Vol. I. In: E. J. DuPraw Ced,), Advances In Cell and Molecular Biology. Academic Press, New York Cin press).

EI g In, S. C. R. and Bonner, J. ( 1970) LI mi ted heterogene I ty of the major nonh I stone chromosomal prote Ins. Bi ocheml stry (in press).

Fambrough, D. M.( 1969) Nuclear protein fractions, (ed.), Handbook of Molecular Cytology. Amsterdam-London, pp, 438-471.

chapt. 18. In: A. Lima-de-Faria North-Ho 11 and Pub Ii sh Ing Company,

Fi rte I, R. A. and Monroy, A. ( 1970) Polysomes and RNA synthesis during early development of the surf clams Sp!sula sol!dlsslma. Develop. Biol. 21: 87-104.

36

Hadler, S., Smart, J. and Bonner, J. (1970) Studies on the mechanism of removal of histones from chromatin by deoxycholate. J. Mal. Biol. {in press).

Jacobson, R. and Bonner, J. { 1970). Further stud-I es of the chromosomal RNA and of the chromosomal RNA-binding protein of higher organisms. J. Mal. Biol. {in press).

Joly, R. A., Sauer, H. H., and Bonner, J. (1969) Preparation of cholesterol-25-3H. J. Lab. Compounds 5: 80-85.

Joly, R. A., Bonner, J., Bennett, R. D., and Heftmann, E. (1969) The blosynthesls of steroidal sapogenlns In Dioscorea florlbunda from doubly label led cholesterol. Phytochemlstry 8: 1709-1711.

Joly, R. A., Bonner, J., Bennett, R. D., and Heftmann, E. (1969) Conversion of an open-chain saponln to dloscln by a Dioscorea florlbunda homogenate. Phytochemlstry 8: 1445-1447.

Joly, R. A., Bonner, J., Bennett, R. D., and Heftmann, E. Ci969) Conversion of cholesterol to an open-chain saponln by Dloscorea florlbunda. Phyto­chemlstry 8: 857-859.

Lezzl, M. (1969) Differential effects of sodium and potassium Ions on the template activity of rat I Iver chromatin. Physlol. Chem. A Physics I: 447-461.

Li, H.-J. and Bonner, J. <1970) On the melting spectra of nucleohlstone. J. Mel. Biol. (In press).

Matthysse, A. < 1970) Organ specificity of hormone-receptor-chromatin Interactions. Bloch Im. Biophys. Acta 199: 519-521.

Matthysse, A. and Abrams, M. <1970) A factor mediating Interaction of klnlns with the genetic material. Blochlm. Biophys. Acta 199: 511-518.

Matthysse, A.G. and Phi I lips, C. (1969) A protein Intermediary In the Interaction of a horrrone with the genome. Proc. Nat. Acad. Sci. 63: 897-903.

Morgan, C. and Bonner, J. (1970) Template activity of liver chromatin Increased by In vivo administration of Insulin. Proc. Nat. Acad. Sci. 65: 1077-1080.

Sadgopal, A. and Bonner, J. ( 1969) The relationship between hi stone and DNA synthesis In HeLa eel Is. Bloch Im. Blophys. Acta 186: 349-357.

Sadgopal, A. and Kabat, D. (1969) Synthesis of chromosomal proteins during the maturation of chicken erythrocytes. Blochlm. Blophys. Acta 190: 486-495.

Sadgopal, A. and Bonner, J. (1970) Chromosomal proteins of interphase HeLa cells. Biochlm. olophys. Acta 207: 206-226.

Sadgopal, A. and Bonner, J. { 1970) Proteins of lnterphase and metaphase chromosomes compared. Blochlm. Biophys. Acta 207: 227-239.

Shih, T. Y. and Bonner, J. (1969) Chromosomal RNA of calf thymus chromatin. Blochlm. Biophys. Acta 182: 30-35.

Shih, T. Y. and Bonner, J. (1970) Thermal denaturatlon and template properties of DNA complexes with purl fled hi stone fractions. J. Mol. Biol. 48: 469-487.

Shih, T. Y. and Bonner, J. (i970) The template properties of DNA-polypeptide complexes. J. Mol. Biol. 50: 333-344.

Slvolap, Y. and Bonner, J. (1970) Association of chromosomal RNA with repetitive DNA. J. Moi. Biol. (in press>.

37

Smart, J. and Bonner, J. ( i970) The selective dissociation of hlstones from chromatin by sodium deoxychoiate. J. Moi. Biol. (in press).

Smart, J. and Bonner, J. (i970) Studies on the role of hlstones In the structure of chromatin. J. Mol. Biol. (in press).

Smith, E. L., Delange, R. J., and Bonner, J. (1970) Chemistry and biology of the hlstones. Physlol. Revs. 50: 159-170.

Tuan, D. Y. H. and Bonner, J. (1969) Optical absorbance and optical rotatory dispersion studies on calf thymus nucleohlstone. J. Mot. Blot. 45': 59-76.

38

Professor: Ray D. Owen

Research Fellow: David B. Dusenbery

Graduate Students: Tamiy C. Douglas, Jeffrey A. Frei Inger, Susan L. Melvin

Research Assistant: Sandra M. Webb

laboratory Assistant: Maude E. Vermaes

Ours is a relatively small research group, concerned with a relatively large range of problems. As wl II be evident from the fol lowing abstracts, our ex­perimental animals range from rotlfers to rabbits, and include birds and mice; our Interests range from behavioral studies to biochemical genetics of a serum protein and the control of graft rejection. Our experimental tools ~re drawn mainly from Immunology, genetics, biochemistry, and radiation biology. Through It al I is a thread of interest In development, mainly in complex organisms and with an empha­sis on the Immune system as a model for developmental analysis.

25. STUDIES ON THE BURSA OF FABRICIUS IN PIGEONS AND ITS CONTRIBUTIONS TO THE IM\IUNE RESPONSE

Investigator:

Support:

Susan L. Melvin

National Institutes of Health, Public Health Service U.S. Atomic Energy Commission

In chickens, development of complete immunological competence requires that both the thymus and the Bursa of Fabrlclus be present in late embryonic and early neonatal life. The thymus-dependent Immune functions Include delayed hyper­sensitivity and graft rejection; the bursa! functions include reactions mediated by humeral antibody. By performing bursectomles in ova, one can further dissect the antibody response with respect to classes of immunoglobulln molecules (7S lgG and 195 lgM) elaborated.

Reconstitution studies of X-irradlated mice similarly indicate 2 cell lines Involved in the immune response. Moreover, they Indicate that at least 2 eel I-types Interact in the sequence of reactions leading to antibody elaboration, Current theories designate the thymlc-dependent eel I as the antigen reactive cell (ARC), important in the recognition of antigen and the induction of the antibody response. The actual antibody-forming precursor eel I (AFPCl is of bone marrow ori­gin In mice and presumably of bursa-dependent origin in chickens.

There exists no precise mammalian equivalent to the bursa and for this reason birds offer opportunities for experimental work so far impossible In mammals. Experiments are in progress to study the development of the bursa, bursal activa­tion In vitro and the possible interaction ben.een bursa! and thymlc cells.

26, A STUDY OF THE EFFECT OF IRRADIATION ON THE PIGEON KARYOTYPE

Investigator: Sandra M. Webb ·.

Support: U.S. Atomic Energy Commission

Pigeons are remarkably tolerant to X-irradiation. In this laboratory pigeons have survived 1750 r whole body irradiation, a dose nearly 3 times that which is fatal to man. The irradiated birds are somewhat runted, have unusual feather structure and she>1 abnormal serum gamma-giobul in patterns on electropho­resis. However, one preliminary experiment has she>1n no significant effect on the rate of primary feather growth in 6 pigeons which received from 1250 to 1750 r whole body X-ray.

It is of interest to see whether effects of high doses of radiation on the pigeon chromosome complement can be detected. The chromcsome squash technique was modified so that squashes could be made from pulp in the qui ils of regenerating primary feathers. Karyotypes of nonnal, unlrradlated pigeons were made, concen­trating mainly on the first 10 pairs of chromosomes, the macrochromosomes. Others of the 80-ch romosane comp lament are i mposs lb le to karyotype accurately. So, tar one i rrad I ated b I rd, wh I ch rece I ved I 750 r, has been compared w I th the no rma I , and no chromosomal abnormalities have yet been observed. •

*

27. MULTIPLE EFFECTS OF ANTl-LYMPHOCYTIC SERUM

Investigators:

Support:

* Susan L. Melvin, Cynthia Johnston

U.S. Atomic Energy Commission National Institutes of Health, Public Health Service

Based on demonstrations that anti-lymphocytic serum CALSl could sub­stantially delay graft rejection, ALS has been used in human transplants and in animal studies of immune rejection. Besides prolonging survival of transplanted skin and organs, ALS treatment has weak suppressive effects on humeral antibody responses and improves growth of transp I anted tumors in hamsters, Human transplant pat I ents treated w Ith ALS show an Increased Incl dence of spontaneous tumors over non-A LS-treated patients. Different preparations of ALS di ff er In these respects and, to date, there are no in vitro tests which correlate closely with the in vivo effectiveness of ALS.

In this investigation, mice that had received graft-prolonging ALS treat­ment were studied for their antibody responses to serum antigens and attempts were made to solubi lize lymphocyte antigens for in vitro assays of ALS preparations. · ALS-treated mice responded to serum antigens in much the same way as control mice al though d I fferences in the ti ml ng of the response may be revea I ed by further studies. ALS yielded a precipitln band against thymic extracts in immunoelectro­phores is. The antigen appears to be a serum contaminant of the thymocyte prepara­tions. At 6 months after receiving their last serum injections, some ALS-treated mice show ·spontaneous tumors. The control mice are tumor-tree. Further work on the antigenic properties of these tumors may elucidate the mechanism of spontaneous tumor induction.

Mayfield High School, Pasadena.

40

Re fe re nces :

Gershon, R. L. and Carter, R. L. ,< 1970) Nature 226: 368-370.

Levey, R. H. and Medawar, P. B. ( 1966) Proc. Nat. Acad. Sci. 56: 1130-1137.

28. AN ATIEWT TO DEVELOP A TECHNIQUE FOR THE IDENTIFICATION OF INDIVIDUAL CELLS PRJDUCING ANTl-BACTERla'HAGE ANTIBODY

I nvestl gators:

Support:

* ** Susan L. Melvin, Marcia Levine , Gerald Felgenson

National Science Foundation National Institutes of Health, Public Health Service U.S. Atomic Energy Conmlsslon

The Jeme plaque technique demonstrates antibody production by Individu­al cells In the center of a focus of hemolyzed red blood cells. Drawbacks of this technique are that erythrocytes have many antigenic sites; they cross-react with bacterial antigens; there exist natural antibodies to red blood cells; and the direct technique reveals lgM, but not other classes of antibodies.

An assay utl 11 z Ing phage wou Id ml n !ml ze these prob lens. We have attempt­ed to develop such an assay by plating lnmune eel Is with bacteria and enough bac­teriophage to clear the plate. A cell producing anti-phage antibody should protect the bacteria surrounding It from Infection and subsequent lysls by the phage.

Early experiments showed a clear difference between cell suspensions from lnmunized and from unlnmunlzed mice. Up to 50% of the cells from Immunized mice were surrounded by stained, roughly circular areas, 2 or 3 cell diameters across, Interpreted to be micro-colonies of E. coll.

Later attempts to duplicate the above results were not successful. No difference was found between eel Is from an lnmunlzed and an unlnmunlzed mouse. The question of the success of the technique remains In doubt.

Further study of this problem may Involve: l) finding more compatible agars, such that eel I growth Is favorable whl le growth of bacteria at the Interface Is enhanced; and 2) Increasing the sensitivity of the assay by using minimal amounts of phage and bacteria to carpet the plate.

29. MAINTENANCE OF POLYt-ORPHISM FOR TRANS FERRI NS IN THE PIGEON

I nvestl gator:

Support:

Jeffrey A. Frellnger

National Institutes of Health, Public Health Service U.S. Atomic Energy Conmlsslon

Transferrln Is a non-heme, Iron-binding protein. In pigeons, It Is found in serum, egg white, egg yolk, pigeon ml lk and probably semen. Only 2 alleles are

* NSF Summer Trainee.

** Division of Chemistry and Chemical Engineering, California Institute of Technology.

41

known at this locus. In our pigeon colony and in the feral population sampled, the 2 alleles occur in approximately equal frequency. The distribution of the genotypes In the population tits the Hardy-Weinberg equi I ibrlum, with no excess ot heterozy­gotes.

The polymorphism has been Investigated developmentally by using matings which produce only heterozygotes. We found that the young squabs, tor several days attar hatching, have only the maternal type of transterrlns In their serum, even though the squabs are fed crop ml lk from both parents and the mi I k contal ns both transferrln types. This indicates that transterrln Is transferred via the egg, not via the ml lk.

The fecundity of the tamale pigeons In the colony was examined. Overal I hatchabl llty of the eggs was 37%. Tabulated according to maternal transferrln ge­notype, the hatchabllltles were: Tta/Tfa, 29.2%; TtB/TtB, 31.4%; TtA/TtB, 46.0%. These differences are significant.

Adult mortality In the flock shews no association with transferrin type. It selection maintains the polymorphism consistently observed at this locus, It appears to act by favoring heterozygoslty for the maternal contribution to the egg, embryo, and young squab.

Several microorganisms can infect adult pigeons asymptomatlcal ly, bwt cause death of large numbers of embryos. We are Investigating a possible differen­tial Inhibition ct microbial growth by egg whites of the different transterrln types.

30. BEHAVIORAL BIOLOGY OF R:JTIFERS

Investigators:

Support:

David B. Dusenbery, Sandra M. Webb

National Institutes of Health, Public Health Service U.S. Atomic Energy Commission

Rotlfers offer some special advantages for biological studies, based on thel r rapidity of reproduction, the I lmlted and fixed number of nuclei Involved In the mature mlcro-metazoan, the ease of developing asexual clones In which particu­lar genotypes can be multiplied without going through the gene-reshuffling pro­cesses of meiosis and fertl llzatlon, and the recent development of a method tor in­duct ng sexual reproduction at wl 11 In Asplanchna brlghtwel 11, which should greatly tac I 11 tate genet I c work.

In spite of past frustrations (Biology 1969, No. 79) we are continuing work with rotiters. Our emphasis Is now on studying their relatively simple ner- · vous system and behavior. This work Is just getting started; our plan is to alter the nervous system (by genetic changes or microbeam Irradiation) and study the effects on behavior.

42

31. THE USE OF ANTIBODIES DIRECTED AGAINST CELL SURFACES TO INTERFERE WITH NORMAL DEVELOPMENT OF THE PIGEON BRAIN

I nvestl gator:

Support:

Tommy C. Doug I as

National Defense Education Act U.S. Atomic Energy Col111\lsslon

An unsolved and chat tanging question is: hO<I do the developing neurons of higher animals reach their proper destinations and establish correct connec­tions? One way of attacking this question Is by Interfering with the .normal devel­opment of the nervous system. This might be done by the treatment of developing embryos with antibodies directed against brain cell surface antigens.

Cel I membranes from cerebel tar tissue taken from 14-day pigeon embryos were Injected with Freund's complete adjuvant Into New Zealand White rabbits. The rabbits received a booster Injection of antigen after I rronth, and were bled 2 weeks later. The resulting antiserum Is to be Injected Into normal embryos which wl II later be examined for behavioral and physiological abnormalities.

A study of the blood-brain barrier of newly-hatched squabs revealed that for the dye trypan blue this barrier Is completely established by the first day after hatching. None of the Injected dye penetrated Into the brain beyond the choroid plexus. Intravenous administration of antiserum Into squabs does not, therefore, appear to be a promising procedure In the above context.

l

l l I t l I ' if-~

! t

f

l I I

r ' !

t l ( I

I

l I

Professor: Max DelbrUck

Visiting Associate: John W. Kauffman

Senior Research Fel IC'ii: David S. Dennison

Research Fel I ows: Robert J. Cohen, Ruth L. Dusenbery, Margarete Petzuch, Ruxton VI I let

Graduate Students: Kostla Bergman, Steven A. Bissel I, Patricia V. Burke, Moises G. Eisenberg, Kenneth w. Foster, E. WI! I lam Goode I I, Algl rdas J. Jesaltls, Charles E. Novitski

Electronic Specialist: Cornelius M. Dekker

Research Assistants: Bertha Jones, Patricia M. Reau

Research Aide: Jeanette Navest

43

The general aim of our work Is the analysis of the sensory transd~cer me­chanisms of the sporanglophore of Phycomyces. Alas, the work reported Is still preparatory to our ultimate hope, an analysis on the molecular level. Autorvated equipment, now operative (32) should yield action spectra of greater accuracy, at various intensities and for different photomutants. The red tal I of the action spectrum has been carefully scrutinized (33) for features Indicative of a metallo­flavoproteln. Polarization dependence (34) has shC'iln that the light receptor Is dlchrolc and oriented. Studies of circadian rhythm (35). hint that the mycellum contains a photoreceptor slml lar to that in the sporanglophore. Earlier suggestive evidence that the 1 lght stimulus can be stored has been vindicated and extended (36). A new procedure has been used (37) to test a hypothesis aiming to explain why the phototropic response Is continuous, in contrast to the growth response to I lght, which is transient.

The puzzle of the avoidance response (38) has been clarified to some ex­tent (with help of friends In Fluid Dynamics). The relative humidity gradients may be the parameter sensed by the organism. A growth response to humidity transients has been found (39).

i'lork with mutants, how to detect recessive ones In good yield (40), how to characterize them (41), and how to study them in heterokaryons (43) Is pursued with new procedures. An attempt to detect differences In the flavoproteln mixture between wild type and a photomutant gave suggestive but equivocal results (42).

The study of growth regulation (44) has led to the discovery of apical dominance, hitherto not known to occur In fungi. Efforts continued to make fast freezing in superfluid helium a workable method for electron microscope work (45).

The last Item (46) does not pertain to Phycomyces, but to a possible model of sensory transducers, the lipid bl layer. It Is hoped to develop gradually a major study of this Intriguing system in cooperation with the Division of Elec­trical Engineering.

44

32. AUTOMATION OF PHYCOMYCES EXPERIMENTS

Investigator: Kenneth W. Foster

Support: McCal lum Fund SI oan Fund tor Bas I c Research

As part of the group's effort to find an In vitro photoresponse system, my goal has been to do precise in vivo characterization of Phycomyoes' photore­sponse.

A semi-automated machine controls the light, relative humidity and temper­ature environment and automatically collects data on Phycomyces growth. In order to autanati ca I ly fol low the growth behavl or of Phycomyces, the sporangl um at the top of a Phyoomyces sporangiophore Is servoed to a constant position. Measurement every 10 seconds of the x, y, and z positions of the servoed stage supporting the sporangiophore provides a growth velocity with less than 10% error. A human ob­server must measure over a one minute period to get the same accuracy. This tech­nique has the additional advantage of keeping the photosensitive and growing region, which ts 0 to 3 rrm belOrl the sporangium, in a fixed position for high magnification observation and uniform I lght stimulation.

Autanatlon also extends to control of stimulus conditions. The changlng of light source from a standard to a test source Is automatically controlled and synchronized with the gra-ith rate data collection. The Intensity of one light source may also be programmed using a circular light Intensity 0.0. wedge to give a slow exponentially increasing intensity, the "sunrise" experiment, or to give positive or negative pulses or steps In intensity, or a coml>lnatlon of these.

The 1 lght program and data col lectlon can be run cyclically together. After the end of a number of cycles an .accurate averaged growth velocity as a func­tion of the cycled light program ls immediately aval lab le. This ls displayed on a cathode ray tube and in seconds may be plotted in a permanent form. It desired al I the experimental data may be stored on digital magnetic tape al lowing detal led computer analysis at a later date on the Caltech computer.

One experiment tor which the apparatus is ideally suited is the collection of growth response act I on spectra. The ettectl veness of a test I I ght I ntens I ty and wavelength ls compared to a standard. The procedure ls to alternate every 5 minutes between the test and standard 11 ght. Ave rag Ing over a number of cycles the rel a-t Ive effectiveness ot the two I I ghts Is eas i ly determt ned. At some test 11 ght in­tens lty there should be no response and this ts the relative quantum efficiency tor the test wavelength.

Other studies that may be carried out with a minimum of set-up time and high precision, are study of stimulus response curves as a function of wavelength and adapting intensity, study of the kinetics of dark and light adaptation, and study of high Intensity saturation effects.

The grow Ing Phycomyces is observed by a vi di con TV camera and displayed at IOOX magnification on a TV monitor al lowing, tor example, observation of the sporangium rotation. If desired, this signal may be recorded on a videotape time­lapse recorder for future analysis.

I I i I ' i

:

I ;-

33. LONG WAVELENGTH PHOTOTROPIC ACTION SPECTRA OF PHYCOMYCES

I nvestl gators:

Support:

Kenneth w. Foste.c, E. Wi 1 llam Goode! I

National Research Councl I of Canada National Institutes of Health, Public Health Service

45

An action spectrum was made of the phototroplc response of a Phycomyces mutant, ~. between 430 and 590 nm. This albino mutant, which contains 50 times less S-carotene than wild type, was grown on a lactate medium which apparently in­hibits phenol polymerization and limits glycogen production. The selection of mu­tant and medium minimizes Internal absorption and scattering. The main action spectra peak Is at 450 nm with a subsidiary peak at 480 nm. From this and other evidence one ls tempted to guess that the photoplgment Is a flavoproteln. If It is a tlavoproteln, there Is no evidence for an associated metal Ion such as Iron. Metal loflavoprotelns have a peak or shoulder above 480 nm. However, If the quantum yield ls constant with wavelength, then the data imply an extinction ratio E450/E550 of about 104, whereas the metallotlavoprotelns, xanthine oxldase and aldehyde oxl­dase, have E450/E550 ot about 3.

Reference:

Rajogopalan, K. V. and Handler, P. (1968) !>'eta! lotlavoprotelns. ~n:

· T. P. Singer (ed.), Biological Oxidations, p. 301.

34. DIOiROISM AND ORIENTATION OF PHYCOMYCES VISUAL PIGMENT

I nvestl gator:

Support:

Alglrdas J. Jesaltls

National Science Foundation National Defense Education Act

It has been found that a Phycomyces sporanglophore (spph) growth response Is elicited more effectively by light polarized perpendicular to Its cylindrical axis than by I lght polarized para! lei to It. This effect could be due to oriented dlchrolc receptor molecules or it could be due to reflection effects at the surface ot the eel I. The latter hypothesis has been strongly discounted by showing that the effect of polarized light is nearly the same tor the wild type and car-10 strains. The car-10 spph is optically relatively clear and colorless. It would give the same growth response to both polarizations If the reflection hypothesis were true, due to a compensating effect ot Internal reflections. The conclusion Is, then, that the polarized light effect arises because the visual pigment ot Phycomyces Is oriented and dlchrolc. Work Is currently under way to strengthen .this conclusion by measuring the polarized light responses of other mutants, car-5 and car-R21. In addition an attempt Is being made to measure the polarization angle (relative to the cylindrical axis) ot maximum response for different wavelengths of stimulating light.

Since the receptor pigment is dichrolc and oriented, Its fluorescent exci­tation and emission should be polarized. A very taint fluorescence excited by high Intensity laser light (I watt/cm2 at 488 nm) has been detected in both the cyto­plasm and cell wall-membrane fractions of spphs. An attempt Is being made to measure both directly and microscopically the polarization of the emitted light in squeezed spphs. Such spphs retain only their eel I wal I and membrane. It a polar­ized emission is detected, then It is strong evidence for localization and orien­tation of the visual pigment In these structures of the cell.

I···. .

,_ .

I I· I

36. EFFECTS OF COLD PERIODS ON THE STIMULUS-RESPONSE SYSTEM IN PHYCOMYCES

Invest I gators:

Support:

Margarete Petzuch, Max De lb ruck

National Science Foundation Stl ftung Volkswagenwerk

The sporanglophores of Phycomyces do not exhibit phototropic responses when growth Is arrested reversibly by cooling to 1°C. Unilateral UV stimuli

47

(254 nm) applied to sporanglophores during cold periods are stored for at least 2 hours and produce tropic responses away from the I ight after warm-up. During the cold period dark adaptation proceeds at a rate which decreases with temperature,

37. PHYCOMYCES PHOTOTROPISM: LOCALIZATION OF ADAPTATION AND THE ROLE OF CELL ROTATION

I nvestl gator: David S. Dennison

Support: National Science Foundation National Institutes ot Health, Public Health Service

Phototroplsm In Phycomyces is due to a dltterentlal growth rate between the i 11 um I nated and dark s I des. However, the growth rate of the ent I re ce 11 changes only briefly fol lowing a change In I lght intensity and approaches a con­stant value If the i llumlnatlon does not change with time. This fact about the growth rate seems incompatible with the observation th~t phototropic bending con­tinues indefinitely in the presence of steady uni lateral i 1 lumination. A study was undertaken to f Ind out why I ntrace I I u I ar growth rate differences pers I st under con­stant uni lateral 11 lumlnatlon.

It was first shown that I ight adaptation is not averaged-out across the eel l's diameter. The eel I is first I llumlnated on one side, using a one-minute pulse of UV Irradiation (285 nm) and then I llumlnated (also UV) from another direc­tion. The second i I lumlnatlon Is oriented so as to straddle the edge of the area of the ftrst UV exposure. The second UV 11 lumlnation thus fal Is partly on portions of the eel I which did not "see" the fl rst UV pulse and partly on portions which did "see" It. If light adaptation is mainly localized, those portions ot the cell that did not "see" the ti rst pulse wi 11 give an exaggerated response. This would be detected by a deviation In the direction of bending relative to the direction ob­served when the first UV pulse is omitted. Such deviations were, in tact, observed and give support to the hypothesis that the changes associated with light adaptation do not propagate from i llumlnated regions to dark regions.

If adaptation is truly local, a possible mechanism for phototropism is the transport of I ight-sensltlve material from dark areas to 1 lght areas, where a con­tinuous light-growth response would be elicited. If the photoreceptor pigment Is located near the cell wall, then the normal rotation of the cell wall due to spiral growth might constitute the mechanism of this transport. This hypothesis can be tested by placing the sporanglophore on a rotating turntable during the uni lateral phototroplc stimulus; at a certain speed the normal rotation ot the cell would be just canceled by the opposite rotation of the turntable and the phototroplc response should be greatly reduced.

Using a unilateral UV stimulus (285 nm) band, 0.25 mm wide, it has been found that both the speed and direction of the phototropic response depend on the

48

turntable speed in the speed range used (36 degrees/minute counterclockwise to 22 degrees/minute clockwise). Although the phototroplc bending speed is relatively lower in the range from 10 degrees/minute, counterclockwise to zero rotation, no

' Instances of total cessation of bending have as yet been found.

38. THE MEDIATION OF THE AVOIDANCE RESPONSE OF PHYCOMYCES

I nvestl gator: Robert J. Cohen

Support: National Institutes of Health, Public Health Service

When an object Is placed a few mm from the growing zone of the sporangio­phore (spph) of Phycomyces, within a few minutes the spph starts to bend <111ay and may continue Indefinitely as In phototroplsm. This behavior Is termed Its avoidance response. It is thought that the spph emits a gas and detects the concentration asyirmetry due to the presence of a barrier on one side. Investigations were made to determine I) whether this concentration asymmetry arises from simple diffusion or diffusion madiated by natural convection, and 2) the effect of controlled air currents.

Because of transpiration of water, the spph Is slightly cooler than ambi­ent. The difference in temperature between It and a nearby object causes a grad.l­ent in the density of air which In turn causes the air near the spph to rise, car­rying with it the diffusible substance. The velocity prof I le Is asymmetric. Cal­culations indicate that It is just possible that this velocity asymmetry may account for the plant's ability to distinguish the presence of an object. Explicit in any natural convection argument Is the direction of gravity. O~vlously, the nature of the currents depends on the relative orientation of spph, object and gravity. How­ever, no difference in avoidance behavior was seen In over 70 separate experiments at various distances of spph to a flat plate in four configurations: I) spph ver­tlca I, plate vertlca I; 2) spph hori zonta I, pl ate vertl ca I; spph horl zonta I, plate horizontal; 3) above and 4) below the spph.

Fast streams of air blow away diffusing material. The critical velocity at which convection becomes more Important than diffusion can be calculated from the Peclet number. As long as one blows (or sucks) above the critical velocity (~ 12 cm/secl, no bending from the barrier occurs; but when this Is reduced (to 2-10 cm/sec) or stopped, normal avoidance behavior appears. This critical velocity corresponds to a diffusion coefficient close to that of water and several other smal I molecules. Blowing or sucking was done both para! lei to the flat plate and perpendicular to it, blowing through a porous ceramic disk acting as a barrier.

Interestingly, a stream of air directed at the spph at a few cm/sec also causes bending Into the wind. Again one can see that diffusion into this stream would cause an asymmetry in concentration similar to that caused by a barrier. Thus this is "anemotroplsm" is not unexpected.

39. EFFECT OF CHANGES IN RELATIVE HUMIDITY ON THE GROWTH RATE OF PHYCOMYCES SPORANGIOPHORES

I nvestl gator: Steven A. Bi sse 11

Support: National Institutes of Health, Public Health Service

The sporangiophore of Phycomyces has been shown to change in growth rate

49

in response to changes In relative humidity CRH) between 100% and 0% at 22-23°C. The experiments were performed In an observation chamber In which the RH could be changed within a few seconds, This chamber has been described elsewhere (Petzuch and DelbrUck, 1970). The four exper~mental programs consisted of cycles of alter­nat Ing the RH every 5, 7, 15, and 20 ml nutes. The data for the I 00% RH phase of the 7-mlnute and 15-mlnute period experiments show an Immediate reduction of growth rate by about 30% and recovery within about 5 minutes. For the 5 and 20 minute one­half cycles, the growth rate is nearly constant during the 100% RH phase. These changes in growth rate are not due to expansion of the sporanglum nor to elongation below the growing zone of the sporanglophore. Currently experiments are being per­formed with a mutant which fal Is to show the avoidance response, to clarify the re­l atl onsh Ip between ·th Is response and the re lat Ive hum I di ty response.

40. MONONUCLEATE SPORES OF PHYCOMYCES

I nvestl gator: Patricia M. Reau

Support: National Science Foundation

Our hunts tor "blind" (nonphototroplcl mutants have so far turned,up only dominant ones. To find recessive mutants we must be able to Isolate and test the descendants of a s Ing le mutagen I zed nucleus. In other words, we need mononl.lj:-1 eated spores. The natural population of Phycomyces spores has from I to 7 nuclei and averages about 3.5 nuclei per spore. In the past we obtained functionally mononu­cleated spores by nuclear kl I I Ing with N-methyl-N'-nltro-N-nltrosoguanldlne. Un­fortunately nltrosoguanldlne Induces not only mitotic lethals (the affected nucleus cannot divide) but also recessive lethals (the affected ~ucleus can divide, but the cell dies if the affected nucleus Is the only one able to divide). The occurrence of both types of lethal events leads to many balanced lethals among the surviving spores. By definition, the spores of a balanced lethal strain must be multlnucle­ate, with each nucleus having undergone at least one lethal mutation.

Are the naturally mononucleated spores different from the multlnucleated ones? To see the nuclei microscopically, a staining procedure was developed. For several hours spores were pretreated In NaOH which partially dissolves the thick cell wal t. Then the spores were fixed in Carney's solution, stained with Feulgen, and mounted In acetocarmlne. By direct measurement, the size of the spore was found to be related to the number of nuclei; smaller spores contal n fewer nuclei. Instead of killing nuclei, which has disagreeable side effects, we should be able to select out mechanically the mononucleated spores naturally present In the popu­lation.

An attempt to sort out the smal I mononucleated spores by sedimentation under natural gravity In a Fico I I gradient was successful. We are now proceeding to mutagenize these spores.

41. CHARACTERIZATION OF THE mad MUTANTS OF PHYCOMYCES

Investigator: Kos ti a Bergman

Support: National Institutes of Health, Public Health Service

Over past

Mutants of Phycomyces with altered responses to light are designated mad. 100 mad mutants with altered phototroplc sensitivity have been isolated In the three years. These mutants are potential tools for elucldatlng the chemical

50

basis of the photosensory system. However since there are many steps Involved In phototropism the mad mutants are a very heterogeneous collection. They must be characterized in defal I to permit a systematic classification.

Simple equipment has been designed to study several sensory phenomena. Mutants are tested to separate them Into classes and to find those that may reward further study. Each stral n w 111 be exam I ned for:

ll Phototroplsm. A system has been devised for measuring bending toward a horizontal 1 lght source as a function of Intensity. In wl Id type sporangiophores the equl librlum bending angle Is constant over a wide intensity range. At very low intensities this angi"e decreases (as measured from the vertical) due to a competi­tion between the gravity and I lght sensitive systems.

2) Avoidance. A sporangfophore placed In total darkness close to a solid barrier grows away from It. This response is one indication of the state of the output system (the differential growth system that must be involved in all tropic res pons es) .

3l Geotropism. Sporangfophores show slow negative geotropism. A special centrifuge Is used to increase the stimulus and make the response more amenabke to study.

So far I have found: a) a mutant that shows no phototroplc response at any I lght Intensity and no avoidance response. b) 5 mutants that show a normal avoidance response but show phototroplc responses only at Intensities 104-106 times the wild type threshold. For 4 of these mutants the equlllbrlum bending angle de­creases near their threshold as a function of Intensity wl-th the same slope as wild type. In one mutant this slope Is 1/5 that of wl Id type.

42. BIOCHEMICAL INVESTIGATIONS OF PHOTOMUTANT PHYCOMYCES

Investigator:

Support:

* Ruxton H. Vil let

Gosney Fund Councl I for Scientific and Industrial Research, South Africa

This work was an attempt to look for blochemlcal differences In photomu­tants of Phycomyces, relative to the wl Id type, which might have some bearing on photosensltivlty. Extracts from a photomutant strain, car-5 mad-117(-), were sub­jected to a fractionation procedure developed by Gerhard Meissner CBlology 1968) for isolating flavoprotelns from the wild-type strain. Absorption difference spectra were measured on fractions from a Bio-Gel column. These were compared with Melssner's results for car-5(-). ·

There appeared to be evidence that a particular flavoproteln might be missing from the ensemble: . for the same yield of protein (OD278 > obtained from the Bio-Gel column for both the photomutant and car-5(-) the corresponding yield of f lavoprotelns U100450> for the photomutant was 48% of that for car-5(-l. Howev­er, th Is was contradicted by curves of LIOD455 and LIOD450 for the photomutant. These showed two chief fractionation peaks as for car-5(-) with a slml lar ratio to that for car-5(-).

* Department of Chemical Pathology, University of Natal, Durban, South Africa.

I i

51

Experiments were also performed to measure the distribution of tlavins In cell tractions of this photomutant strain. There was no significant difference compared with Melssner's results for car-5(-),

43. GENETIC CURING OF BLINDNESS IN PHYCOMYCES

I nvestl gator:

Support:

* Ruxton H. Vi llet

Gosney Fund Councl I tor Scientific and Industrial Research, South Africa

Following the development of a successful microinjection technique tor making heterokaryons, experiments were performed to assess quantitatively the extent of genetic curing of blindness, using a heterokaryon, Cl46*C69. The latter was made by transferring cell-sap from a photosensitive lycopene (red) mutant to a night-blind albino strain. Mycelia of several shades of yellow were selected. The proportions of red nuclei In each mycellum were determined by means of Heisenberg and Cerda-Olmedo 1s technique. A corresponding threshold Intensity for light-perception was measured tor each selected mycellum using the technique of Var ju, Edgar and DelbrUck ( 1961).

From the quantitative results it appears that, although there is ~onsid­erable dominance of the photomutant, there is indeed curing of night-blindness by the admixture of nuclei of a photosensitive strain. The exponential relationship obtained might indicate that a mlssense mutation of a structural gene is Involved giving rise to an altered polypeptide subunit which associates with other subunits In an ollgomerlc form. The active photoreceptor would ~e the homc-oligomer of the wl Id-type subunit.

Heterokaryons between the lycopene mutant and other photomutants have been made and slmi lar quantitative analyses are In progress. The 1 lkel ihood of success with possible biochemical curing ct blindness would tend to be greater it, when wl Id-type nuclei were added to the photomutant, the resulting improvement in vision were large. Moreover, quantitative analysis of the type described here Is a necessary preliminary to work on complementation between photomutants.

References :

Heisenberg, M. and Cerda-Olmedo, E. ( 1968) Mel. Gen. Genet. 102: 187.

Varju, D., Edgar, L., and DelbrUck, M. (1961) J, Gen. Physiol. 45: 47.

44. Pf¥:lCESSES AFFECTING THE GROWTH OF PHYCOMYCES SPORANGIOPHORES

I nvestl gator: E . W I I I i am Goode I I

Support: National Science Foundation

The interactions of the growing zone of the sporangiophore (spph) of Phycomyces with the rest of the spph and with the sporanglum have been analyzed.

The top end of the spph appears to have the rrost active metabolism. The top 6 mm of the spph accounts tor 1/2 of the total oxygen consumption. The oxygen

* Department ot Chemical Pathology, University of Natal, Durban, South Africa.

52

consumption of the whole spph and of this 6 mm long section does not change as the spph elongates from 25 mm to 50 mm. The oxygen consumption of the sporangium is very much I oss than that of the spph. .,

mycel i um. the spph, spph. For

It supplied with water the whole spph can grow Independently from the Similarly, the top 3 or 4 mm can elongate independently from the rest of This section can grow for about 10 hours without the lower part of the the ti rst 4 hours its rate of growth is nearly normal.

Surprisingly, the elongation of the spph is much more dependent upon the sporangium. It the sporanglum is removed, the growth of the spph decreases to 1/10 of the normal rate within 2 hours. 6 to 16 hours after sporanglum removal, the spph produces a branch spph from the former growing zone. The spores in the spo­ranglum appear to synthesize some compound (or compounds) which stimulates the spphs elongation and inhibits its branching. This growth regulation has a striking sim­ilarity to apical inhibition in higher plants.

45. ULTRASTRUCTURE OF PHYCOMYCES SPORANGIOPHORES FROZEN IN SUPERFLUID HELIUM

Investigator: Patricia V. Burke

Support: National Institutes of Health, Public Health Service

Several ultrastructural studies of Phycomyces sporanglophores (spphs) have been done using standard chemical fixation techniques, Orga~el le preservation is satisfactory but considerable disruption and dislocation takes place during fix­ation. I am attempting to improve preservation by using rapid freezing as the pri­mary fixative. The freezing must be sufficiently rapid to suppress ice crystal li­zation.

Superfluid helium was choosen as the coolant. This I iquld has excellent heat transfer properties it boiling is suppressed. A special apparatus was built to drcp the spphs into the liquid through a tube containing warm helium gas. The warm gas prevents the spphs from freezing before they reach the liquid. Boiling of the superfluid helium is suppressed by increasing the helium gas pressure above tho I iquid by 2-3X the saturation vapor pressure. The gas pressure is increased just before the spph enters the I iquid.

Once frozen, the spphs are transferred to I iquld nitrogen and then pre­pared tor electron microscopy by freeze-substitution or freeze-etch techniques. The freeze-substitution work has been done in Dr. Van Harreveld's labs. Freeze­etching is done with a special apparatus designed to accommodate spphs. Early preparations showed ice crystal formation within the spphs. The experiments are being repeated now that a way has been found to warm the helium gas above the Ii quid.

•

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46. ELECTRICAL aJNDUCTION IN LIPID BILAYERS

* I nvestl gators: John W. Kauffman, Carver A. Mead , Max DelbrUck

Support: National Institutes of Health, Public Health Service

Current-voltage characteristics and the conductivity temperature-depen­dence of sphingomyelin bilayer membranes have been determined. The resistances were of the order of 108 ohm-cm2 and exhibited ohmic behavior up to approximately 25 mV fol lowed by increasing conductivity with appJ led voltage. The current Is found to be proportional to a hyperbol le sine function of the voltage. The temper­ature dependence Indicates a thermal Jy activated conduction mechanism. The ob­served behavior closely follows a kinetic model Involving a barrier modified by the applied electric field, the rate-limiting process being the surmounting of the bar­rier by the impinging ions. The model al lows predictions to be made over a wide range of conditions. Further work Is in progress in order to elucidate the nature of conduction across bare I lpld bl layers. In addition alamethlcin-actlvated mem­branes are under Investigation.

Bergman,

PUBLICATIONS

K., Burke, P., Cerda-Olmeda, E., David, Goodell, E., Heisenberg, M., Meissner, and Shropshire, W. < 1969) Phyoomyces.

C., DelbrUck, M., Foster, K., G., Zalokar, M., Dennison, ·D., Bacterial. Rev. 33: 99-157.

DelbrUck, M. ( 1970) Lipid bl layers as models of biological membranes. Jn: F. o. Schmitt (ed.), The Neurosciences: Second Stud¥ Program. The Rockefeller Un Ivers I ty Press, New York.

Goode! I, E.W. ( 1970) Processes affecting growth of Phycomyces sporangiophores. Ph.D. Thesis, California Institute of Technology, Pasadena, California.

Kauffman, J. W. and Mead, C. A. (1970) Electrical characteristics of sphlngomyelin bl layer membranes. Biophys. J, (in press).

Petzuch, M. and DelbrUck, M. (1970) Effects of cold periods on the stimulus system of Phycomyces. J. Gen. Physlol. 56: 297-308.

Vil let, R.H. (1970) Genetic curing of blindness In Phycomyces blakesleeanus: A quantitative assessment of dominance. Nature 225: 453.

* Division of Engineering and Applied Science, California Institute of Technology.

54

BIOPHYSICS

PHYSIOLOGICAL PSYCHOLOGY

CELL BIOLOGY

Alles-Kerckhoff

Morine Station

(First Floor)

•

Professor: Robert L. Si nshel mer

Senior Research Fellow: Roml Jlo T. Espejo

Research Fellows: Graham Darby, Lawrence B. Dumas, Akio Fukuda, Robert T. Heath, Robert F. Mayol, Ramendra K. Poddar, Aharon Razln, Ellen G. Strauss, J. Millar Whalley

55

Graduate Students: Robert M. BenbC1t1, John Lee Compton, John E. Newbold, Robert G. Rohwer, John W. Sedat, Lloyd H. Smith, Anthony J. Zuccarel II

Associate Biologist:· El Jana C. Espejo

Research Assistants: Kathleen Burroughs, Gloria C. Davis, Alma J. Shafer

Research Aide: Barbara Van Gelder

Laboratory Ai de: Jeannette Johnstone

Research upon 0X-speclfic proteins made during Infection has been co-ordi­nated with a thorough genetic study of 0X mutants to provide al a detal led genetic map, bl several self-consistent I Ines of evidence defining the direction of tFan­scrlptlon, and cl recognition and Initial characterization of the proteins speci­fied by the 8 known 0X clstrons.

Further studies of the In vivo 0X mitochondrial RNA (mRNA) have substan­tiated the earlier results Indicating that the mRNA Is matie as a polyclstronic message up to I genome In length, and that Its degradation most often begins from the 5' end before the complete RNA has been synthesized. At the time of maximum phage synthesis, 0X mRNA synthesis amounts to 10% of the total mRNA synthesis of the Infected ce I I •

Studies of the replication of PM2 virus DNA have Jed to the description of 2 Intermediate DNA structures In this process.

47. THE SIZE AND ORDER OF BACTERIOPHAGE 0Xl74 GENES

Investigators:

Support:

• Robert M. Benbow, Robert F. Mayol, Jiii D. Fabrlcant

Earle C. Anthony Fellowship National Institutes of Health, Public Health Service

The order of ~X174 nonsense mutations within 7 of the 8 known clstrons was determined by three-factor genetic crosses. Polyacrylamlde gel electrophoresis In SOS <Mayol, Biology 1969l was used to examine the 0Xl74 specific proteins made after infection by these mutants under nonpermlssive conditions.

Mutations at one end of each clstron do not alter the gel patterns. Mu­tations in the middle of a clstron often give rise to a detectable polypeptide ot altered mob I llty. Mutations at the other end of each clstron cause one (or morel 0Xl74 spei:i fie proteins to disappear from the gel patterns .

• Undergraduate Summer Research Student.

56

The direction of translation as defined by these criteria is V-l-Vll-111-11 (VI-VIII-IV). Strong polar mutations are found in VII and Ill.

Using proteins of known molecular·'Weight as standards in the gel electro­phoresis, the size of the proteins of 7 of the 8 cistrons was determined. The size of each cistron on the genetic map was consistent with the molecular weight thus determined.

48. MULTIPLE LENGTH DNA M:JLECULES OF BACTERIOPHAGE 0Xl74

I nvestl gators:

Support:

Rober+ M. Benbow, Moises G. Eisenberg

Earle C. Anthony Fel lowshlp National Institutes of Health, Public Health Service Rockefe I I er Foundation

One milligram of 0Xl74 multiple length DNA molecules (Biology 1969) was purified by repeated sedimentation through preformed neutral CsCI gradients. Cen­trifugation In the Splnco Model E ultracentrifuge In 3M CsCI at pH 7.5 showed that the product contained less than 3% RF I. In alkal lne 3M CsCI at pH 12.3 several , slower-sedimenting peaks were seen In addition to the major component at 80.5S.

• To verify the structure of each component of the multiple length DNA mole­

cules, equl librium sedimentation with propldium bromide in CsCI was performed. 3 bands were seen, representing nicked molecules, catenanes, and closed circular mole­cules. Spectrofluorometry was used to find the relative amounts of material In each band. After dye was removed by chromatography through a Dowex column, the composition of each band was checked in the Model E ultracentrifuge and by electron microscopy, showing that double length and catenated double rings were the primary multiple length components. Roughly 20 double length molecules were seen for each catenane,

A co60 Y-1 rradiator was calibrated to deliver one single strand break per molecule. By thus artificially nicking the multiple length DNA molecules and mea­suring the sedimentation coefficient of the resulting components In alkaline and neutral CsCI the structure of all major multiple length DNA components was deter­mined.

The infectivlty of these components in spheroplast assays Is currently under study.

49. THE GENETIC MAP OF BACTERIOPHAGE 0Xl74

Investigators:

Support:

* Robert M. Benbow, JI 11 D. Fabrlcant

Earle C. Anthony Fellowship National Institutes of Health, Public Health Service

0Xl74 am, och, op, ts, and cs mutants In 8 clstrons were mapped using two­factor crosses <Biology 1960)-.- am-ts and am-op double mutants were constructed by crossing ultraviolet-irradiated parental phagO-and selecting for doubly conditional lethal progeny. Using these double mutants three-factor crosses of the type am-ts x am or am-op x am 1rnre performed. The tol I o.'11 ng genetic map i ncorporatesboth

* Undergraduate Summer Research Student.

two-factor (Biology 1969) and three-factor crosses.

och 6

ts9 aml6

aml4 tsll6 och 5

nz:

am HSI amlO

I

am6 am27

am3 op9 op6

II

am 23, am 80 am NI

ts 4 am 90 ..

• am 32 il 79

m li y am 9

am89

57

ts 41 D ~SS am87

am H57

tsh 6

High negative interference was seen in al I three-factor crosses. Four­factor crosses of the type am-ts x am-cs, selecting for recombination between the am markers, conf I rmed th Is by show Ing that a 11 4 recomb I nant genotypes - ++++, +++~, ts+++, and ts++cs are generated In sign I fl cant amounts.

58

50. GENETIC RECOl"l31NATION OF BACTERIOPHAGE 0Xl74

Investigator: Robert M. Benbc:m

Support: Earle C. Anthony Fel lc:mship National Institutes of Health, Public Health Service

Two pathways exist for the formation of 0Xl74 recombinants: the host rec A recombination mechanism, and one of the host DNA repair mechanisms. 0X174 forms less than 10% of Its "normal" recombinants in a rec A relative to a rec+ host tor two- and three-factor genetic crosses. I ntroductlooof s Ingle strand breaks Into the parental RF molecule stimulates recombination up to 50-fold in a rec+ cell, but not significantly In a~ A host.

Recombination formation occurs early In the 0Xl74 replication cycle and probably takes place on the membrane. None of the 8 known 0Xl74 cistrons are re­quired tor formation of 0Xl74 recomblnants. However, crosses In which both mutants are defective in gene VI shc:m an abnormally large number of recomblnants.

51. 0Xl74-SPECIFIC PROTEINS: SEDIMENTATION ANALYSIS

I nvestl gator: Robert F. Mayol • Support: National Institutes of Health, Public Health Service

Infection of E. coll with 0Xl74 does not shut off host cell protein syn­thesis. Attempts to detect synthesis of 0X-speclflc proteins have made use of UV­irradlated cells, in which Incorporation into host proteins Is suppressed, while some viral protein synthesis can take place (Burgess and Denhardt, 1969; Gelfand and Hay ash I, 1969). We have observed that double-label Ing procedures al lc:m the detec­tion of \ilX-speclfic proteins without such prior irradiation of the host cell <Mayol and Slnsheimer, 1970). The gene products of clstrons VII (capsid), II (spike), Ill (spike), V (single-strand DNA synthesis), I ( lysis), and the chloramphenlcol-resls­tant Vi <RF replication) have been identified by their mob! lltles on SDS-acrylamide gels. Under these conditions, all phage protein aggregates are disrupted by heat­ing in 9 M urea, 0.1% SOS.

Sedimentation analysis of double-labeled lysates of infected and unin­fected eel Is in 5-30% sucrose gradients (without SOS treatment) shows several dis­tinct 0X-specific species of 2S, 6S, 9S, and 20S, as well as I J4S phage particles. SOS-gel electrophoresis of these regions of the gradients after disruption with urea-SOS shows that the 2S species contains almost exclusively clstron V protein and clstron I protein as (il'X-speclflc components; the 6S species contains primarily cistron II I protein (Greenlee and Sinshelmer, 1968); the 9S species contains cistroh VI I protein; and the 205 species contains a protein whose cistron has not been identified as yet. This protein has also been found associated with RF molecules at late times in infection (Biology 1970, No. 62).

The 6S species probably represents an intermediate in the assembly of the spikes, or possibly is a whole spike component, whl le the 9S species is a capsld subunit, probably a trimer of VI I protein. Similar intermediates have been reported in UV-irradiated cells by Tonegawa and Hayashi (1970).

f<eferences:

Burgess, A. B. and Denhardt, 0. T. (1969) J. Mol. Biol. 44: 377.

Gelfand, D. H. and Hayashi, M. (1969) J. Mol. Biol. 44: 501.

Mayol, R. F. and Slnshelmer, R. L. ( 1970) J. VI rology (submitted tor pub 11 cat I on) . -,

Greenlee, L. L. and Sinshelmer, R. L. (1968) J. Mol. Biol. 32: 321.

Tonegawa, S. and Hayashi, M. ( 1970) J. Mol. Biol. 48: 219.

52. OPAL (UGA) NONSENSE CXJDON MUTANTS OF ~Xl74

Investigator:

Support:

* J 111 D. Fabrlcant

National Institutes ot Health, Public Health Service

59

Opal (UGA nonsense to screen tor nmv c I strons. mutation was then used as an crosses.

codon) mutants of bacteriophage iilXl74 were Isolated In addition, am-op double mutants were made; the opal outside markerinthree-tactor ~-op x ~genetic

Wi Id-type iilXl74 was mutagenlzed with nitrous acid to deamlnate the bases In the DNA. The transition C-+T generates opal mutants when Arg coded by CGA .Js mutated to TGA. Opal mutants were defined as phage which grow on CIT-103 Csu+UGAl but not on HF4714 csu+UAGl or C Csu-). These opal mutants were then characterized by genetic mapping and polyacrylamlde gel electrophoresis.

An am-op double suppressor strain was made by lysogenlzing a su+uGA strain with ~80 d tryp S"Ul'UAG. am-op double mutants were constructed by mating pure amber and opal phage after UV lrradTatlon. Screening for double mutant stocks was done by a double layer technique, plating first with a permissive strain and then with a suppressive strain.

Most of the opal mutants Isolated showed very high reversion frequencies.

53. STUDIES WITH ~Xl74 MUTANTS BLOCKED IN PROGENY SINGLE-STRANDED DNA SYNTHESIS

I nvestl gator: Aklo Fukuda

Support: National Institutes of Health, Public Health Service

Mutation in any one ot the iilX174 clstrons Ill, IV, V, VII or VIII blocks single-stranded DNA synthesis at late period of Infection CLlndqvlst and Slnshelmer, 1967; Funk and Slnshelmer, 1970) and infection with wild-type iilX174 at low tempera­ture C 15°) is also abortive of single-stranded DNA synthesis (Newbold and Slnshelmer, 1970). These observations predict several regulatory steps in the process of slngle­stranded DNA synthesis.

Studies were performed with amber mutants of clstrons 111, IV, V and VI I in an attempt to identify intermediate replicative form <RF) molecules that might accumulate Cas a consequence of mutation) during the period of single-stranded DNA synthesis. These mutants cease progeny RF synthesis around 15 minutes after infec­tion at 36°. At 18 minutes, the Infected culture was pulsed with 3H-thymidlne for

* Undergraduate Summer Research Student.

60

30 seconds, lysed with lysozyme, digested with sarkosyl-pronas~ and sedimented In a neutral sucrose gradient. In al I the IJl,utants studied the majority of the pulse­label was found In RF I I molecules (16S, nick In one strand). However an apprecia­ble amount (10-15%) of the pulse-label was also found in fast-sedimenting fractions (21S-25Sl where RF molecules with single-stranded tal I would be expected to sedi­ment (Knippers et al., 1969). Short pulse-label was not incorporated Into RF I molecules (215, supercoi I).

When analyzed In an alkaline sucrose gradient, the fast-sedimenting com­ponent was found to contain RF molecules with I I near strands longer than unit length. The pulse-label in RF II molecules was also found only In linear strands'. Further­more the results of alkaline CsCI equl llbrlum centrifugation showed that tn both RF II molecules and fast-sedimenting component, only the vlral strand was labeled In a short pulse.

It Is not yet known whether this phenomenon ls observed only In the period shortly after termination of net RF synthesis.

References:

Funk, F. O. and Slnshelmer, R. L. (1970) J. Vlrol. (in press).

Knippers, R., Razin, A., Davis, R., and Slnshelmer, R. L. (1969) J. ~ol. Biol. 45: 237.

Llndqvist, B. H. and Slnshelmer, R. L. (1967) J. Mol. Biol. 30: 69. '

Newbold, J.E. and Slnshelmer, R. L. (1970) J. Mo!. Biol. 49: 23.

54. CHARACTERIZATION OF A SPECIFIC MEMBRANE FRAGMENT ASSCCIATED WITH REPLICATING ONA

I nvestl gator: Robe rt T • Heath

Support: National Institutes of Health, Public Health Service

In recent years numerous reports have I ed to an I ncreas 1 ng awareness that DNA repllca-tion In E. col I occurs In association with the cell's membrane. The rep-1\ca-tion ot lilx\74 ONA also appears to be a host eel\ membrane-associated event \Knippers and Sinshelmer, 19610). Since '<he rep\lcat\n .. 0\-11' \s \)resumabl'I associated with a spec\ tic portion ot the merrorane, two questlons arlse: what ls the nature of the particular portion of the membrane associated with the replicating DNA, and what is the nature of the DNA-merrorane association 1tself1 To examine these ques­tions a procedure has been developed by which cells may be \ysed rapidly and the intact membrane (spheroplast envelope) Isolated by sedimentation. The spheroplast envelopes are then fractionated by a procedure slml lar to that previously reported <Biology 1969, No. 148).

This procedure al lows Investigation of the effects of pH and the Ionic conditions involved In maintaining the integrity of the membrane and the associa­tion of the DNA with It, At a constant Ionic strength ( 1.0 Ml the membrane and the OMA association remain intact at pH 4.5, but as the pH ls increased to 10.5 the membrane ls fragmented Into 2 major fractions. Substructure within these major fractions has been noted. Also as the pH is raised within this range 0Xl74 paren­tal RF Is removed. At pH 8,0, Increasing ionic strength (.01 <I< 1.0 Ml has I ittle effect on the Integrity of the membrane, but the presence oT a non-specific

61

divalent metal Ion chelator effects a fragmentation of the membrane. The posslbi t­lty that Mg++ is specifically required to maintain the structure of the membrane is under I nvestl gat ion. Host ce I I mass-I abe I ed DNA is re I eased from the merlb rane as ionic strength Increases; nascent·-.pulse-labeled host DNA from uninfected cells and '6Xl74 RF from infected eel Is remain associated with the membrane over this range of ionic strength. This association of replicating and newly-replicated DNA with the membrane was not affected by removal of divalent metal ions or the presence of non-ionic detergents; the association ls destroyed at low concentrations of ionic detergents and by proteolytic enzymes.

A particular membrane fraction has been isolated which contains the repli­cating parental tllXl-74 RF and is enriched in nascent host DNA in uninfected eel Is. The chemical composition of this fragment is currently under investigation.

Reference:

Knippers, R. and Sinshelmer, R. L. <1968) J. Mot. Biol. 34: 17.

55. EFFECT OF MITOMYCIN CON LYSIS OF 0Xt74 INFECTED HOST CELLS

I nvestl gator: Akio Fukuda

Support: National Institutes of Health, Public Health Service

•

It was previously shown that host-controlled-reactivation-negative (HCR-) E. coll cells can fully support the '6Xl74 infection proCi'SS even after mitomycln c treatment to inhibit host DNA synthesis CLlndqvlst and Sinshelmer, 1967).

In addition to the above effect it has now been found that when HCR- cells are treated with mltomycln cat 300 µg/ml for 25 minutes at 36° without aeration and subsequently infected with wl Id-type 0Xl74, no lysls of the infected eel Is, as observed by fol lowing O.D.550, is seen for I hour, quite often even 2 hours, after Infection at 36°. Infection without mltomycln c treatment lyses the HCR- eel Is be­ginning at 15 minutes after infection at 36°. The burst size of mltomycln c­treated cells at 2 hours after infection ls 8-12 times that of untreated cells, thus giving phenotyplcal analogy to the Infection with lysis-defective mutants.

No explanation can be given as yet for the effect of mitomycln c. How­ever it serves as a convenient tool to study the 0Xl74 infection process without lys Is comp I I cation.

Reference:

Lindqvlst, B. H. and Slnshelmer, R. L. (1967) J. Mot. Biol. 30: 69.

56. BACTERIOPHAGE 0Xl74 DNA SYNTHESIS IN VITRJ

Investigators: Lawrence B. Dumas, Graham Darby,' Robert L. Si nshelmer

Support: National Institutes of Health, Public Health Service

The nature of the DNA products synthesized on partially double-stranded ONA templates in vitro is dependent upon the temperature of incubation. At 20°C, DNA polymerase from E. coli catalyzes the conversion of these templates to

62

completely helical DNA by fl II Ing In the single-stranded regions, at which time net synthesis ceases. At 37°C the completely double-stranded form of the DNA can serve as template for further synthesis Involving displacement of one of the two strands of the du;i lex wh I le the other strand serves as temp I ate tor the synthes Is of a new JNA strand. The effect of incubation temperature on 0Xl74 DNA synthesis In vitro was invt'.lstigated to further characterize these two kinds of synthesis.

At temperatures below 22-25°C in vitro DNA synthesis by DNA polymerase, using single-stranded circular 0X DNA as template, was I lmlted to one cycle of replication. Al I circular phage DNA was converted to completely double-stranded molecules; then net polymerization ceased. When high levels of Initiator oligonu­cleotides 1;ere used, th·e product complementary strand had several nicks arising from mu~t1ple initiations. The number of initiations per template decreased as the ratio of initiator to template was reduced, Most of the nicks In the complementary stranc cou1d be repaired by polynucleotide-jolning enzyme. However, at 15°C the last nick was less frequently repaired suggesting a unique problem of closure of tne last nick to form a complete circular double-stranded DNA molecule.

At temperatures such that greater than one-fold synthesis could occur (above 22-25°Cl product strands were elongated to several phage DNA unit lengths. The existence of the apparent temperature threshold suggests that a cooperative structural change may be necessary to al ICltl the Initiation and/or propagation of the synthesis leading to extended strands. The activation energy measured for the synthes ls of extended strands was 39+4 kcal per mole. That for the fl rst cycle 'of replication was 17+2 kcal per mole. -Apparently denaturatlon of a small nurrl:>er of base pairs (2 to 4) In the region of the grCltllng point is needed to Initiate and sustain greater than one-fold synthesis.

References :

Mitra, S., Reichard, P., Inman, R. B., Bertsch, L. L., and Kornberg, A. C 1967) J. Mel. Blol. 24: 429.

Goullan, M., Kornberg, A., and Slnshelmer, R. L. (1967) Proc. Nat. Acad. Sci. 58: 2321.

57. NATURE OF THE COMPLEMENTARY STRAND SYNTHESIZED IN VI TR:l FROM ULTRAVIOLET-IRRADIATED CIRCULAR SINGLE-STRANDED DNA

I nvestl gator: Ramendra K. Poddar

Support: Natl ona I I nstl tutes of Heal th, Pub 11 c Hea I th Service

In spite of a large amount of work done on the photochemistry, photoblol-· ogy and repair of ultraviolet CUVl-lrradlated DNA (see review by Setlow, 1968), we lack ~recise knowledge about hClo'I the various kinds of UV lesions Interfere with the replication of such templates. Specifically the question whether UV lesions act as absolute blocks for the polymerization enzyme or simply slow down the polymer­ization process is stl I I open. Since precise physical data about the templates and produc7s are needed to distinguish between these two poss I bl Ii ties, the well-char­acterized E. coll DNA polymerase reaction system CGoullan and Kornberg, 1967) in which The circular single-stranded DNA of bacteriophage 0Xl74 serves as the template for the in vitro synthesis of Its complementary strand, offers an excellent means -.-o clarify the issue. Results of our investigations about the nature of the com­,1ementary strand synthesized In vitro when the UV-Irradiated circular single­stranded DNA of phage 0Xl74 was present as the template in such a reaction mixture

63

are given below.

The I ength of the newly synthes I zed complementary ("ml nus") strands as we 11 as the tempi ate act I vi ty (as measured by dAMP Incorporated In the ml nus strands) was found to decrease with the number of lethal hits sustained by the circular plus strand. However, the mean length of the minus strands, as determined by velocity-sedimentation In alkaline CsCI gradient, appeared to be greater than what one would expect If all the hits were to block polymerization ("chain elonga­tion") with equal efficiency. Approximately only I In 3 or 4 lethal hits seemed to block chain elongation. With an Increase of time, temperature, or amcunt of ol I gonucleotl de "prl_mers" added to I nlti ate polymer! zatlon ("In !ti ators"), the net synthesis of minus strands increased, but their mean length remained more or less constant. It therefore appears that although polymerization can proceed past most of the UV lesions on a circular single-stranded DNA template, some lesions do act as absolute blocks for the DNA polymerase. Experiments are In progress to Identify these blocks as we 11 as to see If any II gase-sea I ab le gaps cou Id be produced durl ng polymerization process. Preliminary results suggest that the lesions blocking polymerization are probably pyrimidine dimers monomerlzable by yeast photoreacti­vatlng-enzyme In presence of visible light.

If the DNA polymerase of E. coll Is the enzyme which catalyzes the"'in vivo conve rs I on of s Ing le-stranded l'JX 174 DNA to double-stranded RF, the above re­su Its would help us understand the mechanism of partial repair of UV-lrradlaftld free l'JX174 by appropriate host cells under conditions of rapid growth (Datta and Poddar, 1970).

References:

Datta, B. and Poddar, R. K. (1970) Molecular and General Genetics (in press).

Goullan, M. and Kornberg, A. (1967) Proc. Nat. Acad. Sci. 58: 1723.

Set low, R. B. ( 1968) Prog. Nuc. Acid Res. Mol. Biol. 8: 257-295.

58. RF I I DNA; RESISTANCE TO DNA POLYMERASE-ASSOCIATED EXONUCLEOLYTIC ACTIVITY

I nvestl gator: Aharon Raz In

Support: National Institutes of Health, Pub I le Health Service

An exonucleolytlc activity associated with highly purified preparations­of E. coll DNA polymerase has been described by Klett, Cerami and Reich (1968) and by Deutscher and Kornberg ( 1969 l. Th Is act I vi ty degrades daub I e-stranded DNA from the 5'-end, producing 5 1 mononucleotldes and ollgonucleotldes terminating In a 3' phosphate group.

The present study Indicates that the progeny RF I I DNA Isolated from E. coli eel Is Infected with l'JXl74 Is resistant to the exonucleolytlc activity associ­ated with E. coll DNA polymerase. A limited endonucleolytic cleavage with mlcro­coccal endonuclease renders the RF 11 molecule susceptlb le to the exonucleolytic activity associated with the E. coll DNA polymerase. It Is suggested, therefore, that the resistance of RF I I DNA to the exonucleolytlc activity is a result of the structure of these molecules at their 5 1 terminus.

64

References:

Klett, R. P., Cerami, A., and Reich_, E. (1968) Proc. Nat. Acad. Sci. 60: 943. -

Deutscher, M. P. and Kornberg, A. ( 1969) J. Biol. Chem. 244: 3029.

Razln, A. and Slnshelmer, R. L. (1970) Proc. Nat. Acad. Sci. (in press).

59. THE IN VIVO 0X MESSENGER RNA

Investigator: John W. Sedat

Support: National Institutes of Health, Public Health Service

The in vivo ~X messenger RNA (mRNA> has been Investigated In an attempt to answer questions about temporal control of transcription, mRNA degradation, and the site (and template) for ~X rrRNA synthesis.

The size of the In vivo ~X rrRNA molecules has been studied by sedimenta­tion of pulse- and uniformly-labeled RNA (from ~X-lnfected cells) through 99% di- • methyl sulfoxlde gradients and subsequent hybridization of fractions to ~X RF; the resultant profi tes indicate a broadly heterogeneous size distribution of both pulse and mass labels with a distinct maximum size of I .7 megadattons and a maximum fre­quency of 0.2 megadaltons. This mRNA size distribution does not change with time after Infection. Little or no RNA capable of hybridization with RF ls found In uninfected cells; no RNA capable of hybridization with 0X DNA Is found In infected eel Is at early or later times.

This data Imply that no temporal control of transcription occurs during 0X Infection, and that the size distribution probably results from rrRNA degradation coupled to transcription and translation. The finding of full genome-sized pieces of 0X rrRNA at early times (4 minutes) after infection together with other data imply that the parental RF Is very likely the template for transcription.

60. THE IN VI TRJ 0X 174 MESS ENGER RNA

I nvestl gator: Lloyd H. Smith

Support: National Defense Education Act National Institutes ot Health, Public Health Service

RNA synthes lzed in vitro using the pure components RNA polymerase and iilX174 am3 rep I 1cat1ve form I <RF I l has been examined. E. coll K12 RNA polymerase was purified using the procedure ot Burgess (1969) through the DEAE-cel lulose step. The DEAE fraction was further purl fled to ~95% purity by KCl-gradlent elution after application to a calf thymus DNA-eel lulose column, then to >99% purity by a single passage through agarose (A-1.5 Ml In I M KCI. Purity estimates were made after electrophoresis from densitometer traces of 0.1% SOS 10% acrylamlde gels.

The final enzyme preparation (designated "AC" enzymel contains the a fac­tor described by Burgess et al. (1970), which was separated from the "core" enzyme by passage through a phosphocet lulose column. The AC and o preparations lacked detectable rlbonuclease and endodeoxyrlbonuclease activities.

-

65

Pure iilX174 am3 RF I was prepared as by Komano and SI nshelmer < 1968). Partially pure RF was a gl ft of M. Eisenberg and J. Sedat. Final RF I preparations usually contained about 5% RF II molecules.

The reaction mixture used tor RNA synthesis contains 0.05 M trls-CI pH 7.9, 0.01 M MgCl2, 0.2 M KCI, 0.0001 M EDTA, 0.0001 M dlthiothreltol, 0.0004 M NaH2P04 and varying amounts of rlbonucleoslde trlphosphates, 0Xl74 RF I, and AC enzyme. The 5 1 terminal nucleotide of the resulting RNA has been examined by Incorporation of Y-32P-labeled ribonucleoslde trlphosphates. Because of the very high specific activities necessary to label the 5 1 terminal nucleotide, TCA pre­cipitation results In high and variable background Incorporation. Reliable data are obtained if reaction mixtures are first fl ltered through Sephadex G-100 (in 0.1% SDSl; the excluded volumes are collected and TCA-preclpltated as usual. Back­ground Incorporation In the absence of DNA is subtracted from the DNA-directed Incorporation. RNA mass synthesized Is usually measured by 3H-UTP Incorporation.

Number average chain length values can be obtained by dividing the moles of mass synthesized by the total moles of y_32p Incorporated (using Y-32P-ATP and Y-32P-GTP Incorporation as the total amount of Initiation}. It Is observed that Ionic strength affects the average chain length. Without KCI present In the reac­tion mixture, an excess of enzyme synthesizes RNA with an average chain length of about 2,000 nucleotides In the time required for more than I-fold synthesis. This value Increases with Increasing KCI concentration, and reaches an optimum ai;ound 0.2 M KCI where the average chain length approaches 5,500 nucleotides (equal to unit iilX length}. At higher Ionic strengths (greater than 0.25 M KCI l the associa­tion of DNA and enzyme begins to be Inhibited.

Under conditions of molar equality between RF ·I and AC, both Y-32P-ATP and Y-32P-GTP are Incorporated to about an equal extent. When an excess of o fac­tor Is added, Y-32P-ATP Incorporation Is stimulated so that about 75% of the end­label ls ATP. However, if the polymerase-DNA Initiation complex is al lowed to form in the absence of trlphosphates with excess AC, and If further complex formation Is Inhibited by rlfamplcln, It Is found that when trlphosphates are added, RNA chains start exclusively with ATP.

Recently, it has been suggested <Bautz et al., 1970) that for certain templates the a-mediated Initiation complexes may be the only polymerase-DNA com­plexes able to Initiate In the presence of rlfampicln. If this ls true for iilX174 RF, a-directed Initiation on RF I occurs with A as the 5' terminal nucleotide. The G-starts 1 n the absence of rl tamp I cl n may represent a rather h I gh I eve I of non-a­medl ated Initiation or Initiation at a low affinity of promoter(s} that cannot Initiate In rlfamplcin. There is probably only one a-mediated ATP initiation site per RF I molecule.

In addition to the 5 1-end label analysis, the size of the RNA synthesized (without rlfamplcinl after phenol extraction has been determined by sedimentation In 99% DMSO <Sadat et al., 1969). After I-fold synthesis, the RNA appears to be fairly homogeneous and of a size about equal to that expected for 0X174 unit length RNA. This RNA can be hybridized only to 0Xl74 RF and not to 0Xl74 viral DNA, demonstrating that the polymerase Is properly transcribing the template asymmetrl­cal ly as In vivo.

The results Indicate that a rlfamplcln-reslstant A Initiation can be ex­amined in the absence of other Initiations and hopefully wl I I produce synchronously­growing chains that can be sequenced.

67

with the antigenicity of whole virus. The capsld particles were prepared by means of a 4 M urea treatment of who I e virus wh I ch was devl sed by Edge 11 C 1969). Rabb It anti sera were then prepared aga Inst both 0X am3 and the caps Id part I c I e and were subsequently compared with respect to,phage Tnactlvatlon and In an lmmunodlffusion system.

lmmunodlffuslon demonstrates that there are 2 or at most 3 antigens asso­ciated with the whole virus, at least one of which Is not present In the capsld par­ticle, and that there are at least 2 antigens associated with the capsld, I of which Is not exposed on the ~X particle. These relationships were determined by means of the cross reaction and differential absorption of the two antlsera by both parti­cles. It Is probabl-e that the capsld particle lacks a spike antigen whereas the whole virus harbors an antigen which Is exposed only when the spike Is removed. It should be noted that one of the 2 bands observed by Rolfe and Slnsheimer (1964) In an earlier study of 0X antigenicity was present only In Impure virus preparations In our system.

In spite of the differences between these sera both contain similar titers of phage-Inactivating antibodies and both Inactivate plaque-formers with Identical "single hit" kinetics. Because the 2 sera were shown by gel di ffuslon methods to have only the anticapsld antibodies In common, one can conclude that at least I caps Id antigen gives rise to a phage-Inactivating antibody. In an earl ler st'udy, Krane (1966) tested temperature-sensitive mutants in the spike component gene~ I I and 111 (and in gene I Vl for the Ir ab 111 ty to synthesl ze serum-block Ing power CSBP) under restrictive conditions. Only the gene Ill mutant, ts79, showed no SBP. Other mutants, Including numerous examples from gene Ill, showedreduced levels, but not the complete absence, of SBP. We have shown that ts79-restrlctlve lysates also lack SBP In assays performed with either the 0X- or-capSIQ-dlrected antlsera. Be­cause ts79 Is known to be a lesion in a spike component, these data have often been cited as evidence that the only phage-Inactivating antibody Is made in response to a spike component. However, It now seems 1 lkely that because the ts79-restrlctlve lysates lack SBP from the unmutated caps Id protein gene VI I, as we_l_I as from gene I II, the complete absence of SBP Is due to some Indirect cause such as greatly re­duced protein synthesis or failure to produce some intermediate to phage maturation which may be the minimum level of organization necessary for antigenicity. It seems I ikely that the less dramatic reductions of SBP by many of the ts mutants tested by Krane to 10-15% of normal levels is indicative of the removal or-one antigenic structure from a pool of several antigens, any one of which can give rise to phage­lnactlvatlng antibodies.

References:

Edge I I, M. H., Hutchison, C. A., 111, and Slnshelmer, R. L. ( 1969) J. Mo I • BI o I • 42: 54 7.

Rolfe, U. and Slnshelmer, R. L. (1964) J. lmmunol. 94: 18.

Krane, S. G. (1966) Ph.D. Thesis, California Institute of Technology, Pasadena, California.

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63. A RADIOIMMUNE ASSAY FOR 0X ANTIGENS

Investigator: Robert G. Rohwer

Support: National Institutes ot Health, Public Health Service

The radioimmune assay of Catt and Tregear (1967) has been adapted tor use with 0X antigens. Antl-0X antibodies are Irreversibly bound to the walls of a plas­tic sclnti I lat Ion vial by maans of a brief Incubation In the presence of .05 M Na tetraborate. The unbound serum Is washed 'IMay and the tube Is coated w I th I 0% bovine serum albumin CB$Al to reduce nonspecific protein absorption later In the assay. A control Is prepared by Incubating a vial only with BSA. Radioactive ¢X antigens can now be added to the tube where they react with the antl-0X antibodies and are bound to the wal Is of the vial. After 24 to 36 hours of Incubation the vial can be washed free of unbound radioactivity and counted.

Because of a high background of nonspecific protein absorption, the assay has proved impractical for use with crude lysates labeled with radioactive protein precursors. Its utility lies In the detection of DNA-phage-antigen complexes. If one labels 0X-lnfected bacteria with a DNA precursor, the only radioactivity bound Is that mediated by the binding of 0X antigenic proteins. With this technique It has been possible to demonstrate the specific binding to the vials of the "RF 11 with tal I" lntennedlate In single-stranded DNA rep I I cation which has been charac1'er­lzed by Knippers et al. Cl969). Such binding Indicates an association of 0X anti­genic proteins with this species of DNA.

References:

Catt, K. and Tregear, G. W. (1967) Science 158: 1570,

Knippers, R., Razln, A., Davis, R., and Slnshelmer, R. L. < 1969) J. Mol. Biol. 45: 237.

64. REPLICATION OF BACTERIOPHAGE PM2 DNA: A CLOSED CIRCULAR DOUBLE-STRANDED MOLECULE

I nvestl gators: Romi 11 o T. Espejo, El I ana C. Espejo

Support: National Institutes of Health, Public Health Service

The DNA ot bacteriophage PM2 exists as a closed circular double-stranded molecule In the mature virus. Since this is a smal I DNA of molecular weight equal to 6 mil lion, the multlpllcatlon of PM2 offered a relatively simple system for fur­ther studies on DNA replication and the slgnl flcance of cl rcularlty and supercol ling.

The most interesting aspect ot our research· on PM2 DNA rep I lcation con­cerns the characterization ot precursors ot the supercol led molecule. Pulse-chase experiments suggest the existence ot two precursors, one which sediments as nicked circular PM2 DNA and another, appearing earlier, sedimenting I.I times taster. This earlier precursor seems to be a molecule with a growing chain since Its sedi­mentation prof/ le upon denaturatlon suggests the existence of single-stranded pieces longer than PM2 DNA, In accordance with. the hypothesis that chain growth In­volves addition of nucleotides to one end of a parental strand. Observation with tne electron microscope of partially purl tied preparations of these molecules shows that they consist of double-stranded rings, the size ot PM2 DNA, with one double­stranded DNA branch of variable length but always shorter than PM2 DNA. Besides

69

the rings with a branch, some continuous molecules I Ike those observed by Calms ( 1963) In autoradlographs, and by others with the electron microscope, were occa­slonal ly found. Even though such molecules were rarely observed (I or 2 per 400 rings with branch), they do not seem to be an artifact; their presence suggests that the rings with a branch could have arisen by specl tic breakage at one terminus of the duplicated region In the continuous molecules.

The purification procedure Is at present being modified in those steps that might produce breakage of the replicative Intermediate in its native state.

Rate ranees:

Espejo, R. T., Espejo, E. c., and Slnshelmer, R. L. (1969) Proc. Nat. Acad. Sci. 63: 1164-1168.

Cairns, J. (1963) Cold Spring Harbor Symp. Quant. Blot. 28: 43.

65. IMPROVED PURIFICATION FDR OS VIRUS

Investigator: El ten G. Strauss

Support: National Institutes of Health, Public Health Service

We have been dissatisfied with the purity and specific lnfectlvlty of 08 virus preparations which were made using the method developed by Strauss and Sinshelmer (1963) tor MS2. The yield of physical particles (as determined by the optical density of the final preparation> was quite good,'but less than 1% of the purified 013 particles were Infective. Also, these OS preparations contained vari­able amounts of a minor protein component when examined by acrylamlde gel electro­phoresis. Therefore we have developed a new and better purification method.

L ys ates of 013 were grONn on an t-p 111 mutant of E. co 11 < M27) wh I ch is sensitive to 013 but resistant to MS2. The virus was precipitated with 10% poly­ethylene glycol and the precipitate resuspended In a small volume. The suspension was clarified by low speed centrifugation and layered Immediately onto sucrose gradients for velocity sedimentation. Samples from these gradients, and from all subsequent steps, were selected and pooled on the basis of lnfectlvlty, rather than radioactivity or optical density.

These pooled fractions were layered directly onto preformed gradients for lsopycnlc centrifugation. The lsopycnlc gradients were poured as are conventional sucrose gradients, but the dense solution was 50% w/v sucrose and 1.25 M CsCI In 99% DzO containing phosphate buffer and EDTA <density = 1.44), whereas the 1 lght solution was 30% w/v sucrose and 0 .5 M CsCI In 99% D20 w I th the same buffer (density= 1.31). After centrifugation to equl llbrlum the virus formed a tight band at a density of 1.37. These conditions were chosen because 013 appears to be stable In CsCI solutions up to 1.0 M, whereas the lnfectlvlty is rapidly Inacti­vated In 4 M CsCI <the concentration used In previous methods).

The recovery at this stage Is about 50% of the original plaque-forming units and more than 99% of the host-specific material has been removed. However, the virus can be further purified by repeating the velocity and lsopycnlc steps; fol lowed by large pore acrylamide gel electrophoresis. These additional steps appear to remove smal I amounts of virus-specific material which are not essential for virus lnfectlvlty.

70

References:

Strauss, J, H. and Slnsheirner, R. L. (1963) J, Mol. Biol. 7: 43-54. -,

PUBLICATIONS

Edgell, M. H., Hutchison, C. A., and Slnshelrner, R. methods of macromolecules and particles. ress in Separation and Purification, Vol.

L. ( 1969) l"odern separation Jn: T. Gerritsen (ed.l, Prog-2. John WI ley and Sons, Inc.

Edge I J, M. H., Hutchison, C. A., and Si nsheimer, R. L. ( 1969) The process of i nfec­tion with bacteriophage l1X174, XXVI 11. Removal of the spike proteins from the phage capsid. J. Mol. Biol. 42: 547-557.

Edgel I, M. H., Hutchison, C. A., and Sinsheimer, R. L. <1969) Large pore "disc" electrophoresis, In: Progress in Separation and Purification, Vol. 2. Wi ley-lntersclence, pp. 1-22.

Espejo, R. T., Canelo, E. s., and Sinshelmer, R. L. (1969) DNA of bacteriophage PM2: A closed circular double-stranded molecule. Proc. Nat. Acad. Sfl. 63: 1164-1168.

• Funk, F. and Sinsheimer, R. L. (1970) The process of Infection with bacteriophage

(.IXl74. XXXI I I. The templates for the synthesis of single-stranded DNA. J. Virology 5: 282-288.

Kiger, J. A. and Sinsheimer, R. L. (1969) Vegetative lambda DNA. V. Evidence con­cerning single-strand elongation. J. Mol. Biol. 43: 567-579.

Knippers, R., Whalley, J, M., and Slnshelmer, R. L. (1969) The process of Infection with bacteriophage 11Xl74. XXX. Replication of double-stranded 0X DNA. Proc. !<at, Acad. Sci. 64: 275-282.

Knippers, R., i<azin, A., Davis, R., and Slnshelmer, R, L. (1969) The process of infection with bacteriophage 0Xl74. XXJX. In vivo studies on the synthe­sis of the single-stranded DNA of progeny 0Xl74 bacteriophage. J. Mol. Biol. 45: 237-263.

Levine, A. J. and Sinshelmer, R. L. (1969) Isolation of chloramphenlcol-resistant protein from ).-Infected eel Is. Proc. Nat. Acad. Sci. 62: 1226-1228.

Newbold, J. and Slnsheimer, f{, L. <1970) The process of infection with bacteriophage (.IXl74. XXXI. The abortive infection at low temperatures. J. ~bl. Biol. 49: 23-47.

Newbold, J. and Sinsholmer, R. L. ( 1970) The process of Infection with bacteriophage 0Xl74. XXXI I. The early steps in the infection process: Attachment, eclipse and DNA penetration. J, Mol. Biol. 49: 49-66.

Newbold, J. and Sinsheimer, R. L. ( 1970) The process of infection with bacteriophage (.IXl74. XXXIV. Kinetic studies of the attachment and eclipse steps of the Infection. J. Virology 5: 427-431.

Sedat, J., Lyon, A., and Slnshelmer, R. L. (1969) Purification of Escherichia coli pu I se-1 abe I ed RNA by benzoy I ated DEAE-ce II u I ose chromatography. J. Mol • Biol. 44: 415-434.

71

Slnshelmer, R. L. (1969) ONA virus maturation. J. Cell Physlol. 74: Sup. I 21-32.

Slnshelmer, R. L. (1970) The Brain of Pooh: An essay on the limits of mind, Engineering and Science Magazl..ne, CIT.

Sinshelmer, R. L. (1970) "Contaglum Vlvum Fluldum," After seventy years. Proc. Kon. Ned. Akad. Wetensch. Serles C, 73: 307.

•

72

Assistant Professor: James H. Strauss, Jr.

Research Fellow: John S. Pierce

Graduate Student: Daniel T. Simmons

Research Assistants: Sandra Kiddy, Ml ldred A. Quisenberry

Sindbls virus ls a Group A arbovlrus. The arbovlruses are a class of animal vi ruses which In nature are transmitted by mosquitoes, and al I the members of this group resemble one another morphologically. The virus particle consists of a nucleocapsld or core surrounded by an envelope or membrane. The core contains the single-stranded RNA of the virus complexed with about 400 molecules of a pro­tein called the core protein. The membrane consists of about 700 molecules of a glycoproteln termed the membrane protein complexed with a variety of phosphollplds and cholesterol, and has the structure of a membrane when examined In the electron microscope. This membrane ls acquired during the last stage of viral maturation when the core buds through the ce 11 surface membrane (whl ch has been modi tied by the virus to contain virus-specific proteins) and becomes enveloped In the process.

We are interested In the virus tor severa I reasons: (I l The virus modi -fies a large portion of the eel I surface membrane during the relatively short in-• fectlon process. This conversion process serves as a simple model of the conver-s Ion process Induced by oncogenl c vi ruses. (2) The virus otters a mode I membrane system. The structure of the virus membrane can be studied and compared wl th the structure of merrilranes of virus mutants as wel I as with the uninfected eel I mem­branes. In addition, synthesis of the virus membrane is rapid and prominent In the Infected eel I and membrane blosynthesls can be fol lowed. (3) The replication of the virus differs in several ways from that of other vi ruses that have been studied. For Instance, the RNA found In the virus ls single-stranded and has a molecular weight of 4 to 5ml11 ion. The major species of RNA made In the Infected eel I, how­ever, ls a single-stranded RNA of molecular weight I .6 ml Ilion. The function of this RNA ls yet to be determined,

66. REPLICATION OF SINDBIS-SPECIFIC RNAs IN TISSUE CULTURE CELLS

I nvestl gators: Daniel T. Simmons, James H. Strauss, Jr.

Support: National Science Foundation National Institutes of Health, Public Health Service

Two predominant species of single-stranded RNA molecules are made in hamster kidney eel Is or in chick embryo eel ls during Infection with Slndbis virus. These virus-specific RNAs have sedimentation coefficients of 265 and 50S and are easy to separate by acrylamlde gel electrophoresis as well as by velocity sedimen­tation. The 50S species corresponds to the complete viral genome and Is the only Ri~A found In the vi rion. Its molecular weight of approximately 4 mi I I Ion has been determined from its rate of migration In acrylamlde gels and from Its sedimentation coefficient after reaction with formaldehyde <Boedtker, 1968). The smaller RNA has a molecular weight of I ,6 ml Ilion as determined by sedimentation In dlmethylsul­foxide (Strauss et al., 1968).

When virus-specific RNA made during Infection Is treated with ribonucle­ase, 3 distinct species of double-stranded RNAs called replicative forms <RFsl, are

73

found. These RFs have sedimentation coefficients of 16S, 20S and 23S, correspond­ing to molecular weights of 3.2, 5.8 and 9.4 ml I I Ion respectively. Al I 3 RFs are released from a single peak of replicative Intermediate CRll which sediments be­tween 23S and 305 and migrates as a sil'lgie peak on acrylamlde gels.

The labeling kinetics of the 3 RFs and the molecular weight data given above suggest that the 165 RF Is a template for synthesis of the 265 single-stranded RNA and that the 235 RF Is a template for 50S RNA synthesis. The 20S RF ls labeled much mores lcoily than either of the other two, and Its rep I lcatlve product(s) has not yet been detected. Further studies on the structure and function of these var I ous RNAs are ncoi In progress.

References:

Boedtker, H. C 1968) J. Mol. Biol. 35: 61.

S-trauss, J, H., Kel Jy, R. B., and Slnshelmer, R. L. (1968) Blopolymers 6: 793.

67. SINDBIS VIRUS-INDUCED HOST CELL PLASMA MEWBRANE CONVERSION

Investigators:

Support:

John S. Pierce, James H. Strauss, Jr.

Damon Runyon Memorial Fund for Cancer Research National Science Foundation National Institutes of Health, Public-Health Service

One consequence of viral-Induced eel lular conversion, though not unique to oncogenlc viruses, Is the acquisition by the eel I of new and/or altered surface antigens. Recently, Slndbls virus and the cells Infected by It have provided a very useful model system for the study of the structure and blosynthesls of a simple membrane. As a consequence of the maturation process the vlrlon acquires Its outer envelope by budding through the plasma membrane of the Infected cell. The envelope Is composed of lipids and a single glycoproteln of molecular weight estimated at 53,000 daltons (Burge and Strauss, 1970; Strauss, Burge and Darnell, 1970). The protein appears to be virus-specific and Is synthesized de novo In large amounts after Infection <Strauss, Burge and Darnell, 1969). The Integration of this protein Into a structure which Is morphologically lndls-tlngulshable from the plasma membrane of the uninfected eel I Is currently being Investigated at times prior to virus maturation. Plasma membrane Is prepared from both primary chick embryo fib rob lasts and from the normal cytoplasmic particles CNCP) which these eel Is continually pinch off into the medium bathing the monolayers. The latter source of membrane Is particularly advantageous since NCP consist simply of cytoplasm en-· closed within a small portion of plasma membrane. Preliminary pulse-labeling ex­periments Indicate that of several proteins resolvable In polyacrylamlde gels, 2 with molecular weights estimated at 22,000 and 9,000 daltons have very rapid turn­over rates. The relationship between these proteins and the single virus-specific protein In the fully converted membrane (e.g., virus envelope) remains to be deter­mined.

References :

Burge, B. W. and Strauss, J. H. C 1970) J. Mel. Biol. 47: 449-466,

74

Strauss, J. H., Burge, B. W., and Darnell, J.E. (1970) J. Mel. Biol. 47: 437-448.

Strauss, J. H., Burge, B. W., and-Qarnell, J.E. (1969) Virology 37: 367.

68. CHARACTERIZATION OF AN INFECTIVITY ASSAY FOR SINDBIS VIRUS

Invest I gator: James H. Strauss, Jr.

Support: National Science Foundation National Institutes of Health, Public Health Service

Many animal viruses have a low specific lnfectlvlty when virus stocks are titored in a plaque assay, with particle to plaque-forming-unit ratios of 100 to 1000 commonly observed. In order to determine this ratio for Sindbls virus, and to eliminate varlabl 1 lty encountered In our assay, a study of a plaque assay for Slndbls was undertaken. This assay uses monolayers of a continuous strain of ham­ster cells <BHK-21). Following adsorption of the virus to the cells, the monolayer, Is overlayed with Eagle's medium containing special Noble agar. The plaques are visualized 2 to 3 days later by staining the eel Is with a vital dye.

Among the variables found to affect the assay were adsorption conditions, eel I density at time of overlay, and the type and concentration of serum used for cell growth and for plaque development. Adsorption was most efficient when the virus was adsorbed to the ce.1 ls from a simple salt solution at 37°C. Under these conditions the number of plaques obtained from a virus suspension rose linearly with time of adsorption for 100 minutes, at which time a plateau was reached. Using the optimum conditions developed, including a 2-hour adsorption period so that timing Is unimportant, the assay Is quite reproducible from sample to sample or from week to week.

Under optimal conditions, up to one-half of the virus particles In our stocks w i I I form a p I aque (the number of virus part I cl es In a stock was determined by a spectrophotometric assay l. Th Is probably means that al I of the virus part I -cles are infective when first formed because our experimental conditions lead to a significant loss of infectlvlty by thermal Inactivation. The half-life of the virus lntectlvlty ls 5 hours at 37°C. Infected cells produce virus at a constant rate from 5 hours after Infection to greater than 14 hours post-Infection at 37°C (and virus stocks are therefore harvested at 11 to 16 hours after Infection), and a 2 hour adsorption period at 37°C Is used In the plaque assay.

The arbovlruses presumably infect a cell by fusion of their membranes with the host membrane (Morgan and Howe, 1968). If so, this fusion mechanism ls highly efficient, rivaled in efficiency only by the complex Infection mechanisms possessed by some bacterlophages.

Reference:

Morgan, C. and Howe, C. ( 1968) J. Virology 2: 1122.

•

PUB LI CAT I Cl\IS

Burge, B. W. and Strauss, J. H. (1970) Glycopeptldes of the membrane glycoproteln of Slndbls virus. J, Mol. B'lol. 47: 449-466.

Strauss, J. H., Burge, B. W., and Darnell, J.E. (1969) Slndbis virus Infection of chick and hamster cells: Synthesis of virus-specific proteins. Virology 37: 367-376.

75

Strauss, J. H., Burge, B. W., and Darnel I, J.E. (1970) Carbohydrate content of the membrane protein of Slndbls virus. J. Mol. Biol. 47: 437-448.

•

76

Professor: James Olds

Senior Research Fellow: Marianne E. Olds

Research Fellows: Muneyukl Ito, Israel Lieb llch, Anthony G. Phi I lips, Michael Ian Phi I lips

Graduate Students: Oavld E. Hiatt, Carol L. Kornblith, Mary Ann Llnseman

Computing Analyst: Jordan Rosenberg

Laboratory Specialist: WI II iam Al Ian

Research Assistants: Elizabeth P. Blankenhorn, David E. Lindsay, Buda J. Martonyi

Laboratory Ass I stants: Robert El bergen, Susan F. Foster

Animal "learning" has been studied by behavioral psychologists with the aim of finding mechanisms which would generalize to human behavior. Empirical studies oriented to overt stimuli and responses have been tried but these have, yielded findings of limited generality.

• Efforts to get behind the specificity of particular experiments to Invari-

ants of animal behavior led In one direction to attempts at theoretical derivation of the mediating mechanisms in the brain which Is treated In these attempts as a black box defined by Its input-output configurations.

It was often easy to create a theoretical underpinning for any given sub­set of behavioral data but the theories were of almost as I lmlted general lty as the empirical data themselves. In such theoretical projects choices were made on rel­atively arbitrary bases from large numbers of equi-posslble mechanisms without any easy way to bring data from relatlvely distant fields of behavior to bear on a given set of choices.

It was generally agreed that direct observation and analysis of the brain might provide the appropriate focal point for mediating the Interaction of dispa­rate data In determining a common theoretical framework. The difficulty lay not in an absence of knowledge about the brain, but rather in a very great sparsity of evidence matching particular brain processes to particular patterns of behavior. The brain sciences themselves had created an Information-rich neuroanatomy which mapped in exquisite detal I the highly organized structures of the brain. This was only loosely I Inked to neurophyslologlcal study of the functional activity and connections of different brain systems. As neurophysiology itself was largely based on the studies of anesthetized animals (In which significant "learning" and behavior control processes were absent altogether) the 1 lnkage of neuroanatomical studies to meaningful behavioral process was almost totally absent. There was some correlation derived from the study of behavior loss after brain damage. Because the brain is redundantly organized so that many different mechanisms are provided as alternates to supply any needed function, lesions could guide suppositions but they did not provide direct coupling between brain and behavior data. What was needed were methods tor direct study of the interaction of behavioral variables and brain variables, preferably In alert and behaving animals.

Direct electric or chemical stimulation of brain In chronic animals ls now the most widely used method for bridging this gap. Developed by W. R. Hess of Zurich for studying vegetative functions In cats, it was extended by N. E. Miller

77

and J. R. Delgado of Yale to study of feeding and other psychologically relevant behaviors, and by J. Olds and P. M. Miiner at McGI II to study of the "rewards" which underly response-learning In animals. Studies currently under way here uti­lizing this method range from drug st.~_dles of the basic neurotransmitter systems In control of positive emotional mechanisms and moods, through studies of neuronal and chemical changes Induced during reward processes, to studies of the behavioral and stimulus correlates of reward phenomena. Generally these studies seek to es­tablish links between electrlcally-induced brain processes and behaviorally-spec­ified processes of learning and performance.

Direct stimulation has the virtue of developing In a positive way the con­sequences of pathologically exacerbated activity In a brain area. Even though a given function may be redundantly represented or may take an organized and complex path through the brain, stimulation at any point within the redundant or complex system ha5 some likelihood of revealing a relation of the brain area studied to the behavioral-dependent variable. One dltflculty Is that the relations revealed by this method are unspecified because of the equivocal effects of electric stimuli on neurons. The effects Include excitation, Inhibition, synchronization, and disorga­nization, to name only the most obvious ones. Another difficulty Is that the rela­tively geometrical stimulus fields do not follCA'I neuroanatomlcal or functional boundaries, and therefore many neurons are activated together that should not go

< together In normal organization. Even more troubling Is the propensity of the method to detect the relevant fiber pathways rather than the more crucial eel I bodies and synapses. The somata of neurons are so spread that the relatively.mod­erate stimulations used do not tire enough of them to Induce effects. These dlftl­cultles have led to the search tor better methods to link brain and behavior.

Electrical recordings from brain during alert b~havlor provides a second significant method for correlating neuroanatomlcal and neurophyslologlcal sciences with the behavioral ones. It circumvents In many respects the dlttlcultlos of brain stimulation: "naturally occurring" excitations and Inhibitions are quite likely observed, and observations are mainly of cell bodies rather than fibers. There are stl II dittlcultles; tor example: first, It Is not absolutely clear that a brain area Invaded by even a relatively small metallic probe retains Its natural­ly occurring activity; and second, organized interactions of afferents and effer­ents within a smal I area are not yet observed. However, findings with this method to date suggest that It does Indicate to some degree tho patterns of organization in the brain during meaningful behavioral processes. The method is currently being applied here to problems of the neuronal correlates of drive, mood and reward, and particularly to learning, that is, to the gradually changing neuron responses trig­gered by auditory and visual signals as these become meaningful. In general these "unit" recording studies made in "chronic" preparations provide I Inks between detected brain processes and behavioral correlates; the detected neuronal activi­ties are clearly less pathological than the Induced brain processes of the stimu­lation studies. The two kinds of studies go wel I together because the older stimu­lation methods provide to some degree a gross scanning for functional local lzations whereas the newer reccrdlng methods provide val ldatlon of the proposed local lza­tlons together with specification of some detal Is of the relations and organized processes Involved.

78

69. NEUROA-IYSIOLOGICAL CORRELATES OF PAVLOVIAN CONDITIONING

I nvestl gator: James Olds

Support: National Institutes of Health, Public Health Service

The study of the neuronal changes that account for learning has been clouded by the problem of rapid Interaction among a manifold of elements. Because of the many feedback loops, the causal succession of events could not be easl ly un­raveled. Even the basic problem of analyzing changes Into those that caused new behaviors and those that resulted from the behaviors Is stl JI unresolved. If it were resolved there would still be the problem of dividing the neuronal antecedents themselves Into an order of priority separating Cal elements with newly opened "synaptic" pathways from (b) elements one or more synaptl c steps beyond the ones with such modified connectivity. The crltlcal points of "learning" In the brain would be at the sites of changed connectivity, however dispersed or localized these might be.

Completed stud I es can be regarded as I ay Ing the groundwork for a search. In one group of experiments, areas yielding clear responses to the "learned" slg­ni flcance of stimuli were studied COids, Mink and Best, 1969; Phi I lips and Olds, 1969). In other studies attempts were made to observe the emergence or disappear­ance of these new responses during repetitive series of learning trials or hablt­uatl on tri a Is C 01 ds and HI rano, I 969; HI rano, Best and OJ ds, 1970; Kam I kawa, Mel Jwal n and Adey, 1964).

In both groups of studies, the responses which appeared during learning could not be clearly separated Into those preceding and those fol la-ilng behavioral responses. Th Is was ft rst because the beg Inn I ngs of the behavl ora I responses were not clearly defined In time, and second because the temporal analysis was not suf­ficiently fine. Furthermore the analysis of new neuronal responses from trial to trial was not adequate to permit the correlation of neuronal activity with partic­ular phases of the learning process. An even greater drawback was that brain re­gldlns were Insufficiently explored. Negative findings were reported from some parts of hypothalamus, preoptlc area and thalamus. However, very large and slg­nlflcant parts of the brain were totally untested. The neocortex, the primary sensory systems and much of the pyramidal and extrapyramldal motor systems were among these, although some work was addressed to the motor systems In other labo­ratories (Evarts, 1966; Travis and Sparks, 1967).

The experiments currently underway are aimed to remedy these difficul­ties by C ll Improved analysis of the temporal order In rapid sequences of neuronal and behavioral events, (2) measurement of time of occurrence of nonspecific and spec! fie overt behavioral processes tor correlation with neuronal processes, (3) refined methods of computer analysis tracing the emergence of new brain responses from trial to trial and the correlation of these with the similar emergence of be­havioral responses, and (4) establishment of a repetitive procedure to permit the relatively rapid screening of a very large number of brain regions tor their In­volvement In learning.

The following observations have already been made: CJ) Many hlppocampal neuronal changes clearly antecede the appearance of learned behaviors coming 50 to 100 msec after application of the condltl~d stimulus and about 50 msec before the appearance of the first nonspecific component of the behavioral response. (2) Mid­brain neuronal accelerations are not so clearly dissociated from the behavioral nonspecl fie responses; ha-iever, these responses occur mainly when uncertainty Is resolved and perhaps do not appear at al I when redundant stlmul I of equal Intensity

k ~--

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r

79

and associative significance are applied. (3l The "primary sensory system" re­sponses <occurring during the first 30 msec after conditioned stimulus application) are not augmented during conditioning procedures, although they are depleted during habituation and perhaps even more so l!<Jrlng conditioning procedures.

Other observatl ons are that some responses derl ved from h I ppocampus appear prior to and during the early stages of behavioral conditioning In the trlal-to­trlal sequence but then disappear after "learning" appears to be completed. Mid­brain responses in general come and go with learned performance. A thl rd group of responses not clearly localized as yet appears some time after behavioral learning Is completed, having perhaps some correlation either with consolidation processes or overlearned performance.

References:

Olds, J., Mink, w. D., and Best, P. (1969) Electroenceph. cl in. Neuro­physlol. 26: 144-158.

Phi 11 lps, M. I. and Olds, J. ( 1969) Science 165: 1269-1271.

Olds, J. and Hirano, T. Cl969l Electroenceph. clln. Neurophyslol. 26: 159-166.

• Hirano, T., Best, P., and Olds, J. ( 1970) Electroenceph. cl In. Neurophys­

lol. 28: 127-135.

Kamlkawa, K., Mcilwain, J. T., and Adey, W.R. Cl964l Electroenceph. cl In. Neurophyslol. 17: 485-496.

Evarts, E. V. (1966) J. Neurophyslol. 29: 1011-1027.

Travis, R. P. and Sparks, D. L. (1967) Physlol. and Behavior 2: 171-177.

Galambos, R., Sheatz, G., and Vernier, V. G. ( 1956) Science 123: 376-377.

70. CHOLINERGIC AND ADRENERGIC EFFECTS ON INSTRUMENTAL BEHAVIOR WITH BRAIN SHOCK, ON NEURAL ACTIVITY IN SUBCORTICAL REGIONS, AND ON BRAIN NOREPINEPHRINE

* I nvestl gators: Marianne E. Olds, A. Yuwel ler James Olds, David E. Lindsay, E 11 zabeth P. BI ankenhorn

Support: National Institutes of Health, Public Health Service

A series of experiments are presently being carried out as part of a broader program Investigating the role of putative transmitters In the region of the central nervous system where electric shock produces positive and negative re­inforcement. The aim of this research Is to obtain evidence regarding the function of these chemicals In the subcortlcal regions Implicated In positive and negative motivation, and therefore, to help close the gap existing between the extensive biochemical and cl lnlcal data on the one hand and the lack of data regarding their functl on In behavl or, on the other hand.

* Brain Research Institute, University of California at Los Angeles.

80

A particularly favorable situation in which to investigate these problems Is provided by the avai labl llty of recent findings concerning regions of the brain which very likely ;>lay a crucial role In the mediation of reward, both positive and negative. Previous research has given a basis for the view that the hypothalamic medial forebrain bundle and sane of Its connections play a central role in the medi­ation of positive re,inforcement, whereas the periventrlcular sys-tem of the mldbraln and the medial hypothalamic nuclei have a critical part in the mediation of negative reinforcement <Olds and Olds, 1962). Recent histochemical studies have revealed that the hypothalamus Is the region with the highest concentration of norepineph­rlne (linden et al., 1966), whereas the mldbraln region ls viewed as cholinerglc and co-extensive with the ascending reticular activating system of the mesencephalon (Lewis and Shute, 1967).

Three methodological approaches have been used to obtain Information on the role of chollnerglc and adrenergic neurons in the brain regions where positive and negative reinforcement can be produced. The fl rst involves the use of elec­trical stimulation of discrete brain regions In combination with Instrumental con­ditioning techniques tor a variety of rewards, Including brain shock. Pharmaco­logical means are then used to alter the level of catecholamines or transmltter­substance and observe effects of such procedures on behavior.

The second approach Is based upon standard biochemical and biologlcal procedures for assaying brain amines and brain ACh, by correlating changes in the levels of these chemicals In the brain with performance of tasks which involve positive and negative brain reinforcement.

The third approach Involves the use of neurophyslologlcal techniques for studying changes In the spontaneous or evoked neural discharges, recorded from sev­eral regions simultaneously, as a function of alteration In the levels of brain ACh and the blogenlc amines. The regions selected tor study are those in which stimu­lation produces instrumental conditioning for brain shock and also those regions In which there is a predominance of a given type of neuron, for example, In the caudate there Is a prevalence of cholinerglc cells, in the strlatum, of dopamlner­gic ones, In the hypothalamus, of noradrenergic neurons.

The results obtained to date show that modification of self-stimulation behavior ls correlated with direct changes In the levels of brain ACh and acetyl­Cho II nesterase (Demi no and 01 ds, 1968). Furthermore it has been cone! us l ve ly sh011n that the nature of the receptor Is muscarlnlc and not nlcotlnlc (Olds and Domino, 1969), and that these effects appear to be specific to regions where electric shock functions as a positive reinforcer (Olds and Domino, 1969). Finally it would seem that ACh is utl ilzed during positively reinforced behavior with brain reward as suggested by decreased levels of brain ACh <Oomlno and Olds, 1970). These data Imply some kind of function of ACh In regions of the brain where positive and neg­ative reward can be produced, although not necessarl ly the same one. It appears likely that such effect Is produced by the action of choilnerglc neurons on a gen­eral arousal system which modulates Indirectly the emotional-motivational regions of the diencephaion.

The catechol aml nes, on the other hand, appear to exercise a more direct action In these dlencephallc and mesencephallc regions. Amphetamine, which through a variety of actions on the metabolism of QOrepinephrine has the net effect of In­creasing aval lab I llty of free noreplnephrlne at receptor sites, has tac! lltatory effects on Instrumental behavior with brain reward which are larger than those to be expected solely from Its well-known psychomotor stimulant properties (Olds and Christenson, 1970; Olds, 1970). It ls also the only compound which can block, though only partially, the depleting effects of alpha-methyltyroslne, a selective

81

Inhibitor of noreplnephrlne and dopamine synthesis (Olds, 1970), A puzzl Ing aspect of the role of noreplnephrlne on positive brain reinforcement Is the observation that Its central application In hypothalamic medial forebraln bundle does not fa-cl lltate Instrumental behavior with bl'aln shock but Instead produces depression and In some cases total blockage of response (Olds et al., 1964). Evidence from studies In col laboratlon with Dr. A. Yuwel !er, Brain Research Institute, UCLA (Olds and Yuwel !er, 1970) correlatl ng I nstrurnental behavior with shock In hypothalamus and amount of noreplnephrlne In the hypothalamus have revealed that In hypothalamus, but not In the rest of the brain, self-stimulation behavior Is correlated with In­creased utl llzatlon of noreplnephrlne, but not dopamine, and not serotonin. Final­ly, the most direct evidence regarding action of the blogenlc amines on nervous activity ls provided by data derived from neurophyslologlcal studies. These con-cl uslvely show that there are selective effects of amphetamine In the brain, In some cases Increased firing of cells Is observed which directly correlates with the behavioral 5tlrnulant properties of amphetamine, whl le, simultaneously, depression of the spontaneous activity of cells Is observed In other regions. The same pat­tern of selective effects on eel I discharges emerges from studies with alpha-methyl tyrosine. It Is hoped that eventually such data will give evidence for pharmaco­logical action of these transmitter-like substances which correlate with the more wel I-known behavioral properties of these compounds.

References:

Olds, M. E. and Olds, J, (1962) Amer. J. Physlol. 203: 803-810.

Anden, N. E., Dahlstrom, A., Fuxe, K., Larsson, K., Olson, and Ungerstedt, u. ( 1966) Acta Physlol. Scand. 67: 313.

Lewis, P. R. and Shute, C. C. D. ( 1967) Brain 90: 521-540.

Domino, E. F. and Olds, M. E. ( 1968) J. Pharmacol. Exptl. Therapeut. 164: 202-211.

Olds, M. E. and Domino, E. F. (1969) J. Pharrnacol. Exptl. Therapeut. 166: 179-188.

Olds, M. E. and Domino, E. F. (1969) J. Pharmacol. Exptl. Therapeut. 170: 157-167.

Domino, E. F. and Olds, M. E. (1970) Effects of amphetamine, scopolamlne, chlordlazopoxlde and ollphenyldantoln on self-stimulation be­havior and brain acetylchollne (submitted tor publication to Psychopharrnacologl al.

01 ds, M. E. and Christenson, T. (1970) Arner. J. Phys lo!. (In press).

Olds, M. E. (1970) I nternat. J. Neuropharmacol. (In press).

01 ds, M. E. and Yuwel !er, A. ( 1970) Effects of tacl 1 ltatory drugs on de-presslon of SS produced by physostlgmlne, chlorpromazlne, tetrabenazlne, alpha-methyl-tyrosine (In preparation).

Olds, J., Yuweller, A., Olds, M. E., and Yun, C. (1964) Arner. J, Phys lo!. 207: 242-254.

Olds, M. E. and Yuwel !er, A. (1970) Effects of self-stimulation behavior on levels of brain NE and DA (In preparation>.

82

71. EVOKED POTENTIALS ELICITED BY ELECTRICAL STIMJLATION IN THE MEDIAL FOREBRAIN BUNDLE

I nvestl gators: Muneyukl Ito, James Olds

Support: National Institutes of Health, Public Health Service

Mlcroelectrode recordings of neuronal discharges In the anterior clngulate area of rat brain show that a close correlation exists between clngulate activation and self-stlmulatlon In the medial forebraln bundle CMFBJ. In order to obtain fur-

. ther evidence for this relationship, evoked potentials were recorded in the clngu­late area while the rat was self-stimulating. One array of multiple macroelectrodes was Implanted In the MFB and closely adjacent regions for tests of self-stimulation behavior. Another siml lar array of macroelectrodes was implanted In the clngulate area tor recording. The preliminary results suggested that spatial distribution and the latency of the clngulate evoked potentials were consistent with the results obtained in the previous mlcroelectrode analysis of this region. The dimensions of the locus effective for behavioral manifestation Cself-stlmulatlon) was compared with that for the clngulate evoked potentials.

Reference:

Ito, M. and Olds, J. (1970) Fed. Proc. 29: 590 (Abstract>.

72. NEURONAL ACTIVITY DURING SELF-STIMULATION

I nvestl gators: Muneyukl Ito, James Olds

Support: National Institutes of Health, Public Health Service

The rradlal forebrain bundle CMFBl, which courses from the paleocortex through the lateral hypothalamus to the mldbraln, In both directions, Is the site of lntracranlal self-stimulation In the variety of experimental animals. The pur­pose of the present experlrrant is to study the behavior of the neuronal electrical activity of various brain structures during self-stimulation. The brain areas examined Include "those which are anatomically related to the MFB and those partic­ipating In the motor performance of the animals.

Multiple mlcroelectrodes were Implanted chronlcal ly In the above brain regions of the rat for purposes of recording. A pair of macro-wires positioned In the MFB served as self-stimulation electrodes. The rats were trained to depress a pedal to start lntracranlal stimulation. The release of the pedal "terminated stimulation. Single unit recordings were made during self-stimulation behavior.

In response to rewarding stimulation (self-stimulation), neurons In the anterior clngulate area and the mldbraln region showed excitation. In particular, "drl ven" uni ts were found exc I us i ve ly In the forms r. These obse rvat Ions confirm the anatomical data on the connections of the MFB. On the other hand, Inhibition was observed in the hlppocampus. No effects were found in the hypothalamus or In the motor cortex.

The neuronal response of the lower brain-stem nuclei of the extrapyrami­dal system ls stl I I under Investigation.

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·.11

83

References:

Ito, M. and Olds, J. (1970) Fed. Proc. 29: 590 (Abstract).

Olds, M. E. and Olds, J. (1963) J, Comp. Neurol. 120: 259-295.

73. lHE ACTION OF CHLORPROMAZINE ON CONDITIONED UNIT ACTIVITY

I nvestl gator: M. I an Ph I 111 ps

Support: ·National Institutes of Health, Public Health Service

The methods which Olds has developed for long term recording of unit ac­tivity in freely moving rats offer new methods of analyzing drug and hormonal ac­tions. First, one can record pharmacological action on the spontaneous activity of neurons simultaneously in different areas of the brain COids and Olds, 1969), and second, one can measure the effects of systematically applied drugs on units during conditioned behavior <Phi I lips, 1969), Since psychiatric medicine uses many drugs to al levlate maladaptions In patients' conditioned behavior, a meanlng­fu I understand Ing of drug act I on shou Id come from the I atter type of study. ,

Chlorpromazlne has been In use over 17 years and despite an enormous number of studies appl led to It, the drug remains an archetype of anti psychotic drugs, without known site and mcde of action. Chlorpromazine has been shown to inhibit the firing of midbrain units (Bradley and Mollica, 1958). This is charac­teristic of sedative and generally depressing drugs In that they are mere effec­tive on multisynaptic connections than on single synapses in a circuit. Many phar­macological effects, though, are not seen unless the substrate Is In a certain functional state, e.g., antlpyretic drugs lower the body temperature In febrl le patients but not In normal subjects. This appears to be the case In the action of chlorpromazlne. On conditioned unit activity, the phenothlazlne did not block the higher firing rate to a tone most appropriate to the an Imai 's motivational state, although it did reduce the movement of the rat and mldbrain unit firing in response to control tones (Phi I lips, 1969). This effect has so far been tested at low doses where the rat, if hungry or thirsty, still eats and drinks. The lack of blocking of the response to the most "significant" stimulus may be considered relevant to the effect that chlorpromazine has on conditioned avoidance. Chlorpromazlne in­hibits the response to a conditioned avoidance stimulus at doses which produce neither ataxia nor sedation but does not block the behavioral escape response to the unconditioned stimulus of the electric shock (Cook and Weidley, 1957). The latter stimulus may be deemed the more "significant" stimulus to the animal. This was tested in a study of avoidance conditioning (Phi !lips and Bradley, 1970) where shock was withheld but the switching noise of an electromagnetic shock scrambler continued to produce the avoidance response. ·

It Is proposed that the action of chlorpromazlne enhances the selective aspects of Input reception or fl iterlng Information such that only the more "Im­portant" stlmull are attended to and the Influence of other stimuli Is Inhibited.

References:

Olds, M. E. and Olds, J. (1969) lnternat. J. Neuropharmacol. 8: 87-94.

Phillips, M. I. (1969) Proc. Amer. Psycho!. Assoc. 77: 875-876.

Bradley, P. B. and Mollica, A. (1958) Arch. Ital. Biol. 96: 168-172.

84

Cook, L. and Weldley, E. (1957) Ann. N.Y. Acad. Sci. 66: 740-748.

Phillips, M. I. and Bradley, P. B. (1970) Science 168: 1122-1123.

74. UNIT ACTIVITY AND CONDITIONED BEHAVIOR

Investigators: M. I an Ph i I I I ps, J ames 0 I ds

Support: National Institutes of Health, Public Health Service

Certain cells in the mesencepha/lc brain stem of freely moving rats have been found to show higher rates of discharge to tones associated with food when the anlmal Is food-deprived than to tones associated with water (Phi II ips and Olds, 1969). Conversely, when the animal Is water-deprived, the same cells respond with Increased activity to tones associated with water compared to tones associated with food. This effect does not depend on the quality of the stlmulus used. A test of tone reversal was made. In this test the response to a food tone of h lgh frequency and to a water tone of I ow frequency Is es tab Ii shed, then the tones are reversed so that the h I gh frequency tone becomes the water tone and vice versa. The result is that the eel rs, after an inltlal loss of discrimination, continue to respond to the I nformatlon of the tone, I.e., that food or water wi 11 be gl ven, and not to the pitch. In contrast, eel Is In the auditory system respond only to the quality of the tone and not to the significance. Further, it appeared that the greater the degree of deprivation the anlmal was experiencing, the greater was the response of the eel ls to the appropriate conditioned stimulus. Thus these eel Is seem to offer a neural Indicator of the motivational state of the anlmal and rep­resent the integrat~d response of the sensory Input with the Internal ml lleu. Whether these eel Is are in fact engaged in that Integration or are merely In the efferent connections of the behavior system cannot be stated at present. However, since the onset of the response has a long latency (50-100 msecl whereas the re­sponse In the auditory pathway (brachium of the Inferior col 1 lculusl had short latency <less than 50 msecl, it would seem feasible that one could find cells which

·show the Integrated response with a latency close to that In eel Is responsive only to the sensory stimulus. Presumably such eel Is would be where Integration of the lnternal state with external Information takes place, i.e., essentially the cellu­lar .locus of I earning. There Is no reason as yet to suppose that such eel Is, if found, w i II be In one b ral n area. On the contrary, It seems pl aus I b I e that they wlll be widespread in view of the fact that even large lesions in the brain do not destroy previously learned behavioral habits.

Every stlmulus which is received In the brain has first to be recognized If it has formerly had significance for the organism. Having been Identified, the Input has to be matched for appropriateness to the internal state at the time of reception. If appropriate or "significant" to the organism, the Information of the stl mu I us w I I I then be incorporated Into the efferent "command" output, perhaps to executive eel Is Involved In the bodl ly responses to the stimulus. The evidence and theoretical notions (Olds, 1969) which have been advanced for the hlppocampus being Involved In memory suggest that this structure may be one which plays a cardinal role at least in the Identification of Input and possibly in other aspects of integration. The hypothalamus is a region yleldlng striking changes In the motlvational state when manipulated by l{'slons, chemical and electrical stlmulatlon. One suspects therefore that the hypothalamus makes an important contribution to the Internal state of the brain and wl II project information to the sites of Integra­tion concerning the internal state. It is in the retlcular formation and adjacent mesencephalic structures, however, that we have found cells showing that Integra­tion has occurred. In the hlppocampal cells we have also found this type of

' I ! f I

85

responsiveness but It ls apparently more labl le. Of the hypothalamic cells from which we have recorded, only a few units In the posterior hypothalamus have shown the response. Our samples are smal I however. Some recent results from our experi­ments and slml lar results collected ·independently In another laboratory (P. Best, unpublished observations) Indicate that Increased or sustained firing of the re­ticular eel Is may not be the only response Indicative of attention. We have found In the small sample so far tested that when a conditioned stimulus Is appl led be­fore an unavoidable electric shock, mldbraln eel Is respond with a decrease In fl r­ing. This suggests that a conditioned stimulus of positive events, such as food for a hungry rat, are responded to with an Increase or sustained rate of discharge, and negative events, such as shock, are responded to by a decrease In firing rate of the mldbraln cells. Further studies are required to establish or disprove this posslblllty; for example, the results of positive and negative stimuli tested In the same animal.

References:

Phillips, M. I. and Olds, J. (1969) Science 165: 1269-1271.

Olds, J. ( 1969) Amer. Psychologist 24: 114-132.

75. THE EFFECT OF CORTISOL ON EXTRAHYPOTHALAMIC NEURJNS

I nvest.1 gators: M. Ian Phi 11 lps, Nachum Dafny

Support: National Institutes of Health, Public Health Service

A negative feedback loop existing between levels of adrenocortlcotroplc honnone <ACTH) secretion from the anterior pituitary and the levels ot hydrocortl­sone (cortisol) In the blood has been demonstrated In several studies. There Is evidence that high blood levels of cortisol also have a neural action. Cortisol In the hypothalamus Inhibited the release of ACTH In experiments with acute cats and rats. This raises the question of what are the neural effects of elevated blood levels of cortisol In unanesthetlzed animals. We have attempted to answer this question by administering cortisol to freely moving rats and recording the effects on unit activity In different brain areas simultaneously. These locations Include the midbraln reticular formation and hlppocampus. The latter two regions of the brain are closely related to the hypothalamus as components of the llnbic­mldbraln circuit. This circuit Is believed to be Involved In the control ot be­havior under changes In motivational states and under various forms of stress which are linked with the release of ACTH. The present study has been made possible by the use of techniques developed by Olds (1965, 1967) tor the long-term recording ot units.

A tota I of I 08 un I ts were recorded from 35 rats, 49 of these were In the mesencephallc reticular formation and 45 In the dorsal hlppocampus. Rats were given cortisol at doses of I, 2, 4, 8, 16 and 64 mg/kg body weight and the effect the hormone had on the spontaneous unit activity compared to pre-established base­line levels was measured. Two kinds of controls were used, one In which no stim­uli were presented and another In which Injections of physiological sal lne were given. The most significant result was that, of the nunber of units sampled, B7% of the mldbraln units responded to the hormone In at least two consecutive doses, whereas only 46.6% ot the units In the hlppocampus were responsive. Mldbraln units showed mostly an l ncrease l n ti rl ng act I vi ty after co rt I sol treatment at I ower doses, and the direction ot effect changed at higher doses where the main effect was of lowered activity. In contrast, hippocampal units responded by about 50%

86

Increased firing and 50% decreased spike discharge after cortisol. Control tests for blood pressure and plasma corticosteroid levels tor these doses of cortisol have been carried out by Dr. Newman Taylor at U.C.L.A. The results suggest that the mldbraln may be Involved directly In an ACTH feedback loop but the hlppocampus has a more indirect role, though the results demonstrate that both areas are In­fluenced by the hormone.

References:

01 ds, J. ( 1965) Excerpta Med. I ntemat. Congress, Serl es No. 87, 372-380.

Olds, J. Cl967J The limbic system and behavioural reinforcement. In: W.R. Adey and T. Toklzane (eds.), Structure and Function of the Llntiic System. Progress In Brain Research, Vol. 27. Elsevier, Amsterdam, pp. 144-167.

76. ONE TRIAL LEARNING WITH ELECTRICAL BRAIN STIMULATION AS RE I NFORCEMENT

I nvestl gators:

Support:

* M. I an Phi 111 ps, Robert Jones

National Institutes of Health, Public Health Service

It has been proposed that the self-stimulation CSSl phenomenon of Olds differs from a conventionally rewarded behavior, such as when food Is the reward, because extinction is very rapid when no brain stimulation Is given (Deutsch and Howarth, 1963). Essentially Deutsch claimed that there was a fast decay of SS effect over time. In keeping with a series of experiments from this laboratory which have tested this premise with different tine Intervals after SS was given (Scott, 1967; Kornblith and Olds, l968J, a behavioral test was devised in which learning was demonstrated 24 hours after a brief, single exposure to brain stimu­lation. The apparatus consisted of a maze with 6 compartments. 30 rats had elec­trodes surgically Implanted Into the hypothalamus. One week postoperatively the rats were tested by al lowing each rat to explore the apparatus for 10 mltutes per day for 4 days. At the end of the trl a I on the 4th day the rat was returned to the compartment which it had entered least and 25 trains of 0.5 seconds electrical brain stimulation were given and the anlmal subsequently removed from the maze. 24 hours I ater the rat was gl ven another tr! al of I 0 ml nutes to exp lore the apparatus. The rats were then tested for self-stimulation In standard Skinner boxes. 22 of the 30 rats had SS rates of I ,000 presses per hour or more and 19 of these showed a s l gn i ti cant l ncrease in the Ir preference tor enter! ng the compartment l n wh l ch they had received brain stlmulatlon. 8 rats had low SS rates and none of these subjects showed a change In preference for entering the compartments on the test day. An unexpected effect which became apparent in this experln-ent has been fur­ther Investigated. Some rats on day 4 before they had experienced SS, shc:Med sig­nificant preference for a compartment In which a rat had received brain stimulation and the recipient rat had high rates of SS., Removal of the olfactory bulbs elim­inated this effect thereby Indicating that olfactory cues are Involved. Control experiments so tar have shown a gradient of responsiveness by naive rats to the odors of another rat, a rat which was stimulated but was not a self-stimulator and a rat which was stimulated but was a self-stlmulator, with Increasing responsive­ness to that order of difference.

* Junior High School Student, Pasadena City Schools.

87

The experiments shCNJ that the reinforcing properties of brain stimulation are pCNJerfu I or durable enough to affect behavior 24 hours after a brief exposure to stimulation and that there are possibly olfactory cues associated with the re-ward Ing effect of SS. .,

References :

Deutsch, J. A. and Howarth, C. ( 1963) Psycho I • Rev. 70: 444-460.

Kornb !Ith, C. and Olds, J. { 1968) J. Comp. Physic!. Psycho!. 65: 488-491.

Scott, J. { 1967) J. Comp. Physlol. Psycho!. 63: 175-183.

77. DEVELOPMENTAL AND HORMONAL ASPECTS OF THE SEPTAL-RAGE SYNDROME

Invest I gator: Anthony G. Ph I 111 ps

Support: National Institutes of Health, Public Health Service

The phenomenon of septa I-rage was fl rst descrl bed by Brady and Naut.p (1953, 1955) and is characterized by hyperactivity, hyperemotionallty and aggres­sion, fol lowing destruction of the septa! nuclei. This finding has had Important Implications for understanding the neuropsychologlcal basis of emotion, but the precise role of the septum is as yet undefined. The following series of experi­ments on developmental and hormonal aspects of septal hyperemotlonality appear to further the understand Ing of th Is subject.

Initially It was discovered that destruction of the septal nuclei at 35 and 45 days of age had no effect on the subsequent emotional lty of the rat, whereas slml lar sized lesions in animals 55 and 65 days of age produced hyperemotlonallty In 100% of the animals tested. The delineation of a transition period, after which time septa! lesions produce extreme hyperemotlonallty, afforded an opportunity to identify the physiological substrates of hyperemotlonal behavior.

Perhaps the most Important event to occur In the rat's development during 45-60 days of age Is the onset of puberty, accompanied by marked changes In steroid hormone levels. This fact, coupled with the recent finding that the septa! area has been shown to take up significant quantities of trltlated testosterone {McEwen, 1970) makes It plausible to assume that this steroid hormone may be a critical fac­tor In the appearance of septa I-rage In post-pubescent ma I e rats.

Consequently, a series of experiments was undertaken In col laboratlon with Dr. Israel Llebllch to determine this possibility. Preliminary results indi­cate that castration at 30 days of age abolishes the effects of septal lesions at 65 days. Intact control I ltter mates receiving the same lesion al I displayed post­operative hyperemotlonal lty. It was also shown that castration after septa I le­sions In mature animals does not reduce the severity of hyperemotlonallty. These findings suggest that steroid hormones play an Important role In the neural devel­opment of the septum.

Re fe re nces :

Brady, J. V. and Nauta, W. J. H. (1953) J. Comp. Physiol. Psycho!. 46: 339-346.

88

Brady, J. V. and Nauta, W. J. H, (1955) J. Comp, Physlol. Psycho). 48: 412-419.

McEwen, B. S. ( 1970) Conference on Current Topics In Neurochemlstry, Cal lfornla Institute of Technology.

78. INHIBITION OF HIPPOCAM"AL UNIT ACTIVITY WITH MOTOR MOVEMENT

I nvestl gators: Israel Llebllch, James Olds

Support: National Institutes of Health, Public Health Service

It ls now well established that patterns of EEG activity in the hlppocam­pus are related to concurrent motor activity In a consistent way over different species. Recent developments Jn recording of unitary activity from freely moving animals have Instigated this study of the relation between unitary activity In the hlppocampus and motor movement In rats. 26 units were observed In 22 animals. 17 of these units were Inhibited during any movement of the animal; 5 accelerated and did not change under these conditions. It is believed that the units showing Inhi­bition during movement are concentrated In the dentate-CA-4 system of the hlppocam­pus.

Re fe re nces :

Llebllch, I. and Olds, J. (1970) Proceedings of the 78th AAA (in press).

Vanderwolf, C.H. (1969) Research Bulletin 126, Dept. of Psychology, University of Western Ontario, London, Canada.

7~. ESTIMATION OF GENETIC DETERMINATION OF THE READINESS TO RESPOND TO ELECTRICAL STIMULATION OF THE HYPOTHALAMUS AS A REINFORCING AGENT

I nvestl gators: Israel Llebllch, James Olds

Support: National Institutes of Health, Public Health Service

Jn this study selective breeding Is being used In an attempt to create a population of relatively poor self-stlmulators and another population of very good self-stlmulators. By self-stlmulators we mean animals which exhibit by clearly defined Instrumental behaviors the rewarding effects of lateral hypotha­lamic stlmuluatlon (Olds, 1962). If the selective breeding Is successful <and evi­dence already Indicates some success> comparisons wi I I be made between the poor and the good groups to find what other characteristics (brain structure, behavioral signs of drive and learning ability, responsiveness to a variety of stimulations, neurophysiology, neuropharmacology, and biochemistry> exhibit concomitant changes.

Reference: '-

Olds, J. (1962) Physiological Review 42: 554-604.

I l

B9

80. HYPOTHALAMIC UNIT ACTIVITY AND FEEDING BEHAVIOR

I nvestl gator: Carol L. Kombllth ·-.,

Support: National Institutes of Health, Public Health Service

Numerous Investigations, using a variety of techniques, have demonstrated the Involvement of the hypothalamus in the neural regulation of food intake. BI­iaterai lesions of the ventromedlal nucleus of the hypothalamus produce hyperphagia (Hetherington and Ranson, 1942), whl le lesions In the lateral hypothalamic region result In aphagla <Anand and Brobeck, 1951). Stimulation of these areas produces opposite effects. Stimulation of the ventromedlal nucleus Inhibits feeding, whl le stimulation of the lateral region produces feeding (Hoebel and Teitelbaum, 1962). It has generally been concluded that the lateral hypothalamic region Is a "feeding center" that can be inhibited by the ventromedial "satiety center."

The present experiment Is an attempt to determine the kind of neural ac­tivity underlying these effects on feeding behavior. The unit activity of hypo­thalamic and more caudal structures wl 11 be recorded from rats under varying de­grees of hunger motivation. Combining lesion methods with unit recordings In awake, behaving animals wl 11 al low correlations to be made between neural activity and extremes of hunger motivation. "

References: •

Hetherington, A. W. and Ranson, S. W. (1942) J. Comp. Neural. 76: 475-499,

Anand, B. K. and Brobeck, J. R. ( 1951) Yale J. Biol. Med. 24: 123-140.

Hoebel, B. G. and Teitelbaum, P. (1962) Science 135: 375-377.

Bl. HYPOTHALAMIC UNIT ACTIVITY AND DRINKING IN RATS

I nvestl gator: Mary Ann LI nseman

Support: National Institutes of Health, Public Health Service

Stimulation of the lateral hypothalamus, either electrically or chemical­ly, has been found to elicit drinking in rats. Lesions of the same areas result In adlpsla. Several investigators have thereby postulated a "drinking center" lo­cated In this region. In addition, units of the supraoptlc and perlventrlcular nuclei of the anterior hypothalamus have been shown In acute preparations to be responsive to intracarotld injections of hypertonlc sal lne, and their Integrity to be necessary to the secretion of ADHRF, a neuroendrocrlnologlcal response to water deprl vat ion.

The purpose of the present study was to observe the responses of single units in these areas, in particular to water deprivation, to drinking and to sub­sequent rehydration. Recording mlcroelectrodes were first Implanted In these and other control brain areas. The animals were then maintained on a 23-hour water deprivation schedule, such that a sort of "conditioned thirst" was bul It up In the animal toward the end of this period. At this time, the animal was screened tor units. Samples of unit activity were then taken at the height of deprivation, dur­ing subsequent drinking, and up to I hour fol lowing drinking. These were compared for changes in rate or pattern of tiring. Comparisons were also made with samples

90

taken during other control behaviors as wef I. At the present time, sample sizes for s peel f i c bra f n areas are as yet too sma ff to form any cone f us fens.

82. UNIT RESPONSES TO THIRST INDUCED BY HYPERTONIC SALINE JN CHRONIC RATS

f nvestf gators: Mary Ann Lf nseman, M. fan Ph ff fl ps

Support: National Institutes of Health, Pubf fc Health Service

fntracarotfd Injections of hypertonfc saline have been shown to after firing rates of neurons of the perlventrfcufar and supraoptfc nuclei of the hypo­thalamus In acute rabbit preparations (Cross and Green, 1959). This raises the question of what happens when no anesthetics are used and the anfmaf is al lowed to move freely. Therefore we studied changes in unit activity Jn chronic unrestrained rats given lntraperftoneal (!Pl Injections of hypertonfc saline. Recording mlcro­efectrodes were permanently Implanted Into hypothalamic, retfcufar, hlppocampal, and brain-stem areas. Rats were placed In the test cage and units were recorded fn 2-second samples every 30 seconds for I hour to give a control basef fne. After I hour an Injection of hypertonfc saffne (2 cc of 4.6% or 2 cc of 9% IPJ was given to Induce thf rst. Un ft activity was slml larly recorded for the next 2 hours, at the end of which time water was offered to the animal. ff the animal drank the water, this was taken to be a behavioral measure of thirst, and another hour of unit activity was recorded as a recovery period. Data was cof fected on paper tape and run on a PDP8 computer for analysis.

To date, about 90 units have been thus sampled. Though sample sizes for a particular brain site are yet smal I, the fof lowing trends are seen. Anterior hypothalamic and reticular units tend to Increase Jn rate Jn response to saline, and to decrease fol fowfng drinking. On the other hand, units Jn lateral, posteri­or and ventromediaf hypothalamic sites and Jn the brain stem, tend to Increase Jn rate Jn response to the Injection, but do not correspondingly decrease fol Jawing dr>i nk f ng. Control responses to Injections of phys I of ogf cal sal I ne have not yet been evaluated.

Reference:

Cross, B. A. and Green, J. D. ( 1959) J. Phys fol. 148: 554-569.

83. OPERANT CONDJTJONJNG OF HIPPOCAMPAL NEURONS JN RAT

f nvestf gators: David E. Hiatt, James Olds

Support: National Institutes of Health, Publfc Health Service

Operant conditioning techniques have recently been successfully applied to several unusual physlofogfcaf systems. For example, heart rate (Trawl f I, 1967; Ml f fer and DfCara, 1967), b food pressure (Of Cara and Mf I fer, 1968), kl dney func­tion (Ml lier and DJCara, 1968) and hlppocampaf theta waves (Black, Young and Batenchuk, 1970) have been shown to be modfffabfe when approprfatefy positively or negatively reinforced. In al I of these cases the animals were paralyzed with curare or flaxedi I to prevent contaminating feedback effects from skeletal muscu-f ature.

91

Of special interest would be the delineation of parts of the brain .which were Involved In such learning. Efforts were begun by Olds (1961l along these tines using recordings of single units In various parts of the brain. Rats under either meprobamate sedation or freetv moving were reinforced with positive brain shock or with food or water for speeding the rate of unit firing. These techniques met with the greatest success In the hlppocampus and reticular formation, and with the least success In cortex, thalamus and hypothalamic areas. In addition, It was noted that It seemed necessary to treat a burst of firing as an event to reinforce rather than single fl rings of the cell.

Because of the success already found In working with the hippocampus and because of the Interestingly differentiated morphology of the hlppocampus, this structure was singled out for the present series of studies. From both anatomical and physlologlcal Indications the hlppocampus Is differentiated Into eel ts which serve Input (dentate gyrusl and output functions (cells of the CA-I areal. Pre­sumably, the Intermediate cells (CA-3) may serve some Information-processing func­tion. The present series of studies might shed some light on the question of whether the cells are Involved differently In the learning process.

Adult male rats are Implanted with several smal I recording electrodes In the gyrl of the hlppocampus where cell bodies of large neurons with high slgnal-to­nolse ratio action potentials may be found. In addition larger probes are Im­planted In parts of the brain where they are known to have either strongly jilOSitlve or negative reinforcing values when stimulated electrically. Some rats are also equipped with a pair of subcutaneous shocking electrodes on the back at the time of the operation. Some days after recovery, the animals are screened to find probes from wh I ch h I gh amp II tu de h I ppocampa I neuron a I sp I kes may be recorded. These rats are further screened for positive or negative reinforcing effects on the stim­ulation probes. Each of these animals Is then trained to behave In a number of arbitrary ways In order to receive positive reinforcement or escape from negative reinforcement, Presumably this preliminary training might help animals learn the experimental task more readl ly, by promoting some response flexlbl tlty. Fol lowing the screening procedure the animals are paralyzed with flaxedl I and maintained by artificial respiration with smal I amounts of COz Infused to ward off effects of hyperventl lation. After al lowing some time for the establishment of a stable base­line of neuronal response rate, the reinforcing stimulus is presented for some time in a random fashion In order to see It the uncorrelated stimulus would bring about rate changes, If It does not, a conditioning phase Is begun, which may take one of four different forms: either attempted acceleration or deceleration of neuronal rate re I nforced by e Ith er pos I ti ve or negatl ve re I nforcement. Re I nforcements are applied whenever the neuronal rates randomly reach certain levels either above or be I ow the ~orma I rates.

Thus far there are strong I ndlcatlons that the phenomenon can be repro­duced under flaxedil paralysis. Neurons In both the CA-I and CA-3 fields of the hlppocampus have been sped up using positive electrical stimulation of the brain as the operant reinforcement, and neurons In the CA-I fields have been slowed by operant use of both positive electrical stimulation of the brain and negative back shock. The dentate gyrus has not yet been explored. Sensitization effects from the initial noncorrelated administration of the reinforcer, have been encountered frequently; both speed-ups and slow-downs In both the CA-I and CA-3 fields result­! ng from e Ith er pos ltl ve and negatl ve re I nforcers have been observed. In add it I on, there have been Instances of no effect resulting during either the sensitization or the conditioning periods. As of now, no clear differentiation of the hlppocam­pus on the basis of this technique Is possible. Hopefully, with methodological Improvements continually being made and with more widespread exploration of the parts of the hlppocampus, clear-cut distinctions will emerge.

92

References:

Trowill, J. A. (1967) J. Comp. Physlol. Psycho!. 63: 7-11.

Ml Iler, N. E. and Di Cara, L. V. ( 1967) J. Comp, Physiol. Psycho!. 63: 12-19.

DlCara, L. V. and Mi lier, N, E. (1968) Psychosomatic Med. 30: 489,

Black, A.H., Young, G. A., and Batenchuk, C. <1970) J. Comp. Physlol. Psycho!. 70: 15-24.

Olds, J. and Olds, M. E. (1961) In: J. Delafrewnaye, A. Fessard, and J. Konarski (eds.), Brain Mechanisms and Learning. Blackwel I, London.

Miller, N. E. and DlCara, L. V. (1968) Amer. J. Physlol. 215: 677.

PUBLICATIONS

Hirano, T., Best, P., and Olds, J. (1970) Units during habituation, discrimination learning, and extinction. Electroenceph. clln. Neurophyslol. 28: 127-135.

Ito, M. and Olds, J. ( 1970) Changes in unitary discharges of cortical and subcor­tical neurons during self-stimulation. Fed. Proc. 29: 590 (Abstract).

Olds, J. <1970) Introductory statement, In: E. S. Valenstein (ed.), Brain Stimu-1 at! on and Motl vat I on: Research and Commentary. Scott, Foresman Comapny ( In press) •

Olds, J. (1970) The behavior in hippocampal neurons during conditioning experiments. In: R. E. Whalen et al. (eds.), The Neural Control of Behavior. Academic Press Inc. (in press).

Olds, J, <1969) The central nervous system and the reinforcement of behavior. Amer. Psychologist 24: I 14-132.

Olds, J. (1970) Pleasure centers in the brain. Engineering and Science 33: 22-31.

Olds, J. and Hirano, T. (1969) Conditioned responses of hippocampal and other neurons. Electroenceph. clln. Neurophysiol. 26: 159-166.

Olds, J., Mink, W. o., and Best, P. (1969) Single unit patterns during anticipatory behavior. Electroenceph. ciln. Neurophysiol. 26: 144-158.

Olds, M. E. ( 1970) Comparative effects of amphetamine, scopolamlne, chlordlazepox­ide, and dlphenylhydantoin on operant and extinction behavior with brain stimulation and food reward. lnternat. J. Neuropharmacol. (in press).

Olds, M, I:. and Christenson, T. ( 1970) Effq_cts of drive and training on extinction after self-stimulation and food reward. Amer. J. Physlol. 219: 208-213.

Olds, M. E. and Uomino, E. F. ( 1969) Comparison of muscarinlc and nicotinic cholinergic agonists on self-stimulation behavior. J. Pharmacol. Exptl. Therapout. 166: 189-204.

93

Olds, M. E. and Domino, E. F. ( 1969) Dlfferentlal effects of chol lnerglc agonlsts on self-stlmulatlon and escape behavior. J. Pharmacol. Exptl. Therapeut. 170: 157-167.

Olds, M. E. and Olds, J. (1969) Effects of anxiety relieving drugs on unit dis­charges In hlppocampus, reticular mldbraln, and pre-optic area Jn the freely moving rat. Jnternat. J. Neuropharmacol. 8: 87-103.

Olds, M. E. and Olds, J. (1969) Effects of lesions Jn media! forebraln bundle on self-stlmulatlon behavior. Amer. J. Physlol. 217: 1253-1264.

Phi I I I ps, M. I. and Olds, J. C 1969) Unit activity: Motivation-dependent responses from mldbraln neurons. Science 165: 1269-1271.

Phllllps, M. I. and Norgren, R. E. (1970) A rapid method for permanent Implantation of an lntraoral fistula Jn rats. Behav. Res. Meth. and lnstru. 2: 124.

94

Professor: Chari es J. Brokaw

Research Assistant: Bruce R. Benedict

Student Ass I stant: Robert K. Lew Is

We are trying to gain an understanding of the bending mechanisms of sim­ple tlagel la and ci I la, by using a variety of experimental techniques and by ex­ploiting the Kerckhoff Marine Laboratory as a base tor obtaining marine animals which provide spermatozoa suitable tor particular experiments. The major advance In the past year has been development of a method tor computer-assisted calculation of the distribution of bending moments on flagella and cl Ila. This method will be applicable to Interpreting results from several different experimental situations. Other work has continued on several projects mentioned in previous Biology Annual Reports: The measurement of mechanochemlcal coupl Ing during the movement of Chaetopterus spermatozoa, the study of the ATPase activity of Isolated sea urchin sperm flagella, and analysis of chemotactlc response in Tubularla spermatozoa.

84. BENDING MOMENTS IN FLAGELLA

Invest I gators:

Support:

* Charles J, Brokaw, Robert K. Lewis

National Institutes of Health, Public Health Service

When a flagellum ls moving through a viscous medium, such as water, the viscous resistance of the medium pushes back on each part of the moving flagellum. These forces tend to bend the flagellum; a tendency which can be expressed as a bending moment at each point along the flagellum. At the points where the flagel­lum Is bending and unbending in order to propagate bending waves along the flagel­lu~, It must overcome this bending moment resulting from viscous resistances. We have developed a computer program which wi 11 compute the values of these bending moroonts for any type of p I ana r bend Ing movement which a t I age 11 um or cl 11 um might execute. This computation al lows us to examine the time course of the resisting moment at a bending or unbending point as It moves along a flagellum.

In a tree-swimming sea urchin sperm flagellum, bending and unbending points start near the head end of the flagellum with a low propagation velocity. Their propagation accelerates and reaches a uniform velocity which Is maintained over most of the length of the fl age I lum. However, our computations show that the resisting moment at these bending and unbending points varies greatly In the region of constant propagation velocity. The bending moment Is zero at free ends of the flagellum, and rises to values of 2-3 times its average value near the middle of the flagel lar length, In a typical case. This Indicates that the propagation velocity of lndlvldual bending and unbending points Is not controlled by the local resistance to bending. Some mechanism for Internal coordination of the bending of different regions of the flagellum appears to be required. The computations also show that the external work done against viscous resistances Is also distributed nonunlformly along the length, with points near the middle of the flagellum doing 2-3 times the average amount of work. These conclusions provide additional impetus for the consideration of "slldlng fl lament" models of tlagel lar bending.

When the head of a spermatozoon becomes attached to a solid object, the bend Ing pattern of its f I age 11 um changes. The beat frequency decreases, the tot a I

*undergraduate, California Institute of Technology.

95

angle of the bent regions Increases, and the propagation velocity continues to accelerate over the whole length of the flagellum. These changes must be a response to an altered distribution of the bending moments resulting from viscous resistances. Comparison of the bending moments comp.uted for attached spennatozoa with those for free-swimming spermatozoa shows only relatively minor changes In the distributions of moments. It Is not yet clear how these changes can be responsible for the changes In bending pattern which are actually observed.

85. UNCOUPLING THE ATPase OF SEA URCHIN SPERM FLAGELLA

Investigators: ·charles J. Brokaw, Bruce R. Benedict

Support: National Institutes of Health, Public Health Service

Sea urchin sperm flagella have been Isolated and suspended In 50% glycer­ol. These Isolated flagella are nonmotlle when transferred to solutions contain­ing ATP, probably because they lack a region near the base of the flagellum which Is required for bend Initiation. However, they dephosphorylate ATP at a rate which Is 1/5 to 1/2 the probable rate of ATP dephosphorylatlon In actively motile sper­matozoa. Addition of 0.3 M thlourea to the ATPase assay solutions, extraction with 0.6 MK.CI, and several other treatments which Inhibit the motl llty of glycerinated spermatozoa, Increase the rate of ATP dephosphorylatlon by 100% or more. This • apparently represents an uncoupling of ATP dephosphorylatlon from other components of the system which generates movement.

The uncoupled ATPase has a higher Km. greater specificity for Mg++ Ion activation, and other altered characteristics In addltlo~ to Increased activity. The activating conditions for the uncoupled ATPase are closer to the conditions required for activation of the motl llty of glycerinated spermatozoa by ATP.

Although uncoupling and extraction from the flagella usually occur to­gether, under some conditions the ATPase activity can be extracted In a less modi­fied form, which ls stll I activated by thlourea. However, the stabl llty of this form has been too poor to allow us to make much progress In determining the molec­ular basis for the differences between the activity ot the In situ and the un­coupled ATPase.

86. CHEMOTACTIC BEHAVIOR OF TUBULARIA SPERMATOZOA

Investigators:

Support:

* Charles J. Brokaw, Richard L. Ml Iler

National Institutes of Health, Public Health Service

During fertl I izatlon In Tubularla and some other marine hydrolds, free­swlmmlng spermatozoa gather around the openings of the female egg-cases. This be­havior has been shown to involve chemotaxls - a response of the spermatozoa to a chemical signal released by the females which changes their swimming paths and causes them to aggregate. Mlcroplpettes fl lied with extracts of the females also attract the spennatozoa.

We have used multiple-flash photll!lllcrography to record the movements of Tubularla spermatozoa In the vicinity of mlcroplpettes fl lied with female extracts. When the spermatozoa turn In response to a chemotactlc stimulus, their flagella

* Department of Biology, Temple University, Phi ladelphla.

96

continue to beat at the normal frequency, but the beat pattern changes to a very asyrrrnetrlcal pattern which presumably causes them to turn. The period of asymmet­rical beating can last for 10-20 beat cycles, and variation In the length of this period is a major factor control I Ing the magnitude of the turn. However, each spermatozoon Is only able to make turns In one direction, relative to its own body, so that chemotactic response often Involves turns which begin as a turn &11ay from the mlcropipette, but continue untl I the spermatozoon has turned through more than 180° and is swimming towards the mlcroplpette.

Most of the behavior of these spermatozoa may be explained if their turn­ing mechanism is activated when they detect a decrease In concentration of the che­motactlcal ly attracting substance.

PUBLICATIONS

Brokaw, C. J., Goldstein, s. F., and Mi lier, R. L. (1970) Recent studies on the mot! lity of spermatozoa from some marine Invertebrates. Symposium on Comparative Spermatology, Rome-Siena (In press).

Mii fer, R. L. and Brokaw, C. J. (1970) Chemotactlc turning behavior of Tubularla spermatozoa. J. Exptl. Biol. 52: 699-706.

Brokaw, C. J. (1970) Bending moments In free-swimming flagella. J. Exptl. Blol. C In press>.

GENETICS

BACTERIOPHAGE GENETICS

CELL BIOLOGY

BIO-ORGANIC CHEMISTRY

BIOCHEMISTRY

BEHAVIORAL GENETICS

Kerckhoff-Alles-Church

(Second Floor)

97

•

•

98

Professor: Sterling Emerson

Research Fellow: Mary B. Mitchel I

Research Assistant: LI la Mendenhall Kort

The subject studied by this group Is genetic recombination In eukaryotes (organisms with true nuclei which divide mltotlcal lyl. Much of the effort Is expended in attempts to Interpret kinds of genetic recombination that have not yet been satisfactorily explained.

Emerson and Kort, In col laboratlon with Professor Clare c. C. Yu-Sun of <ne University of Albuquerque, are accumulating data on recombination between four white-spored mutants of Ascobolus lmmersus. In this example, each of four presumed alleles converts to wl Id type with a frequency of 15% or more, making genotyplc identification of al I segregants feasible without bias due to selective screening.

M. Mitchel I has for a number of years been studying examples of segrega­Tion In Neurospora crassa which appear to be Inconsistent with orthodox Interpre­tation. As reported below, one such example has now proved to be due to a corrbl­nation of Mendelian and maternal Inheritance.

87. INDICATIONS OF GENETIC HETEROGENEITY IN SINGLE-ASCOSPDRE COLON I ES OF NEUROSPORA CRASSA

Investigator: Mary B. Mitchel I

Support: National Institutes of Health, Public Health Service

In some respects the behavior of strains originating from single asco­spores is that expected, If as Is ordlnarl ly assumed, they are, with rare excep­tions, haploid and genetlcal ly homogeneous. Other aspects of their behavior are less readily understandable on this basis. For example, shifts in phenotypic ex­pression during vegetative growth, the capacity for which Is transmissible In crosses, are characteristic of a variety of strains. A more graphic suggestion of genetic heterogeneity was noted during studies Involving observation of large num­bers of single ascospore colonies. The wl Id-type segregants from a particular cross were found to be regularly Inhibited by an amino acid during the early hours of colony growth. However, the mycellal branches within lndlvldual colonies seemed to respond differentially since the effect consisted of a reduction In the nurrber of longer branches, but not In maximum length. The suggested mosalclsm, I Ike the capacity for phenotyplc shift, was transmitted in furtlier crosses.

In attempting to account for the existence of both Mendelian and maternal Inheritance, two sets of determinants, differing at least In mechanism of transmis­sion, are usually postulated. Conceivably, Inconsistency with respect to genetic homogeneity could be a reflection of such a duality.

Reference:

Mitchell, M. B. (1969) Can. J, Bot. 47: 1985-1988.

88. A CHARACTER IN NEURlSPORA SHOWING BOTH MENDELIAN AND MATERNAL INHERITANCE

I nvestl gator: Mary B. Mitchel 1,

Support: National Institutes of Health, Public Health Service

A simple Mendel Ian character, In this ascomycete, Is recognized by 4:4 distribution of c and c+ expression In spore octets from ascl produced by either reciprocal of the cross, c x c+. A maternally Inherited character shows 0:8 or

99

8:0 distribution, reappearing-only from the reciprocal In which the parent express­ing c acts as contributor of the functioning protoperlthecla Clnclplent fruiting bodies formed unisexuallyl. A new character, nova, as It was fl rst observed, ex­hibited properties of both categories. Although not expressed by any parent, nova appeared regularly from 4 spores of each octet when a wt Id strain, S, acted as-­maternal, or protoperltheclal parent In crosses to strains derived from wilds A and C. This result would be expected It expression of nova requires at least two determinants, one maternally Inherited, present In S protoperlthecla, and the other Mendel Ian, contributed by any one of the AC strains. By suitably combining seg­regants from these crosses, nova could be made to appear typically Mendell an or strictly maternal. The observations provide a simple l llustratlon In suppo\f of the view that comprehensive correlations between phenotyplc expressions and genetic determinants are Impracticable, particularly In studies restricted to closel.Y re­lated or Inbred stra Ins.

Reference:

Mitchell, M. B. Cl970) Nature 226: 468-469.

PUBLICATIONS

Emerson, S. <1969) Linkage and recombination at the chromosome level. In: Genetic Organization, Vol. I. Academic Press Inc., New York.

Mitchell, M. B. (1969) Unpredicted growth responses of slngle-ascopore colonies of Neurospora crassa. Canad. J. Mlcroblol. 47: 1985-1988.

Mitchel I, M. B. (1969) Variation In "map distance" In Neurospora crassa. Canad. J. Ml crob I ol • 15: 623-627.

100

Associate Professor: Wi I I lam B. Wood

Research Fellow: Ronald B. Luftlg

Graduate Students: Steven K. Beckendorf, John B. Jamieson, Richard D. K. Josslin, Jane E. Latta, Samuel Ward, John H. WI Ison

Research Assistants: I lga Lielausls, Margaret T. Novitski

Research Ai de: Shirley M. Brown

The reports below describe continuing research Into the structure, func­tion, and morphogenesis of bacteriophage T4, a model system which promises to pro­vide Insight Into the general problem of how complex supramolecular structures are asserrblea under genetic control. The phage Is a nucleoproteln structure of Intri­cate morphology, containing at least 30 different kinds of protein molecules. Con­dltlonal ly lethal mutations affecting phage assembly permit the experimental arrest of the morphogenetic process at any of 45 specific stages. Since many of the as­serrbly steps can be carried out In vitro, In mixtures of Infected-cell extracts prepared using appropriate mutants, It Is possible to apply blochemlcal and genet­ic techniques as wel I as electron microscopy to the problem of how the phage par­ticle Is assembled.

During the past year, the group's work has focussed on the structure, function, asserrbly, and attachment of the tall fibers, which as parts of the phage's adsorption apparatus are particularly amenable to analysis. The genes control I ing the major polypeptide subunits of each half of the fiber have been Identified, and the roles of some of the other genes Involved in tal I fiber assembly have been clarltled. Evidence has been obtained that sites on the phage head are Involved In attaching the finished tal I fibers to the baseplate. In other work less direct­ly related to phage assembly, a novel suppressor of amber mutations has been found to •. arlse at low. frequency within the phage genome, aiidi'he mechanism of phage-In­duced lysls has been clarified.

In the fol lowing reports, defective extracts prepared from restrictive host cells Infected with amber (am) mutant phage are designated by the nurrber(s) of the defective gene or genes In the mutant used for Infection; e.g., 23:34-defectlve extract. Proteins synthesized under the control of mutationally Identified phage genes are designated as P fol lowed by the gene number; e.g., P36. A genetic map of T4 showing gene numbers, relative gene locations, and the phenotyplc consequences of various gene defects is shown In Biology 1965, p, 39, and In the general refer­ences listed below.

References:

Wood, w. B. and Edgar, R. S. (1967) Scientific American 217: 60-74.

Wood, W. B., Edgar, R. S., King, J., Lielausls, I., and Henninger, M. ( 1968) Fed. Proc. 27: 1160-1166.

89. THE SUBUNIT STRUCTURE OF BACTERIOPHAGE T4 TAIL FIBER PRECURSORS

I nvestl gator:

Support:

Samue I Ward

National Institutes of Health, Public Health Service Mary K. Beschonnan Fund

IOI

The characterization of Isolated T4 tal I fibers and 4 lntennedlates in the assembly of these fibers CBlology 1968, No. 25; Bio logy 1969, No. 188) is con­tinuing. In this report the Isolated structures are characterized by their poly­peptide subunit compos~ltlon.

Each purified Intermediate was denatured by heating In the Ionic deter­gent sodium dodecyl sulfate (SOS) and then electrophoresed on acrylamlde gels con­taining SOS. This procedure dissociates the fibers Into their polypeptide subunits and separates the subunits according to their molecular weights. By this method sane of the polypeptides In the fiber Intermediates have been separated and their molecular weights determined. The results are as fol lows (refer to the pathway of fiber assembly In Biology 1969, p. 183 or the figure on p. 103, Biology 1970 for the nomenclature of tal I fiber precursors):

(I) The whole fiber (ABC) contains two large polypeptide chains of moJec­ular weight 150,000 and 123,000 daltons. There are smaller polypeptides as well but these are poorly resolved at present. There are probably 2 copies of each of the large polypeptides In the assembled fiber.

(2) The structure A contains the same 150,000 dalton polypeptides as the whole fiber. Based on estimates of the fiber molecular weight there are probably 2 copies of the large polypeptide in the A fiber. Smaller polypeptides are not yet resolved.

(3) The structures BC' and BC appear to be Identical, containing a major polypeptide of 123,000 daltons and 2 smaller polypeptides of molecular weight 30,000 and 14,000. The assembled BC and BC' fibers probably contain 2 of the 123,000 polypeptides, 4 of the 30,000 polypeptides, and 6 of the 14,000 polypep­tides, based on quantltatlon of the stained bands and on estimates of the fiber molecular weight.

(4) The structure C appears to contain the same large polypeptide as BC and BC'. Smaller molecular weight components have not been resolved.

These results give further Insight Into the structural organization of the tal I fibers and suggest that gene 35, which controls the conversion of BC to BC', may act catalytically in assembly since no detectable polypeptide is added to the fiber In this step.

References:

King, J. and Wood, W. B. ( 1969) J. Mol. Biol. 39: 583.

Shapiro, A. L., Vlnuela, E., and Malzel, J. V. (1967) Blochem. Blophys. Res. Commun. 28: 815.

102

90. THE GENETIC CONTROL OF EARLY STEPS IN T4 TAIL FIBER ASSEMBLY

I nvestlgator:

Support:

Samuel Ward

National Institutes of Health, Public Health Service Mary K. Beschorman Fund

Tail fiber assembly In bacteriophage T4 requires the function of 6 genes: 34, 35, 36, 37, 38 and 57. When complete phage are degraded by treatment with the ionic detergent sodium dodecyl sulfate CSDS) and electrophoresed on acrylamlde gels containing SDS, 2 high-molecular-weight polypeptide bands are found which are miss­ing from slmi larly treated flberless particles. They correspond In molecular weight to the 2 large polypeptides present In the assembled ta! I fiber CBlology 1970, No. 89). The bands formed by these polypeptl des are we II reso I ved from other phage bands, so that they can be easl ly Identified in crude lysates of phage-Infected eel Is. To do this, phage proteins were labeled with radioactive amino acids added to the host eel Is after phage Infection. The crude lysates were treated with SDS and electrophoresed on acrylamlde gels with SDS and the radioactive bands identi­fied by autoradlography or fractionation and sclntl llatlon counting of the gels. Lysates of eel Is Infected with phage carrying amber mutations In each of the 6 tal 1 fiber genes were examined. Amber mutants defective In gene 34 fal I to make the 150,000 polypeptide and mutants defective In gene 37 fa! I to make the 123,000 poly­peptide. Since amber mutations result In synthesis of only a fragment of the nor­mal gene product, these results Indicate that the 2 polypeptides are the products of genes 34 CP34) and 37 CP37) respectively. Amber mutations In genes 38, 36, 35 or 57 do not affect Jhe synthesis of P34 and P37. This Indicates that gene 57 function Is not required for the expression of genes 34 and 37, as had been previ­ously suggested. However, function of genes 57 and 38 appear to affect the solu-bl llty of P34 and P37. In lysates of cells Infected with gene 57-defectlve phage, the P34 and P37 sediment with the eel I debris upon low-speed centrifugation, where­as In wild-type lysates they remain In solution. P37 Is also Insoluble In lysates of eel Is Infected with gene 38-defectlve mutants.

The results presented In this and the previous report, as wel I as those described In Biology 1969, No. 188 are summarized schematlcal ly In the revised pathway of tal I fiber assembly sho-tn belo-t. The various steps are shown by arrows, with the gene(s) Involved at each step shCliln above. The appearance of the various structures Is diagrammed and their dimensions Indicated. The numbers belClil each structure indicate the molecular weights of Its polypeptide constituents; the question marks Indicate that not al I the polypeptides have been Identified.

This work was done In col laboratlon with Dr. Robert J. Dickson, Depart­ment of Bacteriology, University of California at Los Angeles.

103

'-

(insoluble) A

34 57 1 T o

~ 190A

ABC 150,000 Mw 150,000

~~· ? 156° ~ 0

690A

c BC Bc' 'j

(insoluble) 150,000

37 ~ 57,38 IT 0 36 IT 0

35 I l 0

123,000 ... l60A 190A :;.. f90A ?

123,000Mw 123,000 123,000 123,000 •

? 30,000 30,000 14,000 14,000

Asserrbly of Bacteriophage T4 Tai I Fibers

91. THE RJLE OF THE HEAD IN T4 TAIL FIBER ATTA(}JMENT

Invest I gator: Jane E. Latta

Support: National Defense Education Act National Institutes of Health, Public Health Service

The final step In T4 phage asserrbly ls the attachment of 6 tal I fibers to the baseplate. The fibers are seen In electron mlcrographs to be attached by one tip, with the rest of the fiber extending free. However, the fibers wl 11 not attach total Is without heads, and neither of the halves of the fiber wi 11 attach to fiber­less particles, suggesting that attachment may require an Interaction between the phage head and the half of the fiber distal to the baseplate. This suggestion was Investigated using an antl-flberless-partlcle rabbit serum which neutralizes phage by reacting with the tal I baseplate. The neutral I zing activity could be removed by adsorption with Isolated tal Is or an extract containing functional tal Is but no head structures. However, the resulting serum stl 11 reacted with flberless parti­cles to prevent their subsequent activation by an extract containing tal I fibers. This activity of the adsorbed serum could be removed by further adsorption with Isolated complete heads or with any detective lysate tested except those lacking P23 or P31 • P23 is the major head protel n, and P31 Is requl red for convers I on of P23 to a soluble form. These results therefore I ndlcate that antibody directed against P23 can prevent tal I fiber attachment, supporting the suggestion that the attachment process involves direct Interaction of fibers with the head. Further

104

predictions of this model are being explored.

92. ADSORPTION MUTANTS OF T4

Investigator:

Support:

Steven K. Beckendorf

National Science Foundation National Defense Education Act

When T4 adsorbs to bacteria, at feast two phage components, the tal f fi­bers and the baseplate, Interact with the bacterial cell wall. Llpopolysaccharlde (LPSJ extracted from the cell wall with hot phenol can Inactivate T4. Both Intact phage and free ta! I fibers attach to this extracted LPS, but taf 1-flberless par­ticles do not. Therefore, the bacteria! receptor for the tal I fibers appears to be contained In the LPS.

This study Is aimed at answering three questions relating to adsorption. Which T4 gene products are Involved In adsorption? Do both tal f fibers and base­plate attach to specific receptors and are these receptors different? Are both taf I fiber and basepf ate receptors present in extracted LPS?

Host range (h) mutants of T4 can adsorb to and infect resistant (/4) bac­teria to which wild-type T4 cannot adsorb. A number of h mutants have been Iso­lated, characterized, and divided Into 2 major groups. Complementation tests show that the h mutation in al I mutants of the first group Is In tal I fiber gene 37. These phage are ab le to grow on one /4 bacterl um, C327, but not on another, C32, and they have lost the abl llty to Infect E. coll B, a normal host of wl Id-type T4. Mutants In the second group carry an h mutation In one of the baseplate genes 6, 7, 8, or 10. They can Infect C32 and 8,but are unable to Infect C327. The simplest Interpretation of these results Is that there are 2 bacterial T4 receptors, f for the tal I fibers and I for the baseplate.

Many of the baseplate h mutants are Inactivated by extracted E. coll B LPS at a much higher rate than wTfd-type T4. Since the only component of these phage which differs from wl Id type Is their baseplate, It seems f fkely that the baseplate receptor as we! f as the tal f fiber receptor Is present In phenol-ex-t racte d LPS .

A recently discovered temperature-sens I ti ve mutation (tsJ6l In gene 23 seems to change the rate or specificity of T4 adsorption. T4 wfTd type adsorbs to E. col I 8 m:ire rapfdly than does tsJ6. Similarly a baseplate h mutant adsorbs to B much more rap Idly than the tsJ6:t\ doub I e mutant. On the other hand th Is daub le mutant can now infect C327, which the baseplate h single mutant cannot. Thus tsJ6 decreases the rate of adsorption to B and Increases the rate of adsorption to C327. One possible explanation of these results, that tsJ6 changes the charge on the phage, seems to be Incorrect. Both T4 wl Id type andtsJ6 have an lsoelectrlc point of 3.1-3.3. An Intriguing alternative explanation TS that tsJ6 alters the Interaction between tal I fibers and the gene 23 product suggestedln the preceding report (Biology 1970, No. 91).

93. ISOLATION OF A PHAGE-CODED AMBER SUPPRESSOR

Investigators:

Sup port:

* John H. WI Ison, Sandra Kel Is

National Science Foundation Earle C. Anthony Fellowship

105

Stocks of phage strains carrying a single amber mutation normally contain wild-type phage, as a result of back mutation, at a frequency of I in 105 to I In 108. If back mutation were the only mechanism operating, one would expect a stock of multiply-mutant phage carrying several amber lesions to contain wi Id-type phage at a frequency equal to·the product of the revertant frequencies for the individual mutations. However, the frequency of revertant phage (those which grow on a non­permissive host> In a phage strain which carries 6 amber mutations In 4 tal I fiber genes ( 34, 35, 37, 38) Is approx I mate I y I in IO I I • We have begun to I sol ate and characterize these revertants. We expected three possible types: (I) Revertants In which the original amber lesions have been repaired. (2) Revertants containing one or more secondary mutations (suppressors) which permit defective genes to func­tion by allowing amber codons to be translated. (3) Revertants containing one or more additional mutations which al la.; a bypass of ( 1.e., make unnecessary) sane or al I the functions of the genes affected by the amber mutations.

The revertants analyzed thus far have fallen Into two groups. Revertants from the fl rst group yield no amber progeny when crossed to wl Id-type phage. In• addition, they make the products of genes 34 and 37 as analyzed by gel electropho­resis (Biology 1970, No. 90), and block A- and BC 1-speclflc sera equally as well as wi Id-type phage, Indicating that the original amber mutations have been repaired. On th Is bas Is we class I fy them as revertants of the fl rst tyQe. Type I revertants appear at least 1020 times more frequently than theoretically expected, suggesting that they might contain a mutation which drastlcal ly alters the frequency of re­version, perhaps by affecting the fidelity of replication. In 1 lne with this sug­gestion is the observation that several type I revertants give rise to a high pro­portion of progeny with altered plaque morphology. This group has been set aside for further study.

Revertants of the second group when crossed to w I Id-type phage y I e Id prog­eny 1thich contain all the mutations present in the original multiple ant>er parent. However, the revertants have normal ta! I fibers by the criteria applied above, sug­gest! ng that they carry a suppressor mutatl on. It has been demonstrated by appro­priate test crosses to other ant>ers that these revertants do contain a secondary mutation 1th I ch suppresses the amber codon. Al I type 2 revertants Isolated thus far appear to be identical; i.e., no progeny lacking the suppressor have been found in crosses between these revertants. Th Is I ocus has been des I gnated phage suppres5or a (psu~). psu~ suppresses ambers In both early and late genes, suggesting that the suppressor ltsel f is an early function. However, it does not suppress al I amber mutants tested nor does it suppress any ochre or opal mutant yet tested. The pat­tern of suppression of various ambers by psu~ is unlike that for the bacterial sup­pressors, Su- I, -2 and -3.

Work is in progress to map psu~, fl nd other phage-coded suppressors, and define the mode of action of these suppressors. These studies may give insight into the phage-specific modifications introduced into the bacterial protein-synthesizing machinery fol lo1tlng infection.

* Undergraduate, Barnard College, New York, N.Y., on leave at C.l .T., 1969-70.

106

94, THE LYS IS MECHANISM OF PHAGE T4

I nvestl gator: Richard D. K. Josslln

Support: National lnstltutos of Health, Pub I ic Health Service

Under normal conditions, phage T4-infected bacteria lyse at a character­istic time after infection, each Infected cell releasing a few hundred progeny phage. To Investigate the mechanism of this lysls, a selection technique was used to iso­late lysls-defectlve mutants of phage T4. A new class of lysls-defectlve mutants was found which defines a new T4 gene called t. During Infection, t-defective mu­tants synthesize both phage and phage lysozyme, yet fal I either to cease metabolism or to lyse at the usual time. Thus, T4-lnduced lysls entai Is a sequence of at least two events: function of t gene product, resultlng In the cessation of host metabollsm, and al I OH Inge gene-product, phage lysozyme, to degrade the host eel I wall and release progeny phage.

Although the mechanism of t gene product action remains to be determined, some alternatives were ruled out. Neither a metabolic poison nor exogenous lyso­zyme wi 11 phenotyplcal ly revert the t gene defect. These results sh<JH that the normal function of gene t Involves s0rrethlng besides cessation of host metabolism and disruption of the host eel I wal I. To test whether t gene product acts by de­grading host membrane phosphollpid, hydrolysis of host phosphollpfd was measured during T4 I nfectlon. It was found that lysis, although normally accompanied by phospholfpld hydrolysls, can occur in the absence of this reaction. Thus the phosphol Ip Id hydro lys Is observed Is a by-product of the t gene event rather than a requirement for ly~ls. -

PUBLICATIONS

~osslin, R. (1970) The lysls mechanism of phage T4: Mutants affecting lysis. VI ro I ogy 40 : 719 - 726 •

Ward, s. C 1970) An Improved transverse destalning apparatus for acrylamlde gels. Anal. Blochem. 33: 259-262.

Ward, S., Wiison, D. l., and GI I llam, J. J. ( 1970) Methods for fractionation and scintll latlon counting of radioisotope labeled polyacrylamide gels. Anal. Blochem. (in press).

W 11 son, J. H., Lufti g, R. B., and Wood, W. B. ( 1970) Interact I on of bacteriophage T4 tall fiber components with a lipopolysaccharlde fraction from E. coli. J. Mol. Biol. (In press).

i L

107

Professor: Norman H. HorCJiiltz

Research Fel !Oils: Gisela Charlang, El lls E. Golub

Research Assistant: George Horn

Neurospora tyroslnase continues to pose interesting problems In enzyme evolution and regulation. The poss I bl llty that the al lei le tyroslnases S and PR differ In several amino acids, as reported on the follOiilng pages, suggests a rapid rate of evolutlon for this enzyme. This had already been Indicated by the highly polymorphlc state of the enzyme In nature, and It seems to confirm recent calcula­tions by King and Jukes and by Kimura which predict a high rate of amino acid sub­stitution In proteins subject to weak selection. Because the results to date are based on fingerprints only, they should be considered as tentative untl I confirmed by another method.

The possible role of cyclic AMP in the regulation of tyroslnase synthe­sis Is bel ng studied here by Dr. El I ls Golub and at the State University of New York, Albany, by Dr. Jerry Feldman. Feldman first reported an inductive effect of cAMP on tyrosinase In our annual report, Biology 1969. In his summary, Golub . describes experiments which suggest that the mechanism of cAMP induction is dif~ ferent from that of cycloheximlde induction of tyroslnase. The latter probably In­volves preferential blocking of the synthesis of a repressor protein, as was re! cent I y shCJiin in our I aboratory.

The discovery of a substance which appears essential for germination of conldla, and which ts lost from the cells in media of low weter activity, Is de­scribed in the report by Charlang and Horowitz. The suggestion that germination factors such as this may be Important In the ecology of microorganisms In dry or saline environments is a fascinating posslblllty that is being examined.

Re fe re nces :

King, J. and Jukes, T. ( 1969) Science 164: 788.

Kimura, M. ( 1969) Proc. Nat. Acad. Sci. 63: 1181.

Horowitz, N. H., Feldman, H. M., and Pall, M. L. Cl970} J. Biol. Chem, 245: 2784.

95. COMPOSITION AND PROPERTIES OF NEUROSPORA TYROSINASES

I nvestl gators: George Horn, Norman H. Horowitz

Support: National Science Foundation

We previously reported a leuclne-isoleuclne difference In tryptic finger­prints of the allelic tyroslnases Sand PR. Chymotryptlc fingerprints suggest additionally a serine-threonine and an lsoleuclne-threonlne difference between these enzymes, There appear also to be differences in the glutamlc acid (or glu­tamine) and phenylalanine contents of certain chymotryptlc peptides. The appearance of so many substitutions in a pair of al lellc proteins Is decidedly unusual; we are attempting to confirm these findings by column chromatographic methods.

108

Chromatography on OEAE-Sephadex Is now being used as the last step In puri­fication of the enzymes. Previous preparations of both Sand PR showed a diffuse protein band running ahead of the major band In polyacrylamlde gel electrophoresis. DEAE-Sephadex chromatography elimlnates the diffuse band. PR preparations now show a slngle band; S shows a strong major band and a faint, diffuse band running closely ahead of it. These preparations show a consistent difference In the specific activ­ity of the enzymes toward DL-dopa, with PR some 20-40% more active than s. This difference does not seem to be accounted for by the sllght heterogeneity of s, but probably represents a real difference between the enzymes.

lsoelectrlc focusing experiments carried out with the cooperation of Dr. K.-E. Eriksson show conslderable apparent heterogeneity in tyroslnase PR, much less In tyroslnase S, after DEAE chromatography. The same preparations of PR show a slngle band of protein, containing all of the enzyme activity, In acrylamlde gel electrophoresls. We conclude from this that lsoelectrlc focusing is not useful as a criterion of homogeneity. It appears that some denaturation occurs during lso­electrlc focusing, giving rise to new peaks of low specific activity.

The lsoelectrlc point of tyroslnase S, by lsoelectrlc focusing, Is 8.4, In agreement with previous data; that of tyroslnase PR (major peak) is 6.2, consis­tent with earlier qualltatlve findings. In polyacrylamlde gel electrophoresls at pH 8.5-8.9, however, PR moves faster toward the cathode than does S.

References:

Fling, M., Horowitz, N. H., and Heinemann, S. F. (1963) J. Blol. Chem. 238: 2045-2053.

Horowitz, 'N, H., Fllng, M., Macleod, H., and Sueoka, N. (1961) Genetics 46: 1015-1024 •

• 96. GROWTH OF NEUROSPORA AT LOW WATER ACTIVITIES

;I nvestl gators: Glsela Charlang, Norman H. Horowitz

Support: Natlonal Aeronautics and Space Administration

In preparation for a genetic study of water metabollsm in Neurospora crassa, we have investigated the effect of lowered water activity <aw> on germina­tion and growth of wlld-type conldla. aw was controlled by the addition of NaCl, glucose, or glycerol to complete medium. As aw is lowered from approxlmately 0.996 to o.916, the lag period preceding germination Is lengthened from several hours to 6 days or longer, and the rate of growth fol lowlng germination Is Inhibited some 80%. Raising the oxygen concentration to 50% Increases the growth rate but has no effect on germination time at low a.,,. Oxygen Is less soluble in solutions of NaCl or glucose than in pure water. Slmilarly, at low 8w growth is faster at 30° than at 35°, whereas the reverse ls true in normal media, This effect, too, may be re­lated to the solublllty of oxygen. Growth, but not the germination lag, at low a,, is also Improved by increasing the Mg++ concentration from 0.8 mM to 50 mM. This effect Is found In both NaCl-and glucose-containing media.

The duration of the lag perlofr Is strongly Influenced by inoculum size. At aw= 0.936 and 1000 conldla per ml, the lag period is 3 days; with 100 conldia per ml under the same conditions, It Is 14 days. This effect Is apparently caused by the loss of a soluble substance from the cells at low 8w• The substance Is ex­tractable from the medium and from young cultures. It shortens the germination

1'1rnt In rntdla WMOlll 1w'1 MIVI b11n lowarod by ol'l'hll' NICI 01' non•lonlCi §Olll'l'H. 11ol11'lon and ld1n'l'lflo11'lon of tno 1111torl1I 1ro Doing 11naort1kon.

I nv11tl ;1tor1

Support•

11111 i , Oo I 11b

N11'lon1I ln1tltu1'11 of Hoalth, Publlo H1111'11 11rvl0ll

109

C1ropru1lon of fyl'Olln110 In NIUl'OIPOl'I I• 111ool1t1d with llllCUll di ff1r-1ntl1tlon. Both 1vtnt1 can bl trl;;arad by 1t1rv1tlon. Ith• Ilion 1hown (Horowitz et 11., 1970) th1t 11rt1ln 1ntlblotl01 known to Inhibit protaln 1ynth11l1 a1n 1110 d1ropr111 th1 1n1yrn1, Thi• 1ft1ot h• b11n lntarpr1t1d In tol"ll9 of • l'IPldly-t11rn• ln;·ovar r1pra11or wh111 1ynth11l1 11 mora 11n1ltlva to th• 1ntlblotlc than 11 tha 1ynth11l1 of tvl'Olln111. Rtoantly (Slolo;v 1969, No. 165> It h11 b11n 1hown that 1d1no1ln1·3•,5'·cvollc monophotph11'1 (oycllc AMPI d1rapra1101 tyroeln110, Tha pur­po11 of thl1 lnva1tlg11'lcn 11to111plor1 tha n1t11r1 of cyallc AMP Induction, and to d1tarmln1 tho ral1tlon1hlp b1tw11n thl1 tvP• of lndiictlon and Induction by 1t1r­v1tlon or protaln 1ynth11l1 Inhibition.

M111lm1I lnductlan of tvro1ln111 by cvcl lo AMP ocoul'I 1t oonc1ntr1tlon1 of 1·5 11'1>1, with about 2• of tht compound btlng t1ktn up by tht myotllum. Tht r1tlo of tho 111t1nt of lnduotlon by cyclic AtlP to tht 111t1nt of lnd11o1'lon by oyclollt11l­mlde v1rlt1 with th• 1traln u11d from 0.1 to I. No lnduotlon by cyol lo AtlP h11 been obaervtd In mut1nt1 ty•I and ty·2, whloh 1110 cannot b• lnduc:;d by 1t1rv1tlon, but can bo lnductd with oycloht11lmld1. Cyclic AMP lnhlbl'l'f growth 1llghtly, tht growth r11to of newly gtnnln1ttd oonldl1 btln; raduetd 1111 than 10• by Inducing concentretlona of the n11cleotlde, Thi• muat bt compared with 11 50• Inhibition ceu1ed by Inducing conc1ntr1tlon1 of cyclollt11lmldt.

When Induction of fyr01ln111e by cycllc AMP or by 1t1rv11tlon 11 carried out In the pre1enct of varying conc1ntr1tlon1 of c:ycloh111lmldt, It can be 1hown that low level• of the antibiotic prevent Induction of the 1nzy1111 by th11e 1111thod1, Cyclohexlmlde prev1nt1 Induction et 1 concentration of 10 ng/ml end Induct• et 200 ng/ml. This 1upport1 the ld111 that eye! le AMP and cycloht11lmlde act by different routes, end that 1t11rv11tlon Induction eppe1r1 1lml ler to cycl le AtlP Induction.

Cycllc AMP h111 been shown to reverae c1t1bollte repre11lon of Inducible enzy1111s In bacteria. Although It can be shown that sucrose repre1111 the Induction of tyrosln11se, no reversal of this effect by cycllc AMP has been observed.

Cyclic At-'P hes been found to be present In Neurotpore (J, F. Feldman, persona I commun I c11tl on) • Attempts to demonstrate adeny I eye I 111e act I vi tv have been unsuccessful so far, but cyclic nucleotide pho&phodleaterase activity h11& been Iden­tified In mycellal extracts, Further experl1111nts are In progress to determine the rel11tlonshlp between phosphodiesterase activity and the Induction process.

Reference:

Horowitz, N. H., Feldman, H. M., and Pell, M. L, (1970) J, Blol, Chem. 245: 2764-2788.

II 0

PUBLICATIONS

Cameron, R. E., Blank, G. B., and Horowitz, N. H. agar subjected to freezing and thawing. and 6: 16-18.

(1969-1970) Bacterial growth in Cryogenic Technology 5: 253-255

Horowitz, N. H., Feldman, H. M., and Pall, M. L. <1970) Derepresslon of tyrosinase synthesis In Neurospora by cyclohexlmide, actinomycln D, and puromycln. J, Biol. Chem. 245: 2784-2788.

Horowitz, N. H., Fling, M., Feldman, H. M., Pall, M. L., and Froehner, S. C. ( 1970) Derepression of tyroslnase synthesis In Neurospora by amino acid analogs. Develop. Biol. 21: 147-156.

Hubbard,

Leighton,

Le I ghton,

Leighton,

J. S., Hobby, G. L., Horowitz, N. H., Geiger, P. J., and Moro Iii, F. A. ( 1970) Measurement of 14co2 asslml latlon in soi Is: An experiment tor the biological exploration of Mars. Appl. Microbial. 19: 32-38.

R. B., Horowitz, N. H., Murray, B. C., Sharp, R. P., Herriman, A. G., Young, A. T., Smith, B. A., Davies, M. E., and Leovy, C. B. (1969) Mariner 6 and 7 television pictures: Preliminary analysis. Science 166: 49-67.

R. B., Horowitz, N. H., Murray, B. C., Sharp, R. P., Herriman, A.G., Young, A. T., Smith, B. A., Davies, M. E., and Leovy, c. B. (1969) Mariner 6 television pictures: First report. Science 165: 684-690.

R. B., Horowitz, N. H., Murray, 8. C., Sharp, R. P., Herriman, A.G., Young, A. ·r., Smith, B. A., Davies, M. E., and Leovy, C. B. (1969) Mariner 7 television pictures: First report. Science 165: 787-795.

111

Assistant Professor: Daniel McMahon

Research Fe 11 °": Gerhard Gtlpe I

* Graduate Students: John W. Cross Jr., Paul T. Langstroth , Mark J. Mi Iler, Jess I ca Tuchman

Research Assistant: Will lam G. Fry

We have spent the year studying some aspects of eel lular function. Using Chlamydomonas reinhardi we have started to study the functional relationships of the chloroplast and nuclear genomes. We are also studying the mechanism of cell division In synchronously-growing Chlamydomonas. Much of the effort in both of these areas has been exerted In developing methods tor selecting mutants which are defective In macromolecular synthesis or eel I division. We have already Isolated a number of such mutants. Some of these are discussed below. In addition, some facets of the biochemistry associated with these processes have been examined.

Us Ing Dlctyostel I um dlscoldeum, a eel lular s 1 lme mold, we have studied some of the processes which occur when this organism undergoes differentiation. 100,000 Identical amoebae unite and form a sorocarp consisting of approximately equal numbers of each of two cell types. Mutants defective In development wl II sometimes "complement" each other, producing a normal sorocarp, thus making thG organism very useful for the study of lntercellular communication In development. We have been investigating this using the complementary methods of examination of the effects of chemical inhibitors and of the properties of mutants with develop­mental defects.

98. WHAT IS THE FUNCTION OF CHLOROPLAST GLYCOLIPIDS?

I nvestlgator: John W. Cross Jr.

Support: National Institutes of Health, Public Health Service

Chloroplast membranes have been found to contain a group of unique glyco­lipids, which Includes the galactollpids, galactosyl dlglyceride and dlgalactosyl diglyceride, and the sulfonate lipid, 6-sulfoquinovosyl dlglyceride. They are found In all photosynthetic organisms and they have been Implicated in photosynthe­sis. They I ie almost entirely In chloroplast lamellae, and they are synthesized in the chloroplasts. Their biosynthesls during chloroplast development roughly paral­lels chlorophyl I synthesis, and during bleaching or senescence they gradually de­crease, while synthesis also fal Is. Very I lttle is known, hOilever, about the func­tion of these lipids.

The sulfonyl group and the low pK of the sulfollpld make analysis and purification relatively easy. Chlamydomonas synthesizes a single sulfolipld, which was purified and shown to be rapidly Incorporated Into the eel Is from the medium. l am studying the physiological function of these 11,pids by attempting to isolate and study the properties of temperature-sensitive Chlamydomonas mutants unable to synthesize sulfo\ipid.

* Deceased December 12, 1969.

112

References:

Benson, A. A. ( 1963) Adv. in Lipid Res. I: 387.

Goldberg, I. and Ohad, I. ( 1970) J. Cel I. Biol. 44: 563.

0 H

ett,pcc,, ".3• I

~H~CHoce,7NL9 .. ,, 0

~-SULS:OQ,UINO~OSYL 1>1'9LYC.ER.1l>E

99. INVESTIGATIONS OF MITOSIS

I nvestl gator: Mark J. Ml 1 ler

Support: National Institutes of Health, Pub Ile Health Service

There is much cytological data on mitosis but very I ittle biochemical research has been done on this vital process. I have begun pre I I ml nary Investi­gation of the requirements for eel I division using the uni eel lular alga, C. relnhardl, which can be grown synchronously on a 12-hour I ight, 12-hour dark cycle. At the end of the dark period, each cell divides twice resulting in a 4-fold In­crease In the number of ce 11 s.

I ;>Ian to subject synchronous ly-growlng eel Is to different treatments dur­ing the dally cycle to learn at what time different eel lular processes are no longer necessary for the Initiation and/or completion of cell division. I Intend to use antibiotics and eel Is conditionally defective in vital processes,

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100. CHLORAL HYDRATE INHIBITS PROTEIN SYNTHESIS

I nvestl gators:

Support:

* Dani e I McMahon, W i I I i am BI as ch ko

National Institutes of Health, Public Health Service National Science Foundation

II 3

Chloral hydrate is a compound with two diverse effects. It is an effec­tive Inhibitor of spindle function in mitosis for both plants and animals and is also an anesthetic for animals from flatworms to man. It rapidly Inhibits eel I division in C. reinhardi. At the minimal concentrations necessary to effect cell division, chloral hydrate Inhibits protein synthesis as measured in a number of ways: the Incorporation of radioactive amino acids into protein; net protein syn­thesis as measured by the LCN1ry test; and the increase In activity of a specific enzyme. At such concentrations it has no effect on RNA synthesis and an effect on DNA synthesis which is only as great as that of a number of other treatments which inhibit protein synthesis, This and other evidence indicate that it does not exert its effect by generally disrupting eel lular metabol Ism.

Other substances which disrupt the spindle such as colchicine and vinblas­tine do not affect protein synthesis at all. Chloral hydrate's properties qo not appear, therefore, to be the direct result of its actions on the spindle. We are investigating its effects in more detal I to see whether chloral hydrate is qjlle to interrupt protein synthesis in vitro, and the mechanism by which cells become re­sistant to it. Such Investigations may provide help in untangling the mechanism by which chloral hydrate inhibits cell division and induces sleep.

References:

Kaplan, H. M. ( 1969) Fed. Proc. 28: 1557-1569.

Ostergren, G. ( 1950) In: du CNRS, Paris.

~canisme de la Narcose. pp. 77-78.

IOI. THE EFFECT OF CHLORAL HYDRATE ON THE INDUCTION OF GLUTAMATE DEHYDROGENASE ACTIVITY

Investigator: Gerhard GOpel

Support: North Atlantic Treaty Organization

Co I loq. I nternat.

National Institutes of Health, Public Health Service

Glutamate dehydrogenase in Chlamydomonas reinhardi cells has been re­ported to be an inducible enzyme. A new electrophoretic species of glutamate de­hydrogenase appears when dark-grown eel Is are i I luminated. Experiments have shown that In eel Is of C. relnhardi wl Id type, which were grown in the dark and exposed to light, the glutamate dehydrogenase activity increases 2-3 hours after light on­set from + 0 .2 x 10-3 to + 0 .8 x 10-3 enzyme units, i.e., :'.:. 400%.

Chloral hydrate In a concentration of 10 mM added to cultures at the mo­ment of I lght onset completely prevents an activity increase. This concentration of chloral hydrate does not affect the activity of the enzyme in vitro. Thus chloral hydrate not only inhibits the synthesis of protein as measured by amino

* Trainee of the Committee for Advanced Science Training, San Gabriel High School, San Gabriel, California.

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acid incorporation and the Lowry test, but also inhibits the increase in activity of a specific enzyme.

Rcteroncos:

Kates, J • R. and Jones, R. F. ( 196 7J Bi och i m. Bi ophys. Acta I 45: I 5 3- I 58.

Ki vie, P. A., Bart, C., and Hudock, G. A. ( 1969) J. Protozool. 16: 743-744.

102. CANAVANINE KILLING OF CHLAMYOOMONAS REINHARD!

Investigators:

Support:

* Daniel McMahon, Paul T. Langstroth

National Institutes of Health, Public Health Service

We have continued the Investigation of the mechanism by Vlhlch the argi­nine analog, canavanine, kills C. reinhardi. The arginine auxotroph, ~~·may be starved for arginine for up to 2 days without affecting its viability. ·TfiTs treat­ment does, ha.>1ever, reduce the I ncorporatl on of adenine Into DNA and RNA. Addition of canavanine causes eel Is to begin dying immediately. (Death Is defined as the inability of the cell to form a colony when plated,) The Incorporation of adenine into RNA is also additionally inhibited irrrnediately. Its Incorporation into DNA and the incor;ioration of phenylalanine into protein are inhibited only after several hours. Canavanine causes disruption of other eel I functions also. The inhibition of macromolecular synthesis is irreversible. It is not accompanied by any changes in ce 11 u I ar u I trastructure.

The lethal effects of canavanine can be prevented by cycloheximide but not by chloramphenlcol. Its incorporation into protein is also inhibited by cyclo­hexi111ide but not by chloramphenlcol. This is in contrast to results with arginine, whose incorporation is partially inhibited by both antibiotics. Canavanine there­fore'·appears to kill by being Incorporated into proteins. The incorporation rapidly disrupts RNA synthesis. It apparently only occurs In the cytoplasmic (actidione­sensitivel and not the chloroplast (chloramphenico/-sensltive) protein-synthesizing system. These properties make It appear useful for the selection of mutants con­ditional Jy defective in some aspects of macromolecular synthesis.

103. TEMPERATURE-SENSITIVE MUTANTS DEFECTIVE JN THE INCORPORATION OF ARGININE INTO PROTEIN

Investigator: Danie I McMahon

Support: National Institutes of Health, Public Health Service

The results presented above (Biology 1970, No. 102) on the mechanism by which canavanine ki /Is C. relnhardl have al lowed development of a procedure Vlhich selects for mutants which are apparently conditiona/·ly defective in cytoplasmic protein synthesis. Ce/ Is mutagenized with ~-methyl-N'-nitro-N-nltrosoguanidine and then exposed to canavanine sulfate at high temperature contain temperature-sensitive mutants, about 40% of which are defective in the incorporation of arginine into protein.

* Deceased December 12, 1969.

115

Those mutants differ in the kinetics with 1·1h i ch prate in synthesis is in-h i bi ted. Al I of them have some residual synthesis after inhibition has reached a maximum. This residual synthesis can be stopped with chloramphenicol in those cells tested. They are unaffected in the ~ncorporatlon of adenine into RNA but its In­corporation into DNA is inhibited. This inhibition of DNA synthesis also occurs with other treatments which inhibit protein synthesis. The mutant examined in the greatest detai I is inhibited in arginine incorporation within 0,5 hours C<0.08 generation). It shows no defect in the uptake of arginine into the eel I. It is also defective in the incorporation of phenylalanine into protein and remains via­ble for long periods of time at high temperature. These mutants may help unravel tho interrelationships between the two genetic and protein-synthesizing systems in C. reinhardi.

104, THE EFFECTS OF PENCILLIN ON DEVELOPMEIH IN CELLULAR SLIME MOLDS

I nvcsti gator: Jessica Tuchman

Support: National Institutes of Health, Public Health Service

The eel lular slime mold Dictyostel ium discoideum I ives in tho soi I as individual amoebae. When the bacterial food supply runs out, the amoebae form aggregates of up to 105 cells which proceed to carry out a series of developmental changes leading to the formation of a highly organized multicellular organism con­taining two distinct cell types, stalk and spore, in strictly regulated proportion. These organisms possess characteristics (e.g., positive phototaxis) not found in the individual amoebae. Also during this period, many qevelopmental ly control led enzymes increase in amount by as much as 50-fold at precisely regulated times. Studies using actinomycin and cycloheximide have delineated the periods of tran­scription and translation for many of these enzymes. In several cases these periods do not overlap, indicating that this may be an excellent organism in which to study the mechanisms of translational control.

Under laboratory conditions, development can be initiated synchronously in any desired number of cells, and requires only 24 hours for completion. The ab­sence of all cell division during this period emphasizes the developmentally con­trol led changes which do occur.

The drug penicillin G, has an inhibitory effect on slime mold development (Hirschberg and Rusch, 1950). When added to the buffer solution in which the ceils differentiate, 50 ~~penicillin leads to a 3-hour delay in the developmental pro­gram and to the formation of abnormal fruiting bodies. The drug completely halts the developmentally control led increase in uridine diphosphoglucose pyrophosphorylase activity. This enzyme increases beginning at 12 hours after the initiation of development. Its mRNA is synthesized starting at the 4th and ending by the 12th hour moth, Ashworth and Sussman, 1968). When ce I ls are exposed to peni ci 11 in only during the transcriptive period, enzyme activity reaches tho control level. However when the drug is present during the 14-30 hour period, inhibition is com­plete.

scribed 1970).

Alkaline phosphatase, another developmentally control led enzyme is tran­between 4 and 10 hours and accumulates betlveen 18 and 26 hours (Loomis, It is unaffected by pcnici II in at this concentration.

\vork is under way to determine the effects of penicillin on related de­velopmentally control led enzymes concerned with polysaccharide and eel I wal I

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synthesis and to elucidate its site and mode of action.

References :

Hirschberg, E. and Rusch, H. P. ( 1950) J. Gel I. Comp. Physiol. 36: 105.

Roth, R., Ashworth, J.M., and Sussman, M. (1968) Proc. Nat. Acad. Sci. 59: 1235.

Loomis, W. F. ( 1970) (in press).

PUB LI CAT IONS

McMahon, O. and Langstroth, P. ( 1970) Canavanine ki 11 ing of Ch lamydomonas reinhardi. Fed. Proc. 29 (Abstract): 905 .

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Professor: Arie J. Haagen-Smit

105. CXlNSERVATION AND AIR POLLUTION,

Investigator: Arie J. Haagen-Smit

Support: Genera I Funds

In his capacity as Chairman of the State Air Resources Board, Dr. Haagen­Smit was instrumental in ;:>romoting extensive control measures. This year has been characterized by a major change in attitude of the automobile industry. Stimulated by strict regulations end a progressively more $tringent schedule of control, we witnessed the removal of lead from gasoline, a necessary prerequisite for the ulti­mate control of automobile emissions. Decrease in emissions of hydrocarbons and carbon monoxide from motor vehicles has continued, and increase in oxides of nitro­gen has come to a halt. Dr. Haagen-Smit has continued to participate in an advisory capacity to the National Institute of Environmental Science and the Atomic Energy Committee and chaired a task force for the President for advice on al r pollution matters.

PUBLICATIONS

Haagon-Smit, A. J. (1970) Presidential Task Force Report, Cleaner Air for the Nati on (in press).

Haagen-Smit, A. J. (1970) Man and his Horne, B. Y. Morrispn Me11orial Lecture.

118

Research Associate: Geoffrey L. Keighley

Senior Research Fo I I ow: Peter H. Lowy

Research Fe I I ow: Nata I i e S. Cohen

f<esearch Assistants: Hay G. Keighley, Maria Rouklove

f'esearch Aides: Josie E. Jarami I lo, Walter K. McClelland

Red cells have fixed life times. As old cells are removed from the cir­culation in animals they are replaced at such a rate that under normal conditions the number is held relatively constant. If large numbers are removed at once, as by hemorrhage, the rate of replacement is temporarily increased. The total number of red cells can be increased experimentally, tor instance by exposure to hypoxia or high altitude. There are also genetic factors and many hereditary anemias are recognized.

Erythropoietin is a substance, found in the plasma, which participates in and regulates erythropoiesis. We have sought to purity and characterize erythropoi­eti n and exp lore the ways in which it works.

106. ERYTHROPOIETIN AND ERYTHROPOIESIS

I nvost i gators: Geoffrey Keighley, Peter H. Lo>iy, Natalie S. Cohen

Support: l'aNonal Institutes of Health, Public Health Service

We have found that subcutaneous injections of 0.2 to 4 mg of serotonin crcatinino sulfate stimulate erythropoiesis in our usual assay animal, tho ex-hyp­oxic polycythemic mouse. The serotonin precursor L-5-hydroxytryptophan stimulates, but•D-5-hydroxytryptophan and the serotonin catabolite 5-hydroxyindoleacetic acid do not. Se rot on in a I so sti mu I ates eryth ropoi es is in ad rena I ectomi zed or hypophy­sectomized, but not in nephrectomized, mice.

Murray ( 1965) has shown by ink penetration that serotonin blocks blood circulation in the kidneys of rats for 2-3 hours. He confirmed this in mice and observed in addition that the adrenal circulation was not affected. Serotonin is knO\vn to reduce the 02 tension in a variety of animal tissues. Since low 02 ten­sion is considered to bo the primary stimulus of erythropoiesis, an erythropoietic effect of serotonin is logical. While it is not yet known how or where the animal senses the low o2 signal, there is much evidence that tt1e kidney plays a role in the generation of e ryth rope i et i n.

The observation that, in the mouse, serotonin lowers 02 tension in tissues and that it interferes with blood circulation, especially through the kidney, sug­gests that serotonin increases erythropoietin production in the animal by causing severe hypoxia specifically of the kidney.

The effects of eryth ropoi eti n on rat bone marrow eel Is in vitro have been studied by Goldwasser and his col leagues (ftrinda, 1969) and by Dukes ( 1968). We have applied their methods to mouse bone marrow, uti Ii zing increases in 59Fe incor­poration into heme as an index of cellular response to erythropoietin and other substances. We have compared the in vitro responses of bone marrow from mice in various hematological states.

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Bone marrow trom ex-hypoxic polycythemic B6D2F1 female mice Vias refrac­tory to erythropoietin (0,05 to 0.2 u/ml) in vitro. These mice are the preferred ones for the standard in vivo erythropoietin assay, which measures increased 59Fe uptake into the circulating RBC, because ct their extreme sensitivity to very smal I quantities of erythropoietin (0,05 u/20 gm mouse). Perhaps the in vivo response of those mice involves the participation ct an extramedul lary component which is absent in the in vitro system.

We have determined that bone marrow from genetically anemic mice (W/WVJ does not respond to erythropoietin in vitro (0.05 to 0.2 u/ml). These animaTs­respond poorly to erythropoietin in vivo, and normally maintain rather high circu­lating titers ct the.hormone.

References:

Murray, S. M. ( 1965) J. Path. Bact. 90: 409.

Gordon, A. S., Cooper, G. W., and Zanjani, E. D. (1967) In: P.A. Miescher (ed.l, Seminars in Hematology, Vol. 4. Grune and Stratton, p. 337.

Hrinda, M. E. and Goldwasser, E. ( 1969) Biochim. Biophys. Acta 195: 165-175.

Dukes, P. P. ( 1968) Ann. N.Y. Acad. Sci. 149: 437-444.

PUBLICATION

Lowy, P.H., Keighley, G., and Cohen, N. S. (1970) Stimulation by serotonin ct erythropoietin-dependent erythropoiesis in mice. Brit. J. Haematol. (in press).

120

Professor: Herschel K. Mitchel I

Gosney Research Fe I I ow: Mo gens Westergaard

Research Fe 11 ow: David T. Su II i van

Graduate Students: Alan B. Blumenthal, W. Jack Driskell, John E. Geltosky, Paul S. Meltzer, William D, Seybold, Vlil/iam A. Thomasson, Sandra Winicur

f{esoarch Assistants: Ursula Weber Tracy, Loveri za A. Sarmiento

The interests and research activities of most of the members of this group are directed toward problems concerned with the biochemistry of the regula­tion of developmental processes. However, the group is not mission-oriented in terms of a common, we/ I-defined objective. Rather we hope that an environment is provided that permits a maximum of individual orginality, self-direction and re­sponsibility. For these reasons a concise summary of a "project" is not realistic even though several of the individual projects summarized here have obvious inter­relations.

107. PHENOL OX/DASE FUNCTION IN DRJSOPHILA

Investigators:

Support:

Herschel K. Mitchell, Ursula Weber Tracy, Loveriza A. Sarmiento

National Science Foundation Gordon S. Ross Foundation

Previous evidence has been consistent with the contention that phenol oxi­dase is deposited as a structural component during construction of the cuticle of Drosophila larvae. For hardening and pigmentation of cuticle it is then necessary t./iat one or more substrates be transported from epithelial eel Is through a thick layer of chitin to the enzyme. To gain further insight into this process 2-3 3H­tyrosine was injected into larvae and animals were taken before puparium formation, at puparium formation and at I hour intervals for the next 4 hours. Sclerotization occurs at puparium formation, and pigmentation for the fol lowing 3 to 4 hours. Ex­tracts were made for a chromatographic analysis of metabolic products of the tyro­sine, and cuticle sections were made for autoradiography.

It was observed that virtually nothing happened to the tyrosine if it was injected just before puparium formation, but If Injected a day earlier, products tentatively identified were tyrosine-0-phosphate, tyramine, dopa, dopamine and N­acetyl-dopamine. The last 3 were at minimal levels just after puparium formation, presumably due to use in sclerotization. The existence of a large reservoir of bound tyrosine was also indicated.

Radioautographs of cuticle sections showed an accumulation of label at the epithelial cell-chitin interface at puparium formation with a progressive accu­mulation throughout the chitin layer at 2 and 3 hours. Finally at 4 hours tho label was very heavy in the outer half of the chitin layer and relatively weak on the in­side. The results are inter~reted to mean that substrate is continuously supplied from the epithelial ce I ls through at least the first 3 hours of sclerotization and tanning. Substrate apparently moves directly through the chitin matrix to points of oxidation for ~rotein cross-linking. Internal pressure is high inside the ani­mal during this period and this may ~rovide a positive force in movement of water and dissolved solutes. There is a weight loss of about 25% at this time.

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These results agree with the notion that sclerotization and tanning re­sult from movement of substrate to predeposited phenol oxidase but neither the 1<hol e pattern of substrate format\ on nor the exact position of the enzyme activity is yet established. '

I 08. SOME PROPERTIES OF DYS IN

Investigators:

Support:

Herschel K. Mitchel I, Loveriza A. Sarmiento, Ursula lveber Tracy

National Science Foundation Gordon S. Ross Foundation

Oysin is a substance initially purified from normal Drosophila pupae, which is lethal to second instar larvae through prevention of puparium formation. It is lethal to third instar larvae just after puparium formation and has no ad­verse e ftect on pupae or adu I ts evon at high concentrations.

Since the last report (Biology 1969, No. 195) it 1<as observed that indi­vidual organs of larvae (fat body, brain, salivary glands and imaginal disks), sev­eral hours after injection, contain up to 1,000 times as much dysin activity as control animals. Results suggested a virus-like reproduction but this does not seem to be the case. Attempts to label dysin 1<ith 3H-thymidine or 3H-urid~ne were totally negative. Highest purity dysin preparations were examined with a variety of preparative methods tor electron microscopy and no viral particles have been ob­served. It may be noted that our Oregon R 1<ild stock does contain the virus-like particles in eel I nuclei, particularly in mid-intestine, that has been described by the Schneiderman group (Akai et al., 1967). However, there is no correlation between the presence of these particles and dysin activity. \Ve assume for the present that the dysin-induced increase in activity is due to release of preformed material.

Progress has been made in dysin purification to the extent that the best preparations contain about I x 109 units per mg. That is, I mg is sufficient to ki I\ half that many larvae, by injection but not by feeding.

Purified dysin is only slightly retarded on columns of agarose 150 and thus the active material is very large or aggregated. The material is inactivated by heat, protease or high concentrations of urea or guan i dine ch I ori de. It is not inactivated by nucleases. A protein character is indicated tor the active sub­stance but definitive analysis must await further purification.

Reference:

Akai, H. et al. ( 1967) Science 157: 810.

109. BIOCHEMISTRY OF PHENOL OXIDASE PRECURSOR Al IN DROSOPHILA

Investigator: Paul S. Meltzer

Support: National Science Foundation

Phenol oxidase in Drosophila has been shown to be a complex, developmen­tally regulated system (Horowitz and Fling, 1955; Mitchel I and \1eber, 1965; Geiger and l•litchell, 1966; Mitchell, 1966; Mitchell, Weber and Schaar, 1967), Several features of Drosophila phenol oxidase suggest that it is a potentially valuable

122

system for the analysis of the developmental regulation of enzyme activity and for the analysis of cuticle biogenesis. In Orosophi la, phenol oxidase is a latent en­zyme. Activity appears in homogenates on standing and is dependent on the inter­action of several proteins. The active enzyme is unusual structurally, being an insoluble aggregate of very high molecular weight. It is inhibited by tho quinones produced in the phenol oxidase reaction. The in vivo enzyme probably has structur­al as wel I as catalytic functions. Of the 5 precursors which have been identified and designatod S, P, A1, A2 and A3, Ai seemed a likely candidate for isolation and characterization since it has a well defined assay on acrylamide gels.

A simple quantitative assay for A1 has been used to develop a purification scheme which yields material of a high degree of purity. Ammonium sulfate precip­itation, heat treatment, G-200 chromatography, G-100 chromatography, preparative isoelectric focusing, and preparative acrylamide gel electrophoresis have been suc­cessfully used under conditions which block the tendency of the fresh homogenate to convert A1 to tho active enzyme. Large scale preparation of Ai is novi in prog­ress. Preliminary results indicate that A1 is a lipoprotein with a nominal molec­ular weight of 80,000 and an isoelectric point of 4.8. Microheterogeneity in molec­ular weight and isoelectric point has been observed. A1 has no phenol oxidaso activity itself and appears to require the Sand P components to produce oxidase activity.

References:

•

Horoviitz, N. H. and Fling, M. (1955) In: \~. D. McElroy and B. Glass (eds.), Amino Acid Metabolism.

Mitchell,~· K. and Weber, u. (19651 Science 148: 964.

Geiger, H. R. and Mitchell, H.K. (1966) J. Insect. Physiol. 12: 747.

l~itchel I, H. K. ( 19661 J. Insect Physiol. 12: 135.

Mitchell, II. K., lfober, U., and Schaar, G. ( 1967) Genetics 57: 357 .

110. CHITINASE ACTIVITY DURING DR:lSOPHILA DEVELOPMENT

Investigator: Sandra Winicur

Sup port: National Institutes of Health, Public Health Service

Before both larval molts in llrosophi la melanogaster, the chitin in the cuticle is digested to a significant degree by chitinase, an enzyme in the molting fluid. The production of chitinase appears to be a cyclic phenomenon during the Drosophila life cycle when rreasured per mg of soluble protein or per mg of wet vieight. A spurt of chitinase activity appears just before each molt, about twice as much before the second as before the first molt. Chitinase drops after the first molt, and begins to rise again a few hours before chitin degradation becomes visi­ble in electron micrographs. The level of enzyme activity reached just betorG the second molt (about twicG that before the first) drops after the molt, not reaching a minimum unti I just before puparium tormQtion.

The activity ;ier individual larva however shows a som:iwhat different pic­ture. It starts to rise sl011ly after hatching, peaks slightly at th0 first molt and continues to rise through the second molt to a point mid way between the second molt and puparium formation, starting to fal I again at about the point whore chitin

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is no longor boing synthesized for the pupal case. This indicates that some of the rnGasured chitinase activity may be due to the reversible activity of a chitin syn­thetasc, which would be active during the second instar.

There is indication that chitinase activity is due to more than one en­zyme. Two tractions totaling about 80% of the activity can be separated by ammo­nium sulfate precipitation, one below 30% saturation and one between 60% and 70%. Three peaks, totaling 40% of the activity, have been separated on a OEAE-Sephadex column at pH 8.3, two major peaks and a minor one. On isoelectric focusing, how­ever, al I the enzyme activity is discerned in a single peak, with a pI of about 3.7, with about 60% recovery.

The enzyme is stable, maintaining complete activity for at least 3 vieeks in the refrigerator, and loses no activity on freezing.

The absence of a requirement for any loosely bound co-factor is demon­strated by the recovery of 100% activity by dialysis, concentration by ultrafil­tration or by running the enzyme over a Bio-Gel P-10 column, vihich has a molecular weight exclusion limit of about 1,000.

Reference:

Mitchell, H.K., Weber-Tracy, U. M., and Schaar, G. (1970) J. Exptl. Zool. (submitted for publication).

I I I. STUDIES ON DROSOPHILA PHENYLALANINE HYDROXYLASE

Investigator: John E . Ge I tos ky

Sup port: Nati ona I Defense Education Act

The metabolism of aromatic amino acids and their derivatives in insects is quite complex, for these compounds serve a multitude of functions in the animal. Tyrosine, one of these substances, is involved in cuticle formation, pigmentation and perhaps in nerve eel I physiology. In a system such as this, regulation is probably very strict. One interesting aspect of this system is the control mech­anisms for the synthesis of tyrosine.

Little work has been done on the synthesis of tyrosine; however, there is some evidence that this compound arises from a hydroxylation of phenylalanine via phenylalanine hydroxylase (Gilmour, 1965). An investigation of this process in Drosophila has been undertaken. ~onversion of phenylalanine to tyrosine was first demonstrated by injection of IC-phenylalanine into larvae and, following a short incubation period, subsequent detection of the newly synthesized tyrosine by paper chromatography of the larval extract. Once it was established that the ani­mal had the capacity to utilize phenylalanine in its synthesis of tyrosine, a search for the activity in eel I free extracts was made. Assay systems were devel­oped to detect tyrosine. Paper chromatography of aliquots from the reaction mixture and subsequent analytical processing proved to be vGry sensitive, however the pro­cess is extremely slovi and cumbersome for assaying large numbers of samples. The procedure can be used when experiments on tissue distribution of the enzyme are done. A rapid and sensitive fluorescence assay (Waalkes and Undenfriend, 1957) ~lith modifications (P. Geiger, personal communication) is now routinely used.

Passage of eel I extracts over Sephadex G-25 renders the protein fraction inactive for synthesis of tyrosine. Addition of the included fraction (low

124

molecular weight compounds) yields normal activity. This sort of experiment imp/ i­cates the presence of a cofactor in the system. It has been sho.-in with the rat liver system that a pteridine serves as a cofactor for the phenylalanine hydroxyl­ase. The particulars of the overal I process have been worked out and it appears to proceed as fol lows (Kaufman, 1959):

Phe + o2

+ reduced pteri dine ; t TPNH

Tyr + H2o +oxidized peteridine

A synthetic pteridine, 6,7-dimethyl-5,6,7,8-tetrahydropterine was tried in the Dro­sophila system and excellent activity was achieved. A study of the regeneration of the pteridine and ho.-i it works in conjunction with the hydroxy lase wi 11 be under­taken later.

Characteristics of the enzyme activity in crude extracts have been studied. Kinetics, pH optimum, activity vs. substrate and cofactor concentration curves, effect of heavy metals and effect of sulfhydryl reagents (Cleland's reagent enhances activity) have been derived. Variations in enzymatic activity have been noted in animals throughout many stages in development, indicating that control mechanisms are operative. Work to determine what these controls are is in progress.

Rofc rences:

Gilmour, lJ. (1965) In: The Metabolism of Insects. 01 iver and Boyd, Edinburgh and London, p. 127.

Waalkes, T. P. and Udenfriend, S. (1957) J. Lab. Clin. Med. 50: 733.

Kaufman, S. (1959) J. Biol. Chem. 234: 2677.

112. PROTEIN GRANULES OF DROSOPHILA LARVAL FAT BODY: ORIGIN OF THE PROTEIN

l.n vest i gator: vii I Ii am A. Thomasson

Support: National Institutes of Health, Public Health Service

Work has continued on the protein granules which appear in the fat body of the fruit fly Drosophila melanogaster during the last day of larval I ife. Locke and Col Ii ns have presented evidence that much, perhaps al I, of the protein in apparently similar granules in the butterfly Calpodes eth/ius is preformed protein sequestered from the hemolymph, rather than protein synthesized in situ at the time of granule formation. Two lines of evidence prove that in Drosophila also essen­tially all protein ln the granules is preformed.

In the first series of experiments, larvae of an age kno.;n to be accumu­lating granules were injected with a mixture of 14c-amino acid and 31-i-laboled pro­tein previously isolated from Drosophila larvae. 6 hours later the granules from these animals were isolated and the 2 isotopes counted. In both experiments gran­ulos were significantly laboled with 3H, while no 14c counts above background could be detected. In tho most favorable expe~iment, the proportion of free amino acid incorporated could have been no more than 2-3% of the incorporation of preformed protein.

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In the other experi men ta I series I arvae were injected short I y before the first visible appearance of granules with 2 nm of cycloheximide, an amount known to effectively suppress protein synthesis. This treatment had no effect whatsoever on the appearance of the granules, proving that very I ittle synthesis of any protein needs to take place during formation of the granules. However, injection of cyclo­heximide into younger animals simultaneously with ecdysterone <which induces pre­cocious formation of granules) blocked the action of the hormone.

Reference:

Locke, M. and Col I ins, J. V. (1968) J. Cell Biol. 36: 453-483.

113. PKOTEIN GRANULES OF DKOSOPHILA LARVAL FAT BODY: INDUCTION IN VITKO

Investigator: Wi 11 i am A. Thanasson

Support: National Institutes of Health, Pub I ic Health Service

It has not been clear what factors cause the appearance of protein gran­ules in the fat body of the fruit fly Drosophila melanogaster during the last day of larval I ife. Elegant I igation experiments have shown that similar granule!> in the butterfly Calpodes ethlius are induced by the molting hormone, ecdysone (Collins, 1969). Experiments in which tat bodies were cultured in adult hosts have suggested that the Drosophila granules likewise could be induced by ecdysone, but could also be induced by a product of the ovary (Butterworth and Bodenstein, 1968).

vih i le the observation (Biology 1969, No. 197l that precocious formation of granules could be induced by injection of ecdysterone into larvae supported the belief that ecdysone is the true inducer in Drosophila, it seemed that use of an in vitro organ culture system would give more completely unambiguous results, elimi­nating any possibility that the effects observed were secondary to a primary action of the hormone on another organ.

The culture medium first used was Schneider's medium plus 15% heat-inacti­vated fetal calf serum. When this medium is supplemented with as little as 10 mg/ml of ecdysterone, granules consistently form in the incubated fat bodies. These gran­ules share with those induced by the injection of ecdysterone in vivo the properties of being frequently larger but less numerous than those occurring naturally. The interpretation of the experiments is clouded, however, by the tact that granules appear about half the time in the absence of added hormone.

Suspecting that this might be due to ecdysone-like substances in the serum, I turned to the development of a defined medium, and soon found that the addition to Schneider's medium Of virtually any protein at concentrations in excess of 20 mg/ml would permit satisfactory survival of fat bodies for several days. Albumin was routinely used in the experiments.

It was surprising to find that in this medium granules would form consis­tently in the absence of hormone, though hormone caused the granules to form more quickly, and in some cases to become larger and more numerous.

These experiments therefore demonstrate that, while ecdysterone can act directly on the tat body to induce granule formation, neither it nor any other fac­tor in the insect hemolymph is necessary tor appearance of the granules. Consider­ation ot the time at which granules first become visible in vivo agrees with the

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conclusion that ecdysone does not normally induce their appearance.

The observation that fetuin, a sialoprotein from fetal calf serum, pre­vents the appearance of the granules in vitro remains unexplained. Addition of large amounts of hormone does not overcome this Inhibition.

Ro fe re nces :

Collins, J. V. (1969) J. Insect Physiol. 15: 341-352.

Butterworth, F. M. and Bodenstein, D. ( 1968) J. Exptl, Zool. 164: 251-256.

114. ELECTROPHORETIC HETEROGENEITY IN HUMAN INSULIN

Investigator: William D. Seybold

McCa 11 um Fund Gordon S. Ross Foundation

Mirsky and Kawamura (1966) showed that crystalline insulin from a number of species appears heterogeneous when analyzed by disc electrophoresis. It is not known if this heterogeneity has physiological significance or is an artifact pro­duced by the conditions commonly used in the purification of insulin.

lnsulln has been partially purified from the serum of a single individual by gel filtration and subjected to disc electrophoresis, Fractions were assayed by a double antibody radio-immunoassay. Insulin prepared In this way has been sho,.m to consist of at least 2 and probably more components. Preliminary experiments using electrofocusing gels also indicate a heterogeneity in serum insulin.

Experiments are in progress to determl ne if the heterogeneous forms are , being generated as a result of the isolation procedure.

Reference:

Mirsky, A. and Kawamura, K. ( 1966) Endocrinology 78: 1115.

I I 5. THE MOLECULAR ORGANIZATION OF THE SYNAPT I NEMAL COMPLEX

Investigator: f>logens Westergaard

Support: Gosney Fund

When chromosomes undergo homologous pairing during meiotic prophase - a process which is a conditio sine qua non for normal sexual reproduction and genetic recombination in eukaryotes - a synaptinemal complex is formed between the bivalents (Moses, 1956). This complex, which can be observed only in electron micrographs, consists of two lateral components, approximately 1,000 ~apart, connected by a central region. In the ascomycete Neo~tlel la it has recently been shown that in formaldehyde-fixed eel Is, the lateral component can be specifically tagged with metal I ic si Iver when stained with ammoniacal si Iver ions at pH 8-9. From this pH­dependent staining reaction it is proposed that the lateral component contains a basic protein, different from the chromosome histones (Westergaard, 1970). The experiments carried out during the tenure of the tel lowship have been aimed at

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finding a biological system, from which strictly synchronized meiotic eel Is can be harvested in quantities sufficiently large to make it ?OSsible to isolate and char­acterize this basic protein. The myxomycete Didymlum i ridis was chosen as a pos­sible source of such a material becau~e it has been reported that strictly synchro­nous meiotic divisions take place in the young fruiting bodies before spore delim-i nation (Carroll and Dykstra, 1966).

Experiments done here have defined conditions for growth of the organism on a semidefined liquid medium to form an abundance of microplasmodla in shaking cultures. When spread on agar plates the p!asmodla yield fruiting bodies, but op­timal conditions for a high yield and adequate synchrony remained to be established.

References:

Moses, M. J. ( 1956) J. Biophys. Biochem. Cyt. 2: 215-217.

Westergaard, M. and von Wettstein, D. ( 1970) Compt. Rend. Trav. Lab. Carlsberg 37: 239-268.

Carroll, c. and Dykstra, R. (1966) Mycologla 58: 166-169.

PUBLICATIONS •

Eakin, R. T. and Mitchell, H. K. ( 1969) A mitochondrial dihydroorotate oxidase system in Neurospora. Arch. Biochem. Biophys. 134: 160-171.

Harding,

Scott, w.

Wi II i ams,

R. W., Huang, P. C., and Mitchel I, H. K. ( 1969) Photochemical of the carotene Id b I osynthetl c pathway in Neuros'pora. Arch. Biophys. 129: 696-707.

studies Biochem.

A. by

L. in

and Mitchell, H.K. (1969) Secondary modification of cytochrome c Neurospora. Biochemistry 8: 4282-4289.

G. and Mitchell, H.K. (1969) Mutants affecting thymldine metabolism Neurospora. J. Bacterlol. 100: 383-389.

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Professor: Seymour Benzer

RosGarch Fe!IG'ls: Thomas E. Hanson, Ross B. Hodgetts, Yoshiki Hotta, John R. Merriam

Graduate Student: Ronald J. Konopka

Research Assistants: Karen B. Searcy, Lydia C. Yuan

Research Aide: Eveline Eichenberger

Laboratory Aide: Lynda A. Macleod

The behavior of an organism is in large part determined by its genes, which dictate the development of the various components of the nervous system, their precise interconnections, and the biochemical features involved in their function. Alteration of specific genes may be used as a microsurgical tool to dis­sect the specific steps in these processes. Our research group is uti Ii zing the fruit fly, Drosopn i la melanogaster, as a model system. Urosophi la has a detailed nervous system and a broad repertol re of behavior, yet reproduces rapidly and I ends itself readi Jy to manipulation of its genetic material.

Our main interest has been focused initially on the visual system, in which mutants with defective phototaxis and optomotor response are analyzed. Given such a mutant, the cause of the detect is investigated by electrophysiological, an­atomical, and biochemical techniques; by genetic methods, the chromosomal site of the mutation is localized. Thus, a series of mutants has been shown to have blocks at various steps from the Initial photoreception mechanism to later, more central, events required tor the normal phototactic response to occur. Another aspect of behavior under study is the internal biological clock mechanism which controls circadian rhythm of eclosion and locomotor activity in Drosophila. Hore too, mu­tations are being used in an attempt to dissect the elements of the clock mecha-tii sm.

Genetic techniques can also be applied to creating composite individuals having scmo parts mutant while the remainder of the individual is normal, providing the equivalent of transplantation surgery tor testing detective parts. This makes it possible to determine the site of action of genetic defects and also to construct "split-brain" flies to study tho interaction of parts of the nervous system.

116. ISOLATION AND GENETIC MAPPING OF BEHAVIORAL MUTANTS IN DROSOPHILA

Investigators:

Sup port:

Joon R. Merriam, 'Karen B. Searcy, Eve! lne Eichenberger, Barbara Steviart*, Kev! n R. O'Hara**, Pau I T. Ch ri stensenT, Seymour Benzer

Nati ona I Institutes of Hea I th, Pub Ii c Hea I th Service National Science Foundation

129

Mutations were induced by treatment of male flies with ethyl methane sul­tanate. These were mated to attached-X females, so that X-linked mutations could be observed directly in the progeny males. The progeny were fractionated by a two­dimensional countercurrent distribution method to separate normally phototactic flies from ones with aberrant behavior. Thus, normal files give repeated positive responses toward light, but tend not to move away from light; "blind" flies move equally whether to or from light; sluggish mutants move very little in either di­rection. Flies in the aberrant fractions were mated individually to produce prog­eny Ii nes for retesting, In this way, over 500 strains have been obtained that show hereditary behavior abnormalities. These abnormalities include poor photo­taxis, poor optomotor response, sluggish activity, flightlessness, susceptilli lity to mechanical shock, poor geotacti c response, and other syndromes.

• To map the locations of the mutations on the X-chromosome, females were

pre pared that were heterozygous for the behavi ora 1-mutati on-bearing X-ch romosome and a chromosome containing several morphological markers havl ng no effect on phototaxis. The male progeny populations were fractionated by countercurrent dis­tribution according to movement from light and to light; then etherized and scored for crossover classes. The entire countercurrent profile characteristic of a par­ticular mutant vias thus employed as a behavioral marker. Reciprocal crossover classes showed one or the other of the parental profi !es, except for crossovers occurring in the interval containing the behavioral mutation, which showed a mix­ture of the two parental profi !es. Thus, the location of the mutation on the map is determined.

By combining these measurements with tests for complementation between pairs of mutants having similar map positions, 7 cistrons have been defined on the X-chromosome having to do with various aspects of visual function. The number of independent occurrences of mutations so tar in the various cistrons ranges from I to 16. For mutants of the "easily shocked" phenotype, 7 different locations have been established. Since all but one of the latter represent single occurrences, it appears that a very large number of genes may be involved in the "easily shocked" syndrome.

References:

Benzer, S. ( 1967) Proc. Nat. Acad. Sci. 58: 1112.

Merriam, J. R. and Benzer, S. ( 1969) Genetics 61: Suppl. P S40 (Abstract).

* Summer Assistant, University of Calltornla, San Diego, ** NSF Summer Trainee.

·:·Undergraduate, California Institute of Technology.

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117. NEUROPHYSIOLOGICAL ANALYSIS OF VISUAL MUTANTS OF DROSOPH I LA

Investigator: Yoshiki Hotta

Nati ona I Science Foundation

Among the X-linked mutants of Drosophila selected for abnormal behavior over 35 strains have been found to show defects in initial events in the visual chain detectable by electroretinogram <ERGl analysis. The ERG is the corneal sur­face potential change induced by a light stimulus, and contains two major compo­nents: a corneal negative wave due to receptor depolarization, and a positive spike of short duration that appears to be due to an induced nerve discharge.

One cistron close to the tip of the X-chromosome is essential for tho presence of normal receptor potential. 6 independently-arising mutants affecting this cistron have been isolated. 5 of them produce essentially no ERG. One of the members is temperature-dependent, so that ERG and behavior recover after i ncu­bati on at I ow temperature for more than 36 hours. This ci stron seems to be re-1 ated to the coup I ing mechanism between I ight absorption and membrane permeabi I ity change. 2 other cistrons are found to affect the presence of the positive spike. One includes the classical tan mutant, while the other is a newly-found one which lies near the forked gene. Mutation of either of these cistrons abolishes the positive component 1n the ERG. These cistrons are thus closely related to the pro­cess that fol lows after receptor eel I depolarization. Another 2 clstrons have been found in which mutations produce highly abnormal ERGs with smal I amplitude and long delay. These are associated with extensive degeneration of the rhabdomeres of the photoreceptor ~ells. Another cistron has been found for poor phototaxis which differs from the others in that the optomotor response of the f I i es remains fairly normal, thus showing that their visual system is still functional, but selectively altered with respect to the phototactic response. The ERGs of such mutants show an absence of the changes associated with dark adaptation in normal f"ies.

RGferences:

Pek, W. et al. ( 1969) Nature 22: 351.

Hotta, Y. and Benzer, S. (1969) Nature 22: 354.

Cosens, D. J. and Manning, A. (1969) Nature 224: 285.

118. GENETIC SURGERY OF THE VISUAL SYSTEM OF DROSOPHILA BY THE USE OF MOSAICS

I nvosti gators: Yoshiki Hotta, Seymour Benzer

Sup port: National Science Foundation

Given a mutant having abnormel visual behavior and a defect in the ini­tial part of the visual path1<ey detectabh> by meens of the electroretlnogram (ERG>, it does not necessarily fol low that the primary cause of the defect is in the vis­ual receptor system. For example, the receptor might not function due to the ab­sence of some necessary substance in the circulating hemolymph. This could be tested if one could transplant a mutant eye toe normal fly. Although that is not feasible, the equivalent experiment can be done by producing genetic mosaics. There

I

131

are several methods for doing this in Drosophila. We have been using a strain that contains an unstable ring X-chromosome which is often lost in the initial stage of development, so that one portion of the body becomes mutant male <XO) while the rest is normal female()()(). By intro'i:Jucing a recessive ERG mutant gene to an X­chromosome containing also visible markers such as y (yellow body color), w (white eye), and f (forked bristles), it is possible to score the parts of the body for sex by their appearance. The division line of male and female parts in gynandro­morphs is arbitrary, so that one can choose individuals containing one mutant eye and one normal eye. If the primary cause of visual defect is in the eye itself, the mutant eye wi 11 have an abnormal ERG.

Mutants of 5 different cistrons on the X-chromosome have been tested by this method. Al I have been found to be autonomous, i.e., a mutant eye (marked by white color, which by itself produces no ERG defect) always produces a defective ERG, whl le the normal eye of the same mosaic fly always has a normal ERG. Mutants of two of these cistrons show degeneration of receptor structures. By histologi­cal examination of mosaics in which part of the eye is mutant and part is normal, one can determine the division line of phenotype (degeneration) and genotype (by marker gene) and tell whether they coincide. The results so far obtained show without exception that the two division lines are always the same, and therefore it is concluded that the specific degeneration originates within the photore;::eptors themse Ives.

References:

119.

Sturtevant, A.H. (1929) Z. wiss. Zool. 135: 323.

Garcia-Bellido, A. and Merriam, J. R. (1969) J·. Exptl. Zool. 170: 61.

ELECTRON Ml CROSCOPY OF VISUALLY DEFECT I VE MUTANTS OF DROSOPH I LA

Investigators: Lydia C. Yuan, Seymour Benzer

Support: National Science Foundation

Various non-phototactic mutants have been compared with normal flies in respect to the detailed structure of the rhabdomeres and eel I membranes of the photoreceptor cells, the distribution of screening pigment granules, and the syn­aptic structure in the lamina, where the photoreceptor axons terminate.

In two classes of X-linked mutants, the photoreceptor cells are almcst normal in appearance when the adult fly first ecloses from the pupal stage, but as the fly ages, there is a gradual dissolution of the rhabdomeres, fol lowed by de­generation of the retinula cells, disappearance of synaptic structures in the lam­ina, and loss of the capitate projections which attach glial cells to the photore­ceptor axons. The disorder resembles the genetic syndrome of retinal dystrophy in man and other mammals (Dowling and Sidman, 1962). An interesting feature Is that 6 of the 8 photoreceptor ce 11 s degenerate earl i er, leaving the 2 eccentric ce 11 s relatively intact.

In another set of X-1 inked mutants, characterized by absence of receptor potential in the electroretinogram, the rhabdorreres are quite normal in appearance, but unusal structures are found in the photoreceptor cell membrane. In this group, the ommochrome pigment granules of the photoreceptor eel Is do not show the light­induced migration toward the rhabdomeres that is typical of normal flies (Kirschfeld,

132

1969), with the exception of the eccentric eel I number 7, in which the pigment appears normal. There are also signs of abnormality in the primary pigment cells.

In a third-chromosome mutant, characterized by i nab i I i ty to properly s us­tai n membrane current in response to continuous light (Cosens and Manning, 1969) the light-induced pigment granule migration is also absent, except in cell numbe; 7. Abnormally large amounts of glycogen-like granules appear to accumulate in the secondary pigment eel Is, as might be expected if this mutant suffered from a detect in energy metabolism.

References:

Dowling, J.E. and Sidman, R. L. (1962) J. Celi Biol. 14: 73.

Kirschteld, K. ( 1969) Kybernetik 6: 13.

Cosens, D. J. and Manning, A. (1969) Nature 224: 285.

120. ISOLATION OF RETINULA CELLS FROM DROSOPHILA

Investigator: Thomas E. Hanson

Support: National Science Foundation

Some behavioral mutants with detective v1s1on have lesions affecting the function ot the retinula cells. Isolation ot these cells would allow biochemical comparison of mutant·and non-mutant eel Is, directed at the molecules involved in the photochemical reactions ot vision, the coup I ing of these reactions to membrane potentials, and the adhesion of eel Is to each other to produce the precise organi­zation of the retina.

• For the isolation of retlnula cells a batch of 20-50 grams of I ive flies is vibrated, thus breaking off the heads, which are separated from other body parts by wet screening. The heads are soaked in water, to loosen the attachment of re­tinula cells to pigment cells, and are then crushed. Retinula cell bundles, 10 µ x 75 µ, each containing the set of photoreceptor eel Is of one ommatidium, are released along with muscles, fragments of brain and other tissue. This material is passed through increasingly fine meshes ot nylon screen to obtain a traction containing pure retinula cells in a yield ot 30-50%. Fractions are also obtained which can be further purified to produce pure brain tissue and relatively pure muscle tissue. By a modification ot the procedure, retinula eel I ghosts may be obtained which appear to contain primarily the photoreceptive rhabdomere membranes and, in a vesiculated form, the cytoplasmic membrane. These membranes retain their original adhesion in bundles ot 8. Various separation techniques are being used to resolve retinula eel I proteins and in particular those proteins associated with membranes. Preliminary tests indicate that several proteins are specific for re­tinula eel Is as compared with muscle and brain. Comparison ot retinula eel I ghosts with whole eel Is indicates a larger proportion ot some proteins and a loss of others. Proteins from cells Of'wi Id type and vision-detective mutants are being canpared.

121. ULTRASTRUCTURE OF THE DEVELOPING VISUAL SYSTEM OF DRJSOPH I LA

Investigators: Lydia C. Yuan, ThOlll<!S E. Hanson, Seymour Benzer

Support: National Science Foundation

133

The compound eye of the fly is in effect a crystal I ine nervous structure in which the basic unit is an orrrnatidium containing 8 photoreceptor cells. From each ommatidium, 6 of these photoreceptor cells send axons to the first optic gan­g! Jon, tho lamina, making synaptic contact with 6 different pairs of second order neurons in a precise pattern which is repeated exactly from one orrrnatidium to tho next, such that each pair of second order neurons is connected with 6 different orrrnati di a <Truj i 11 o-Conoz and Me lamed, 1966; Brai tenberg, 1967). This pattern of connections neatly compensates for the fact that the photoreceptor eel Is within one ommatidlum receive light from different directions in space, because it results in each pair of second order eel Is being connected with 6 photoreceptor eel Is having a common axis. The final effect is an accurate point-to-point projection of the visual field onto the lamina CKirschfeld, 1967). Our anatomical studies on Dro­sophila confirm the general features of the above observations, which have been made on other fly species, and provide a basis for examining visually defective mutants.

An impressive feature of the eye of the fly is the symmetry of its upper and lower halves. Thus, while the photoreceptor eel Is of a given ommatidium are arranged in a precise, asymmetrical, trapezoidal pattern, this pattern has one form (spin) above the equator, and the opposite spin below the equator CDietrich, 1909). We find the equator in Drosophila to be variable; there araoccaslonal dislocations in the line dividing ommatidia of opposite spin. In 10 eyes mapped every one had at least one dislocation, these dislocations appearing at different points in dif­ferent individuals. They represented, however, only smal I perturbations in the symmetry of the upper and lower eye halves. To study the developmental lineage of the two sots of ommatidia of opposite spin, gynandromorphs were made in which eye color markers provided a distinction between tissues derived from male and female developmental eel I lines. If ommatidia of opposite spin derive from different an­cestral eel Is, the color dividing line in mosaic eyes should correspond to the equator. In tho data from over 50 mosaic eyes, no Ii ne cou Id be drawn across ·O-he eye that was not crossed by some mosaic patch. In eye color mosaics induced by somatic crossing over Becker ( 1957) noted a tendency for the boundaries of patches to sometimes fol low an approximately equatorial path, but since he did not observe the ommatidial spin, the equator was not clearly defined. In no instance did the mosaic dividing line of our gynandromorphs coincide exactly with the equator defined by the two spins. Thus, the spin syrrrnetry is apparently not clonally transmitted during eel I division, as are the genes tor pigmentation. It seems to be estab-1 ished at a relatively late stage of eye development, after the position of omme­tldial precursor eel Is has been fixed within the eye.

To study the process of the formation of connections of photoreceptor ax­ons to the lamina, cross-sections for electron microscopy have been made et various points along the optic stalk, which attaches the imaginal disc of the eye to the larval brain. The axons at the distal level of the stalk are found to be grouped in closely-packed, rosette-shaped bundles of 8, corresponding to the bundles de­scribed by Waddington and Perry (1960) for incompletely developed sets of photo­receptor eel Is. At some point more proximal to the developing second order eel Is in the brain, the axons in a bundle should disassociate in order to produce tho adult pattern. Further examination of the proximal half of the optic stalk, at various stages of development, will be necessary to fol low the development of these

134

connections.

Re fe re nces :

Trujil Jo-Cenoz, O. and Melamed, J. (1966) J. Ultrastruct. Res. 16: 395.

Braitenberg, V. (1967) Exptl. Brain Res. 3: 271.

Kirschfeld, K. (1967) Exptl. Brain Res. 3: 248.

Dietrich, W. ( 1909) Z. f. wissenschaftl iche Zoo I. 92: 465.

Becker, H.J. (1957) Z. f. indukt. Abstam. und Vererb. 88: 333.

Waddington, C.H. and Perry, M. M. (1970) Proc. Roy. Soc, Lond. Sor. B. 153: 155.

Horridge, G. A. and Meinertzhagen, I. A. ( 1970) Proc. Roy. Soc. Lond. Ser. B. I 75 : 69 •

122. TWO-DIMENSIONAL COUIHERCURRENT METHOD FOR ISOLATION OF OPTOMOTOR-DEFICIENT MUTANTS

I nvosti gator: Seymour Benzer

Support: National Science Foundation

The program of isolation of non-phototactic mutants has yielded ones with defects at various steps Jn the photoreceptor process. Phototaxis operates by the fly orienting itself to equal lze the I ight received by both eyes. In order to ob­tain mutants affecting connective pathways in the visual system, it is desirable to

•use a more complex stimulus that requires integration to produce a response, such as the optomotor response to moving black and white stripes.

In one form of optomotor response, f Ii es in a hori zonta I tube, when ex­posed to a striped pattern moving along the axis of the tube, walk in the direction opposite to the movement of the stripes, thus accumulating at one end of the tube (Hecht and Wald, 1934). A plastic holder was constructed which holds two sets of plastic test tubes, mouths fadng each other, in such a way that one set can be slid over the other as a unit. This apparatus is laid horizontally over a moving hor;zontal bolt of opaque and transparent stripes i I luminated from below. Flies are placed in the starting tube and tapped down to one end. If the stripes are moving toward this end, most of the fl !es, if normal, move against the pattern to the opposite end, but blind, non-optomotor, or otherwise defective flies do not move properly. After 30 seconds, the sliding frame Is shifted, thus separating responders from non-responders. Tapping the apparatus now brings al I the ti ies back to the starting end, the responders being in tube no. I while the non-respond­ers rema;n in tube O. The sliding frame is returned to its original position. Tho cyc10 is then repeated 4 more times, producing in 2-1/2 minutes a countercur­rent distribution of the population according to optomotor response. A closely re1ated method has been devised by G1ltz'-( 1970).

To obtain further fractionation in a second dimension, each of the frac­tions is now run again, separately, tor movement with the patter~ of st;ipes. In this way, a two-dimensional chromatogram for optomo;or respons7 1s obtained. When a mutagenized population is run, the bulk of the flies end up 1n one area

135

corresponding to frequent movement against the pattern and rare movement with the pattern. SI ugg i sh mutants move Ii tt I e either way; non-optomotor mutants give equa I responses in both directions. Among the latter, some are simply blind due to de­fects in the peripheral visual system, detectable in the electroretinogram. Others, however, show a normal phototactic response, indicating that the visual apparatus is functional and suggesting that more central integration is at fault.

References :

Hecht, S. and W<Jld, G. ( 1934) J. Gen. Physiol. 17: 517.

Gtltz, K. G. ( 1970) J. Exptl. Biol. 52: 419.

123. "EASILY SHOCKED" - A NEW MUTANT PHENOTYPE IN DF\DSOPH I LA

* Investigators: Paul T. Christensen , John R. Merriam

Support: National Science Foundation National Institutes of Health, Public Health Service

In the course of isolation of non-phototactic mutants, certain mu1'ants are found whose apparent lack of phototaxls is due to the fact that the flies, when tapped down to the end of the tube, become immobile for several minutes•. Closer inspection sha11s a regular syndrome of events following the shock. In the mutant PC 75, for example, the legs and proboscis extend, the wings are held up­right, and the abdomen curls inward, culminating in ejaculation or defecation (in males) or occasional extrusion of an egg (in females). The appearance while shocked is one of extreme rigidity, reminding one of catatonia or grand mal epi­lepsy in man. The visual behavior of these flies in the unshocked state is normal, as is their ERG.

By virtue of the selection procedure used, the mutants so far isolated are all X-linked. Genetic mapping reveals them to be located at various regions of the chromosome. There are regular differences in degree of shock<Jbi lity and other aspects of the phenotype between the different mutants, including dependence on temperature.

124. PHOTOTAXIS OF DF\DSOPHILA LARVAE

Investigator: Seymour Benze.r

Sup port: National Science Foundation

It has long been kna11n that fly larvae are phototactic, although unlike adults they move away from I ight rather than toward it (Loeb, 1918). Tests per­formed in a chamber constructed from glass plates (inner dimension 7 cm long x 2 cm wide x 0.1 cm high) showed a good response for third instar larvae of the normal C-S strain to fluorescent illumination incident to one end. To determine whether the larval photoreceptor is related to photoreception in the adult fly, this test was applied to larvae of mutants that exhibit no phototaxis as adults: JM 11, which sho1~s no receptor potential; tan, which has a defective positive spike in the olectroretinogram; sine oculls, which has no adult eyes or ocelli. In every case, larval phototaxis was observed. Since exposure of normal larvae to I ight can

* Undergraduate, California Institute ot Technology.

136

entrain circadian rhythms, another mutant of interest was the arrhythmic mutant RK442. Larvae of this mutant were also found to show negative phototaxis, indi­cating that tho light arrhythmlcity is not duo to lack of the receptor involved in larval phototaxis.

Reference:

Loeb, J. ( 1918) Forced movements, tropisms, and animal conduct. Lippincott, Philadelphia.

125. CIRCADIAN RHYTHM MUTANTS IN DROSOPHILA

Investigator: Ronald J, Konopka

Sup port: National Science Foundation

Mutants have been isolated that affect the circadian rhythm of eclosion in Drosophila melanogaster. One mutant, RK 330, has a froe-.running period of 19 hours at 25°C, and another, RK 442, is essentially arrhythmic under these condi­tions. Both are X-chromosome linked. The temperature sensitivity of the mutants was determined by measuring tho period of the free-running eclosion rhythm at 25°C and 20°C. The increase in average period length of the rhythm of RK 330 males was less than I hour when tho temperature was lowered, indicating that the rhythm is temperat1,re-componsated. The RK 442 males were arrhythmic at 25°C and 20°C,

The mutants were mapped by crossing each mutant to virgin females homozy­gous for several recessive markers, mating the progeny to each other, and determin­ing the oclosion profile of each class of parental and recombinant males, Each mutant was mapped with respect to the markers y2, sc, v, f.y+ in one experiment, and with respect tow, sn3, m in another. Both mutations-were shown to be located near the tip of the X-chromosome.

• The mutations were tested for recessivity and for complementation by con-structing female flies heterozygous for the RK 330 mutation alone, the RK 442 muta­tioh alone, and the RK 330 and RK 442 mutations in the trans position. The periods of the free-running adult activity rhythms were determi~ The results showed that the RK 330/+ flies had n period of 22 hours, indicating partial recessivity, while the arrhythmic RK 442/+ files had a period of 24 hours, shO\~ing complete recessivity. The period of RK 330/RK 442 flies was 20 hours. This result shows that the RK 330 and RK 442 mutations do not complement and therefore presumably represent different mutations ln the same functional gene (cistron).

126. CIRCADIAN ACTIVITY RHYTHMS IN SINGLE INDIVIDUALS OF DROSOPHILA MELANOGASTER

Investigators: Yoshiki Hotta, Ronald J. Konopka, Seymour Benzer

Support: National Science Foundation

The much studied circadian rhythm of eclosion of Drosophila is measured on a large population, each adult fly eclosing from the pupa only once. Thus, for a rhythm to be observed in the population, tho individual biological clocks must be synchronized. An arrhythmic mutant could result from a detect in either of tho two mechanisms. It is, therefore, necessary to huve other measures of ongoing circadian rhythm that can be fol IO\ved in an individual fly. This is also essenti~I

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for testing rhythm in mosaic individuals.

Genera I I ocomotor activity sh.~s a .circadian rhythm in many insects and other animals, and many devices have been used for Its measurement. For the present purpose, darkness ls necessary; it is also desirable that visible I ight can be applied at wi 11 tor phase shift experiments without disturbing the continuous mea­surement of activity. In order to have a large number of gadgets to perform mea­surements on many flies simultaneously, an inexpensive design is important.

An activity meter was bui It by using the tact that infrared I ight does not affect the rhythm. A light from a miniature 24-volt lamp, operated at 6 V AC to increase the relative content of infrared, is passed through a gelatin filter transmitting only wavelengths greater than 820 nm into a dark plastlc box. Inside is a chamber 5 mm wide x 40 mm high x 2 mm thick, made from two microscope slides and a grooved space, in which a fly and food are placed. Behind the transparent fly chamber are two small sll icon solar eel Is (having maximum sensitivity in the infrared) located.at the upper and lower thirds of the chamber. The silicon cells are connected in a series-bucking arrangement, so that the outputs of the two eel Is cancel as long as the 1 lght incident upon them is identical; this' is true when the fly is in the middle third of the chamber. When the fly moves up or down, project­ing its shadow onto one or the other of the solar eel Is, the output becomes either positive or negative. It· is therefore possible to monitor the opproximate location of the fly as wel I as its frequency of movement. With the series-bucking arrange­ment, the output is relatively insensitive to fluctuation of I ight source intensity, eliminating the need for a DC power supply. The output is fed into an inexpensive Fairchild µA 741 C llnear operational amplifier whose output is sufficient to drive a conventional pen recorder to obtain a continuous long-term record. Since it is unnecesssry for the present purpose to record the location'of the fly, the output of the amplifier is used to trigger a si I Icon-control led rectifier (SCR), which drives a multi-channel event recorder. In this way it is possible to record the activity of 20 or more fl las separately on a single rol I of paper.

This method has been successful in demonstrating that the normal fly has c. regular circadian rhythm of activity in constant dorkness, lasting as long as 11 days. Mutations so far studied which produce arrhythmic and abnormal periods (with respect to ecloslon) have similar effects on the locomotor activity rhythm of indi­vidual flies. Flies with head and body parts mosaic with respect to rhythm geno­type have been successful, ly monitored to determine the locatlon of clock-control I ing centers.

127. LOCALIZATION OF THE SITE OF ACTION OF A RHYTHM MUTATION WITHIN THE FLY

I nvesti g<itor: Ronald J. Konopka

Sup;iort: National Science Foundation

The mutant RK 330 has a shorter than norma I period for its nett vi ty rhythm. In order to localize the site of action of this mutotion within the orga­nism, gynandromorphs were constructed in which the female parts of the body were normal while the male parts carried the RK 330 mutation linked to the indicator markers yellow, singed, and miniature. The free-running activity rhythms of the gynandromorphs were anolyzed, und correlations drawn between the part of the body that wus male and the expression of the RK 330 mutation. It was found that the express ion of the mutation was governed by the sex of the head of the fly; if the head was male, the rhythm was mutant, but if the head was female (heterozygous for

138

f{K 330), the period of the rhythm wns that of an RK 330/+ heterozygote.

Some flies with mosaic heads wore also run; those flies gave complex activ­ity pnttorns that were neither mutant nor normal. Although tho number of flies was insufficient to properly interpret the patterns, the result supports the conclusion that tho site of nction of the mutation is in the head of the fly or in nn internnl tissue of cormion origin.

128. PRODUCTION OF fVOSAICS FOR AN AUTOSOMAL MUTATION

Investigators: Ross B. Hodgetts, Yoshiki Hottn, John R. Merriam

National Science Foundntion

Sexual mosaics for mutations on the X-chromosome are easily produced. However, we wished to apply the method to tho analysis of autosomal mutations, in particular, to the ebony mutation (ell) which is located on the third chromosome and produces an abnormal electroretinogram CERGJ. The key to our approach was the feet that females homozygous for the gene claret nondisjunction (cand) produce some eggs which, during embryogenesis occasionally suffer tho loss of whole chromosomes (Lewis und Gencarel iu, 1952). This usually results in lethality except in the case of X-chromosome elimination. Wo therefore made use of a stock carrying a translo­cntion of the normal ebony gene (e+) from tho third to the X-chromosome. Females homozygous for cand anifOTI, and carrying the trans location, were mated to appro­priately marked ell males. Among the progeny, some were obtained in which the X­chromosome bearing the translocation was lost during development, as shCN1n by the uncovering of recessive markers (white eyes, forked bristles) in part of the adult body. Loss of the trans location also results in uncovering of tho ell gene in the same tissues. Analyses of the eloctrorotinograms and body color distributions of these gynandromorphs are underway.

'Re fo rence:

Lowis, E. B. and Gencarel la, W. (1952) Genetics 37: 600.

129. S-ALANINE METABOLISM IN WILD TYPE OR'.JSOPHILA ANO TWO MUTANTS, EBONY AND BLACK

I nvosti gator: Ross B. Hodgetts

National Science Foundation Jane Coffin Chi Ids Fund for Medical Research

The mutant ebony has a defective eloctrorotinogram (ERG). Since Jacobs ( 1966) hns shown it t~abnormal in its utilization of S-alanino, tho metabolism of this substance wus investigated. Amino acid analysis clearly demonstrated that tho mutant suffers from a 7-fold excess of $-alanine in the hemolymph ubout 8 hours after ?uparium formation, and a 70% excess shortly after eclosion. Microinjectlon ex?eriments fol lowed by puper chromatography revealed the fol lowing udditional facts. When wi Id-type white prepu?ae we-re injected with 14c-l)-alanlne, the radio­nctivity remained in the free Limino acid form unti I the time at which tanning of the pupal cuticle became visible. After this time, it became incorporated into mate­rial which remained nt the origin of the chromatogram. However, with ebony flies, unable to tan the cuticle, incorporation of S-alanine into insoluble material did not occur. The injected label was eventually degraded and probably released from the

139

animal as 14C02. These results support Jacobs' finding that ebony is unable to in­corporate S-alanine into the pupal case (Jacobs, 1966). His suggestion that this defect might lead to an elevated level of the amino acid in the hemolymph was found to be true. However, microinjection of_ S-alanine into wl Id type animals 3-8 hours after puparium formation, or just priorro eclosion, failed to produce ebony be­havior in the resulting adults. Thus no clear relation between S-alanine metabolism and the behavior of Drosophila was evident.

Analysis of the S-alanlne level in black, a mutant 1;hose bodily phenotype is very similar to that of ebony, but which has normal visual behavior and ERG, revealed that the mutant is apparently unable to manufacture S-al.anine. Thus, its pupal case is white. _By feeding mid-third Instar larvae of this mutant on a med­ium supplemented with $-alanine, It was possible to obtain normal, tanned, pu;ial cases.

The experiments on these two mutants and the strong correlation between the appearance of tanning in wi Id type flies and the incorporation of $-alanine in­to insoluble material suggest thats-alanine ls involved in the tanning process. Since no intermediates were detected on the chromatogram, it is possible that the molecule goes directly from hemolymph to the cuticle, where it may play a role in cross-linking the cuticular proteins, as suggested by Jacobs (1968).

References: •

Jacobs, M. E. (1966) Genetics 53: 777.

Jacobs, M. E. (1968) Biochemical Genetics I: 267.

130. CATECHOLAMINE LEVELS IN ADULT DROSOPHILA

Investigator: Ronald J. Konopka

Support: National Science Foundation

To investigate the pathway of tyrosine metabolism in Drosophila and to determine which catecholamines might be important in nervous function, homogenates of adult flies were analyzed for the presence of noradrenalin, adrenal in, dopamine, and its precursor, dopa. White-eyed mutant flies were used in order to avoid inter­ference by eye pteridines in the fluorescence assay. No noradrenalin or adrenalin could be detected; their levels were below 0.05 ng/mg. This is in contradiction to measurements reported by Tunnlcl iff et al. in which this precaution was not taken.

The level of dopa in adult flies was less than 0.1 ng/mg, while the level of dopamine was found to be 4.6 ng/organism. The dopamine levels in adult tan and ebony flies were also determined; the level in tan is 0.6 that of wi Id type-;-6nd the level Jn ebony is twice wl Id type. These ratios also hold for the dopamine levels in very late, ready-to-emerge pupae. Further investigation into the possible role of dopamine as a neural transmitter or effector in Drosophila may yield some clue to the chemical nature of the visual and behavioral .defects in tan and ebony flies. --

Reference:

Tunnicliff, G., Rick, J. T., and Connolly, K. (1969) Comp. Biochem. Phys! ol. 29: 1239.

•

140

131. CATECHOLAMINE METABOLISM IN DR'.ISOPHILA

Investigator: Ross B. Hodgetts

Support: National Science Foundation Jane Coffin Chi Ids Fund for Medical Research

Abnormalities in neural transmitter systems might be expected to cause behavioral anomalies, and catecholamines are implicated as such transmitters. The catecholamines are known to be Involved in tanning of the pupal cuticle in insects, "process which converts the soft, white larval cuticle to the hard, brown pu;ial case (Karlson and Sekeris, 1964), and In the production of melanin, a black pig­ment in the adult cuticle (Hackman, 1964). Since two behavioral mutants, tan and ebony, also suffer from abnormalities in their adult body pigmentation, their catecholamine metabolism was investigated.

Measurements were made on tan, e~gny and wi Id-typo animals at the time of puparium formation. Microinjecti0i1"""of C-labeled tyrosine, dopa and dopamine was accomplished using a device designed by H.K. Mitchel I. After a period of in vivo incubation, the animals were squashed onto a sheet of Whatman 3 mm paper which was then deve I oped by descending chromatography. The paper was cut into strips and scanned In the strip counter. We were unable to detect any tyrosine hydroxy lase activity in wi Id typo Drosophila, although this enzyme does convert tyrosine to dopa in thG blowfly, Cul I iphora (Karlson and Sekeris, 1964). By contrast, there was substantial dopn decarboxylaso activity, which supports an earlier observation (Lunan and Mitchel I,. 1969). Furthermore, evidence of dopamino-N-acetylase was provided by tho discovery that the major product resulting from dopamine injection had chromatographrc properties similar to N-acetyl-dopamlne. No evidence of the enzyme which converts dopamine to norepinephrino could be found in those experi­ments, which is consistent with Konopka's inability to detect any norepinophrine in Drosophila <Biology 1970, No. 130). Finally, thGre was no obvious difference be­tween tan, ebony and our wi Id type strain upon injection of dopa or dopamine . Konopka has found abnormalities in dopa and dopamine levels in the mutants, but these differences, whi lo not due to gross enzymatic abnormalities during puparium formation, may result from regulatory failures at other times.

In conclusion, the tanning pathway as describGd for Cal liphora seems to hold for Drosophl la. However, the first step involving the convGrsion of tyrosine to dopa may either be inoperative at puparium formation, or altered in some way. The mutants tan and ebony both possess the enzymes necessary for the conversion of dopa to dopamine and dopamine to N-acetyl-dopamine.

ReferenCGs:

Karlson, P. and SGkeris, C. E. ( 1964) In: M. Florkin and H. S. Mason (eds.), Comparative BiochGmistry, Vol. 6, Chap. 4. Academic Press, New York.

Hackmein, R. H. ( 1964) In: Vol. 3, Chap. 8.

M. Rockstein (ed.), The Physiology of lnsecta, Academic Press·, New York.

Lunan, K. D. and Mitchell,~. K. ( 1969) Arch. Biochem. Biophys. 132: 450.

·q--

141

132. COMMUN I CAT I ON OF DIRECT I ON IN THE HONEY BEE

* * * Investigators: James l. Gould , Michael A. Henerey , Michael C. Macleod

Ford Foundation Notional Institutes of Health, Public Health Service

As shC1<rn by von Frisch ( 1946), forager bees, on returning to the hive from i'l food source, perfonn a "waggle dance" containing parameters related to the distance and direction of the food source from the hive. Recently, Wenner and Johnson (1967) have questioned the notion that recruits utilize such information, and have ;ierfonned experiments that Indicate that under certain conditions odor cues alone are sufficient to impart the food source's whareabouts to recruits.

A series of experiments was designed to test whether direction informa­tion can be uti Ii zed by recruits in the absence of odor and visual cues. The ex­;ioriments were performed, taking extensive precautions to avoid olfactory cues, at a flat, desert-I Ike area in eastern Oregon, with marked bees that were Individually observed while dancing in the hive. The results showed that recruited bees were able to locate the food source indicated by the dances of recruiting foragers in preference to an equivalent location in the opposite direction, thus supporting_ von Frisch's hypothesis that direction information can be corrrnunicated by tho · dance.

References:

von Frisch, K. ( 1946) Osterr. Zoolog. Zeitschr. I: I.

Wenner, A. M. and Johnson, D. l. ( 1967) Science 158: 1076.

Gould, J. l., Henerey, M.A., and Macleod, M. C. (1970) Science 169: 544-554.

PUBll CATIONS

Gould, J. l., Henerey, M., and Macleod, M. C. (1970) Communication of direction in the honey bee. Science 169: 544-554.

Merriam, J. R. and Benzer, S. (1969) Genetic mapping of behavioral mutants in Drosophila melanogaster by countercurrent distribution. Genetics 61: Suppl. P S40 (Abstract).

Merriam, J. R. (1969) Autonomy of mutant bristle frequency in gynandromorphs with different sc alleles in Drosophila melanogaster. Amer. Naturalist 103: 672-675.

* Undergraduate, Californlo Institute of Technology.

142

Professor: W i 11 i am J. Dreyer

Senior Research Fe/ low: Wi 11 iam R. Gray

Research Fellows: Marion T. Lai co, David S. Papermaster, Erkki I. Ruoslahti, John E. Smart

Graduate Students: Charles R. Bi rdwel I, Zoltlln A. T6k"1s

Associate Biologist: Inge S. Frahm

Laboratory S;iecialists: David M. Schemberger, Ronald W. Siemens

Research Assistant: Umavathi Ramaswamy

The research carried out in this laboratory covers a broad spectrum of biochemical a;>;>rooches, some very biological and some very chemical. The general theme which unifies most of these projects is the study of the role of biological membranes in the organization of I iving organisms. The laboratory is particularly well equipped to study proteins and ;ieptides and emphasis tends to be placed on studies of such molecules.

One important result of these studies has been the discovery of a grou;i of peptides, "miniproteins," which are present in large amounts in each of the mem­branes studied to date. The abundance and similarity of the miniprofoins obtained from different membranes suggest that these peptides play a fundamental role in the structure and functi-0n of biological membranes.

There is also considerable interest in the study of the molecules involved in receptor and recogn /ti on functions of membranes. Perhaps the most concrete knowledge of the sensing and recognition processes by eel I surfaces, derives from <the study of antibodies, known to play such a role in the detection and ki I ling of foreign graft tissues and abnormal cancerous eel Is. Studies of antibody molecules ~see previous Annual Reports) have led to some surprising conclusions in relation to the chromosomal events which underlie cellular differentiation in the immune system.

The immune system represents but one example of a more general phenomenon of chemospecific recognition, and is a fairly recent evolutionary development. It seems probable that it has arisen from a widely occurring system such as is used for the recognition of one ce 11 type by another during the comp lex process of ce 1-1 u lar differentiation and organogenesis. Little is known about the chemistry of these highly specific cell interactions, and it would be of great interest to study the molecules involved in cell surface specificity, to see whether they possess some of the remarkable structural features found in antibodies. The investigation of chemoreceptors, whether in the membranes of immune lymphocytes or in other cells possessing equally high specificity, poses some very difficult technical prob !ems. It does seem important to develop new methods for the study of eel I surfaces, for fractionating mclecules contained in or on membranes, and for the analysis of such molecules after they have been obtained in pure form. Tho study of the membranes responsible for the detection of light i•.n the retina has proven to represent a par­ticularly simple model system for the study of receptor-membrane structure and function. It is hoped that methods being developed in relation to this program can be applied at a later date to more complex systems. Histochemical and immunolog-i ca I techniques ; r. comb; nation with the e I ectron microscope seem pa rt i cu I ar I y relevant in this regnrd.

The development and application of automatic instrumentation together with data processing has continued to pay rich dividends In relation to the ease

143

and efficiency with which this type of work can be carried out. Tho totally automat­ic amino acid analysis system has been·-..uti Ii zed virtually around the clock with many benefits to our research program. The automatic sequential degradation equipment has also been put to interesting uses.

Our concern with the need for simpler, more sensitive, and more rapid techniques has led Dr. Gray to spond the past year exploring possible applications of mass spectrometry.- Some extremely promising results have been obtained.

133. ISOLATION OF FUNDAMENTAL POLYPEPTIDE SUBUNITS OF BIOLOGICAL MEMBRANES

Investigators:

Support:

Marion T. Lai co, Erkki I. Rues lahti, David S. Papermaster, WI 11 i am J. Dreyer

National Institutes of Health, Public Health Service American Cancer Society

In order to further characterize the molecular components responsibl.e for the common functional roles of cellular membranes, we have developed technique~ for tho isolation and structural analysis of membrane proteins of diverse origins. This study included a comparison of membranes from human and bovine rod blood cells, bovine liver mitochondria and bovine retinal rods.

Each of the membranes studied was analyzed by polyacrylamide gel electro­phoresis in the presence of sodium dodecyl sulfate (SOS), By this technique all 3 membranes showed a major peptide component or "mlniprotein" that migrated with a mobility equal to that of insulin (molecular weight 5700), while tho larger protein components seemed to differ in each membrane. Furthermore, apart from rhodopsin, the miniprotein was the only prominent polypeptide component seen in the rod mem­brane. Lyophi I ization, drying, or de! ipidation of membranes resultod in a ;iattern showing mainly high molecular weight bands on the gels.

Fractionation of the membranes on a calibrated Sepharose 66-SDS gel ti 1-tration system yielded a mini protein peak at a position corresponding to a molec­ular weight well below 10,000. While this fraction contained only a portion of the total amount of protein in the sample, gel electrophoresis showed that the higher molecular weight fractions also had miniprotein present as reversible polymers of a subunit.

A sample of miniprotein was stripped of its carbohydrate and the molecu­lar weight of the protein moiety was determined on a calibrated P-100 column in the presence of SOS. Part of this fraction behaved as low molecular weight material, giving a wide peak in the molecular weight range of 4,000-6,000.

The peptides present in high molar amounts in the peptide maps of whole membranes were all present in the peptide maps of subti lisin digests of the mini­protein preparations from those membranes. Further, the peptide maps of miniprotein from mitochondria and red cell membranes were extremely simi tar, indicating that the mini proteins from these sources may be products of sim! lar if not identical genes.

Terminal group analyses (Ruoslahti, Laico and Dreyer, 1970) showed that the miniprotein represents a mixture of several different sequences, and that they

144

cannot be proteolytic degradation products. The ubiquitous distribution and the abundance of the mini protein fraction in different membranes make it an ideal can­didate for the long-sought-aftor corrrnon protein subunit of biological membranes. Similar results have recently been reported for Neurospora structural protein (Munk res, Swank and She i r, 1969 l.

Although evidence is lacking concerning the detailed structure and mode of assembly of natural biological membranes, we believe that the ab! lity of the mini proteins to undergo reversible polymerization in vitro may reflect a self-as­sembling system which is responsible for the formation and growth of the biological membranes in vivo. Furthermore, the simplicity of the retinal rod rrombrane, con­taining only rhodopsin and miniprotein as major polypeptide components, along with the fact that there are large amounts of the miniproteln fraction in each of these membranes, leads us to propose that miniproteins play a fundamental role in tho cooperative phenomena, ion permeability and electrochemical events believed to occur in those biological membranes.

References:

Munkres, K. D., Swank, R. T., and Sheir, G. I. (1969) lnternat. Symp. Autonomy and Biogenesis of Mitochondria and Chloroplasts. Aust. Nat. Acad. Sci., Canberra (in press),

r'uoslahti, E. I., Laico, M. T., and Dreyer, W. J. <1970) Cin preparation).

134. PARTIAL CHARACTERIZATION OF THE FUNDAMENTAL PROTEIN SUBUNITS OF CELLULAR MEMBRANES

Investigators: Erkki I. Ruoslahti, Marion T. Lai co, Wi I I lam J. Dreyer

Support: National Institutes of Health, Public Health Service

Biological membranes provide the structural basis for several important cellular functions. Tho control led movement of ions across the membrane results in the capability of excitation and transmission of impulses ln the nerve and muscle eel Is. Tho eel l membrane is also the site of receptors for various solutes, hor­mones and other effector substances. One of the most important aspects of the mem­brane structure are tho molecular mechanisms behind the interactions that occur be­tween eel ls during differentiation and growth. lt is possible that studios on the nature of these normal interactions may also throw more light on tho question of the molecular basis of malignancy.

Fractionation of red blood cells and mitochondrial membranes on SE-Sepha­dex resulted in a fraction which contains the mlniprotein subunits of the membrane. More than 30% of the membrane protein is found in this traction.

No N-termlnal or C-terminal amino acids have been detected in the m1n1-protei n fraction. End-group analysis after selective cleavage of the minlproteins at arginine residues showed that tho fraction was a .mixture of several different amino acid sequences. Further fractionation of the mlniprotoins resulted in iso­lation of a glycopeptide fraction with 6G% carbohydrate. Fingerprints and amino acid analysis indicate that the peptide portion may be an example of a single mini­protoin. Several other fractions with fingerprints much less comp! icated than those of the whole miniprotein fraction were obtained. These results suggest that mem­branes contain a number of distinctly different peptides, each about 50 amino acids in length. Further fractionation and structural analysis wil I be required,

145

however, before a definitive statement can be made concerning the chemical proper­ties of these fundamental membrane subunits.

Reference:

Lciico, M. T., Ruoslahti, E. I., Paperrnastor, D. S., and Dreyer, W. J, ( 1970) Proc. Nat. Acad. Sci. (In press).

135. A PROPOSED MODEL FOR THE GENERATION OF GRADED POTENTIALS IN ROD OUTER SEGMENTS: IONIC GRADIENTS AND RESTORATION OF DARK ADAPT A Tl ON BY Na-K-Mg-ATPase

Investigators: David S. Papermaster, Wi 11 iam J. Dreyer

Support: National Institutes of Health, Public Health Service American Cancer Society

The photoreceptor elements of the retinas are localized in a specialized receptor cell organelle - the rod outer segment. We have sham (Biology 1969, No. 205) that the menbranes of this lame I lar structure are composed predominantly ,of 2 proteins, rhodopsin and miniprotein. To date, the only demonstrated alterations in rod structure that occur when I ight is absorbed by 11-cis-rotlnal in the acti\113 site of rhodopsin is a els-trans isomerization ot retinal, a slight change in con­formation ot rhodopsin and decreased dark current across the outer menbrane of the rod. In an attempt to couple these phenomena and provide for amplification of the signal input by light, we postulate that light-induced conformational changes in rhodopsin are transmitted to ordered miniprotein molecules- in the rod lamellae. A concerted mini protein conformational shift alters the penneabi lity of the lamellar sacs. The resultant ion shifts cause a hyperpolarization of rod plasma membranes and generation of a graded potential.

The source of energy tor this amplification process could be remote from the site of absorption of a photon, as seems required by tho paucity of molecules other than rhodopsin and miniprotein.

This energy might derive from the process of restoration of tho original ionic gradient, a function currently proposed in many cells tor a Na+-K-Mg++-acti­vated ATPaso. We have tound this enzyme in high specific activity in purified rods. The enzyme is nevertheless present in smal I molar amounts, relative to rhodopsin and mini protein. If this enzyme does play an important role in the rod outer segments, it might be expected to be localized in restricted regions of the sac or rod outer membrane. Attempts to localize this enzyme by histochemical techniques with the electron microscope are now in progress. Wo hope that these and related irrrnuno-histochemical techniques wi I I serve as prototypes for the localization of a variety of membrane functions.

References:

Hagins, W. A., Penn, R. D., and Yoshikami, S. (1970) Biophysical J. JO: 380.

Toyoda et al. ( 1969) Vision Res. 9: 453.

Dreyer, W. J., Lalco, M. T., Ruoslahti, E., and Papennaster, D.S. (\970) Fed. Proc. 29 (abstract).

146

Laico, M. T., Rouslahti, E., Papermaster, D.S., and Dreyer, W. J. (1970) Proc. Nat. Acad. Sci. (in press).

I 36. PROTEINS DEf<I VED FROM THE SURFACES OF E~BRYON IC CELLS

I nvcsti gators:

Support:

* Charles R. Bi rd1·1el I, Jeffrey M. Sch redder

Nationa I Institutes of Hca I th, Pub Ii c Hoa I th Service

The EDTA method for dissociating eel Is has been found to release proteins from eel I surfaces (Moscona, 1968). A revised method has been used here to dis­sociate different bovine and chick embryonic tissues: brain, heart, liver and ret­ina. Al I of these tissues (from both animals) give 2 proteins by this procedure with molecular weights of 35,000 and 42,000 as determined by SOS gels. These are the 2 major protein bands on these gels. The goal of this research project is to isolate and to characterize these proteins.

The investigation is now being done on 10-day-old chick embryonic brain coils. These are easy to dissociate and can be obtained in reasonable quantities. The main technique which has been used thus far to isolate the proteins of interest has been column chromatography, but preparative gel electrophoresis is now being used us we I I •

The question one would like to answer about these proteins is whether they are homogeneous or heterogeneous. If they are homogeneous, they may be part of the hexagonal struct~res which are found at tight junctions between membranes and which can be remcved by EDTA <Revel, 1967). If they are heterogeneous, they may play some sophisticated role in differentiation. In either case, once the pro­teins are isolated, sequencing several residues at the N-torminal ends of these molecules should help answer those questions.

Ro fe rences :

Mos con a, A . A . ( I 9 68) Deve I . Bi o I . I 8: 250 .

Revel, J.P. and Karnovsky, M. J, (1967) J. Cell. Biol. 33: C7.

137. STUDIES ON MEMBRANE PROTEINS OF E.COLI

Investigators:

Support:

* John E. Smart, Richard S. Newman, Andrew H. Weigel

National Science Foundation

If miniprotoins are the major polypeptide components of all normal cellu­lar membranes, and/or if miniproteins are encoded in the mitochondrial DNA, then the evolutionary derivation of miniproteins takes on additional interest. For this reason, we have investigated the occurrence and propertie$ of membrane proteins in bactcri a.

" Whole bacteria (W31 IQ) and bacterlal membranes (prepared according to Saedler and Starlinger, 1967) have been analyzed for their protein constituents by sos polyacrylamide gel electrophoresis. Whole bacteria and b<'lcterial membranes have also been fractionated on SE-Sephadex according to Lai co et al. ( 1970). This

* Undergraduate, California Institute of Technology.

147

procedure yields a number of protein fractions, one of which is not bound to the column. SOS polyacrylamide gel electrophoresis of this fraction Indicates that it is a homogeneous protein of molecular weight between 30,000 and 40,000. If we assume that there is one histidine resl~ue per molecule the theoretical molecular weight (based upon amino acid composition) is ca. 34,000. The presence of a sin­gle N-terminal amino acid, approximately the expected number of peptides upon di­gestion with trypsin, the high content of glycine (ca. 20 mole%>, tyrosine, and phenylalanine, and relatively low content of histidine (ca. 0.3 mole %l, lysine, and arginine al I indicate that the protein is homogeneous. Characterization of this and other protein components of bacterial membranes is continuing.

References :

Laico, M. T., Ruoslahti, E. I., Papennaster, D. S., and Dreyer, W. J. ( 1970) Proc. Nat. Acad. Sci. (in press).

Saedler, H. and Starlinger, P. ( 1967) Mclee. Gen. Genetics 100: 190.

138. STUDIES ON MEMBRANE PROTEINS OF WILD TYPE (74-A) AND EQKl' (mi - I ) NEUR'.JSPORA MITOCHONDRIA

Investigators: John E. Smart, Wi 11 iam J. Dreyer

Support: National Science Foundation National Institutes of Health, Public Health Service

Laico et al. (1970) have recently reported on the occurrence of m1n1pro­teins (molecular weight ca. 5,000) which seem to account for more than 50% of the molar amounts of al I peptides or proteins present in all membranes which they have investigated. Because of the presence of such large amounts of mini proteins in each membrane, they have suggested that these miniproteins might play a fundamen­tal role in the several functions which occur as common denominators in biological membranes, and might in fact be the true subunit of structural protein fractions reported by many other workers <Criddle et al., 1962; Lenaz et al., 1968).

Woodward and Munkres (1966) found that the mitochondrial structural pro­tein of the maternally inherited, respiratory-deficient mutant (mi-I) of Neuros;iora differs from that of wild type (74-Al by a single amino acid replacement, trypto­phan to cysteine. Their work with several eel lular membranes suggested that a com;ionent(s) of the mitochondrial structural protein is common to other eel lular components (presumably membranes) and that this component Is encoded in the mito­chondrial DNA.

Are miniproteins encoded in the mitochondrial DNA? As one possible ap­proach to this question we have investigated the occurrence and properties of mini­proteins in wi Id type (74-Al and .QQ)sy (mi-ll Neurospora mitochondria.

Fractionation ct purified mitochondria by SE-Sephadex chromatography according to the methods of Laico et al. ( 1970) yiel.ds a protein fraction which is not bound to the column. This fraction contains a major protein which exhibits a molecular weight noar 5,000 upon SOS polyacrylamlde gel electrophoresis. The map of peptides obtained from a subti llsin digest of these mini proteins is too complex for a homogeneous protein of ca. 5,000 molecular weight. The peptide patterns are surprisingly similar to those obtained from subtilisin digests of miniproteins from bovine red b Iced eel I stroma and mitochondria. Several of the peptides have been analyzed for amino acid composition. No significant differences have been observed

148

between poky and wild type minlproteins. In particular, maps of subtilisin peptides show no differences when stained for tryptophan. This result is incompatible with the genetic alteration suggested by previous work and we feel one must look for other explanations of the molecular consequences of the poky mutation.

This work was done In collaboration with Dr. Dow O. Woodward, Department of Biological Sciences, Stanford University.

Re fe ranees :

Criddle, R. S., Bode, R. M., Green, D. E., and Tisdale, H. H. ( 1962) Biochemistry I: 827.

Laico, M. T., Ruoslahti, E. I., P<ipermaster, D. S., and Dreyer, W. J, (1970) Proc. Nat. Acad. Sci. (in press).

Lenaz, G., Haard, N. F., Si I Iman, H. I., and Green, D. E. ( 1968) Arch. Blochem. Biophys. 128: 293,

Woodward, D. 0. and Munkres, K. D. (1966) Proc. Nat. Acad. Sci. SS: 872.

139. A NEW SYSTEM FOR IDENTIFYING PROTEINS

Investigators: Wi I liam R. Gray, Wi I liam J. Dreyer

Support: National Institutes of Health, Public Health Service

Some ti rre 'far (?) in the future we shou Id be ab I e to take a minute samp I e of a protein and determine its amino acid sequence by automatic machines which feed the results directly to a computer. This would then calculate the folded structure in three dimensions, and how the protein would react with other molecules, large and small. In short it will have fully identified the protein. We wish to report l;ome minute steps along this heroic path.

Part of the program in our laboratories has been the fractionation of erythrocyte rrembrane proteins. Last year we reported the purification of a protein "K" by extraction of human erythrocyte ghosts with di lute EDTA solution. Its func­tion was quite unknown. Examination of a few milligrams in the automatic protein "sequenator" showed that its amino terminal sequence was Gly.Lys.Val.Lys.Val.Gly. Vol .Asn.Gly.

Subsequently we have compared this sequence with the amino terminal se­quences of al I proteins I isted in the "Atlas of Protein Sequence and Structure." It was found that glyceraldehyde-3-phosphate dehydrogenase CGPCH) from pig muscle started with the sequence Val .Lys.Val .Gly.Val .Asp.Gly, extremely similar to protein K except for the lack of the first two residues. We were then able to find many other identities and near identities between the amino acid compositions of tryptic peptides from the two proteins.

The level of agreement reached was so high as to make tho identification almost certain. Ful I confirmation was obtained by carrying out enzymatic assays on protein K - it was found to be about 95'.t·as active as crystal line human erythrocyte GPCH.

This may seem a bizarre way to identify a protein, but as more sequences become available for comparison and it becorres even simpler to obtain partial

149

sequence Information, it promises to become a valuable tool in some types of pro­tein studies. It is perhaps unnecessary to add that the principle is not limited to amino terminal sequences, but would work as well with any partial sequence such as a hexapeptlde. The whole search procas:Jure can be carried out by computer pro­grams similar to those used by Fitch to search for homologies within and between proteins of known structure.

This work was done in collaboration with Dr. Michael J. A. Tanner, De­partment of Biochemistry, University of Bristol, England.

140. WORK AT JPL SUPPORTED BY THE PRESIDENT'S FUND

Investigator: W i 11 i am R. Gray

Sup port: President's Fund

The President's Fund at Caltech wes es tab I !shed to encourage col I abora­tion between researchers at universities in Southern California and those at JPL. We received a grant from the fund to enable us to work extensively with the ana­lytical mass spectrometer in the Space Sciences Division, under the guidance of~ Dr. H. G. Boettger. The objective of the research was to try to assess how useful mass spectrometry would be tor analyzing amino acid sequences of proteins. We • finished the year cautiously optimistic.

Several groups of workers have previously shown that mass spectrometry can be used for sequencing peptides of up to about 8 residues, with occasional suc­cess on larger ones. It is a long way from peptides to proteins, however, and wo were interested particularly in questions such as whether the mass spectrometer would be faster, more sensitive, more reliable, or simpler, than the wet chemical methods which are the mainstay of protein sequencing at present. It wi II probably come as a shock to many chemists to learn that the mass spectrometer was u rather poor loser in terms of sensitivity. The reason for this lay principally in the nature of peptide spectra, rather than in the absolute sensitivity of the instru­ment. To determine the sequence of a peptide one needs to Identify fragment ions which contain successively larger segments of the chain, usually commencing at the amino terminal end. Unfortunately the intensity of such fragments drops off severe­ly as one goes along the chain. The result is that sensitivity suffers - the spec­trometer can detect less than 0.1 nmole of an acetyl amino acid, but it requires about 150 nmole to give a gCXJd spectrum of a tetrapept!de derivative. This problem is discussed further in a subsequent report CBiology 1970, No. 142).

We have worked mainly with peptides that have been modified by acetyla­tion and permethy lation, procedures developed principally by Lederer and his col­leagues. These procedures render the peptide more volatile, and also simplify the fragmentation pattern, so that interpretation of the spectra is much simpler. In all we have examined over 100 peptide derivatives. Somo synthetic ones have been used extensively in studies of chemical modifications, reaction conditions, etc. Many others were peptides of known sequence isolated in the course of work on var­ious proteins. Complete or partial sequences were established on some smal I pep­tides isolated from tropoelastln (Biology 1970, No. 143), in para! lei with wet chemical methods. In addition we have examined many peptides from the antibiotic alamethicin, and established most of its structure by this means.

Perhaps the most interesting samples examined, however, have been the terminal sequences of proteins (Biology 1970, No. 141). These represent a break­away from the traditional way of first isolating a peptide, then modifying it, and

150

then examining its spectrum. It is in this general direction that we shal J con­tinue moving, hoping to develop methods that are designed with proteins in mind . ' rather than pept 1 des.

14 I. MASS SPECTROMETRY OF PROTEIN AMI NO TERMINAL SEQUENCES

Investigators: WI I I lam R. Gray, Ursino E. del Val le

Support: President's Fund National Institutes of HGalth, Public Health Service

Proteins are too large to evaporate without decomposition in a mass spec­trometer. It is ;iossible, however, to determine their amino terminal sequences in a relatively straightforward way. We have obtained satisfactory spectra from as Jitt10 as I mg of protein, and the use of chemical ionization mass spectrometry may ;iermit us to get away with much Jess material than that (Biology 1970, No. 142).

The essence of the method is a series of simple procedures that are de­signed 7o give a very hydrophobic derivative of the amino terminal peptide, at the sooie time rendering al I other peptides very water soluble. (J) Acetylate the ;iro­tein, Diocking al I free amino groups, including the amino terminus. (2) Digest with a protease of broad s;iecif!city such as subti lisin. A mixture of perhaps hun­dreds of peptides is produced, the amino terminal peptide being the only one which lacks a tree amino group (the digestion creates amino groups at every point of cleav­age). (3) Permethylate under highly basic, but nonaqueous conditions CCH3.SO.CH[ Na+ in anhydrous dimethylsulfoxide). Methyl groups are introduced on al I peptide bonds, hydroxyls, and ~arboxyls giving neutral hydrophobic products; tree amino groups, however, ore converted to quaternary ammonium iodides which are very water­solub le. After permethylation the amino terminal peptide can be Isolated by a simple extraction into chloroform, al I other peptides remaining in thG aqueous lay­er. Mass spectra of the chloroform-soluble material are then obtained without fur-

• ther purification.

An example is shown below. In this case the acetylation was done with a mixture of normal and deuterium-labeled acetic anhydride. This gives rise to twin peaks separated by 3 mass units whenever a fragment ion contains the amino terminal acetyl group (CH3.CO- plus CD3.CO-l. This labeling greatly assists one in deducing the amino acid sequence from the spectrum In many cases. Proteins hav­ing no free amino group (e.g., cytochrome c which is acetylated in vivo) can also be studied by this method; in those cases no doublets are found, but strong singlet peaks are obtained in their place.

8ecause it is applicable to smal I amounts of protein, with or without amino groups, the method promises to be very useful for preliminary characterization of proto ins. If we can separate smal I peptide derl vat! ves on a gas chromatograph we may nave a powerful tool for investigating mixtures of proteins, and fol lowing their purification.

' l-

151

313 186

--~Ace1yl-Me2 Asp I• -CO ·1· Me Leu or Me I le ------tot•~-- Me Val ----1 • -co .

100 Leu+J/e

158

80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400

426 553 682 713 1--- Meleu or Melle Me2 Thr "j' oc~ -CH30H

I'

x2.5 x 2.S'

I I

539

400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700. 720 Electron impact mass spectrum of the amino terminal peptide from human Bence-Jones protein Mi I. Horizontal scale, mass number; vertical scale, intensity. These­quence of the peptide shown is: Asp.I le.Val .Leu.Thr.

142. CHEMICAL IONIZATION MASS SPECTROMETRY OF PEPTIDE DEkl VATI VES

Investigator: W i 11 i am R. Gray

Support: President's Fund National Institutes of Health, Public Health Service

Mass spectrometry is usually thought of as an extremely sensitive ana­lytical tool; samples in the submicrogram range are often sufficient for structural analysis. For peptides, however, there is a severe I imitation in conventional mass spectra which prevents the full sensitivity from being achieved. We have tested severa I peptides with a newer type of mass spectrometer ( "chemi ca I ionization") which promises to overcome this limitation.

The sample is normally present at a vapor pressure of about 10-6 mm of Hg in as clean a vacuum as can be obtained. Electrons with an energy of 70 ev (typically) arc tired at the sample molecules, and sometimes knock out one of the orbital electrons, producing a molecular ion CW). This may undergo rapid decom­position into daughter ions and neutral fragments. The positively charged Ions are removed from the ion source and separated on the basis of their mass to charge (m/el ratio. After electron impact ionization, peptide derivatives give spectra which are composed largely of low mass ions, corresponding to the first I or 2 amino ar.ids in the chain. Peaks which contain information about tho 3rd, 4th and 5th amino acids are usually hundreds of times less intense. To obtain an inter­pretab lo spectrum therefore requires hundreds of times as much material as can be detected, making tho system less sensitive than conventional wet chemical methods. This is clearly shown in the spectrum In the previous report, which is an unusually favorable case Conly a 150-fold drop by the ti tth residue!)

152

In chemlcal ionization the sample vapor pressure is sti 11 maintained at about J0-6 rrrn of Hg. Instead of a hard vacuum, however, there is a background of reagent gas such as methane at a pressure of I mm of Hg. Under these conditions there is I ittle direct ionization of tho sample. Instead, tho reagent gas becomes partly ionized and produces an equilibrium mixture of simple ions such as CH5+ and C2H5+. On collision with a sample mcleculo a proton may be transferred, giving a "pseudomolecular ion" CM+ HJ+. Ionization in this manner is much more chemically specific than is electron impact, and there is much loss energy imparted. Fragmen­tation of the pseudomolecular ion then occurs, and the daughter ions are separated and analyzed as with the electron Impact system. With peptides the cleavages occur almost exclusively at peptide bonds, and there is not a great decrease in the inten­sity of the high mass peaks. This then appears to be an extremely promising ap­proach to sequence analysis, and should enable the high sensitivity of mass spec-t romot ry to be rea I i zed for peptide work.

This work was done in collaboration with Leonard H. Wojcik and Joan H. Futral I of the Department of Chemistry, University of Utah, Salt Lake City.

Reference:

Field, F. H. (1968) Accounts of Chemical Research I: 42.

143. STRUCTURE AND FUNCT 101" OF TRJPOELAST IN

Investigator: WI 11 iam R. Gray

Support: President 1 s Fund l~ational Institutes of Health, Public Health Service

Tropoelastin is the soluble precursor of the protein elastin which is a vital structural component of elastic tissue such as arteries. Failure to convert file precursor into the finished product results in early death due to rupture of blood vessels. The conversion process is an oxidative one, lysine residues of tro­poolastin being oxidized to allysine (a-aminoadipic semialdehydel. Condensation of 3 al lysine residues with I unmodified lysine residue then results in the forma­tion of extremely stable cross-links (desmosine and isodesmosinol which create an insoluble rubber from the previously soluble protein (see Biology 1968, Nos. 7, 118). We have studied the peptides produced by trypsin digestion of tropoelastin at the arginine and lysine residues, of which there are about 3 and 19 respectively in the structural unit of 34,000 rrolecular weight. It is not yet clear whether the protein subunit is of this size, or whether it is composed of 2 such units joined end-to-end as a result of genetic duplication. The results of our study nre very suggestive with regard to the nnture of the structures involved in desmcsine for­mation.

There is a strongly nonrandom distribution of the lysine residues. 50% of them are contained in a series of smal I peptides which otherwise account for only 8% of the molecule. When it is recalled that those small peptides are almost all preceded by lysine as wel I, the distribution is even more dramatic. One alter­native is that al I of the lysines are present in pairs such as -Lys.A.A.Lys- and -Lys.A.A.A.Lys- . The other is that the're are more highly bunched sequences such as -Lys.A.A.Lys.A.A.A.Lys-, sti 11 involving three-fourths of the lysine residues. The compositions, sequences and quantities of the smal I peptides suggest to us that the latter alternative is mere probably correct.

153

The poptides are extremely rich in alanine; glycine is almcst complete­ly locking, although it accounts for almost one-third of the molecule as a whole. 2 of the peptides, Ala.Ala.Ala.Lys and Ala.Ala.Lys, are repeated at least 3 times in the molecule, and poss lb ly 4 times; t1'e others are only present once. We sug­gest that these two peptides ore contiguous in the tropoelastin molecule, so that the structure may be represented diagrammatically as

f--KAAKAAAK l\j\/\/\/\,KAAKAAAK l\j\/\/\/\,KAAKAAAK--j

where the letters Kand A are the standard one-letter symbols for lysine and alanine respectively. The repeated structure could, of course, be more extensive than that shown. In our model the cross-links are envisaged as being widely spread along the protein, so that there Is ample space for the Intervening peptide chains to fold into compact structures. The overal I unit would be I Ike 2 rubber springs on short strings, with 2 loose ends. Knots between the segments of string on different units then produce o 3-dimensional network having the rubber-like properties of elastic. The 3-fold repeat of the two peptides is of particular interest, since 3 is the minimum number of linkage points that are required to form a network - 2 cross-links per monomer would only produce a I inear structure. It is also of interest to note that the desmosine plus isodesmoslne content of mature elastin is fully consist~nt with this proposal. We are testing this model by studying larger fragments ot the molecule, to see whether Rand T sequences are in fact contiguous. •

This work was done in collaboration with Dr. Lawrence B. Sandberg of the Department of Surgery, University of Utah, Salt Lake City.

144. THE DEVELOPMENT OF ADULT MEMBRANE MARKERS ON THE RED BLOOD CELLS

Investigator: lo I tdn A. HlkE!s

Support: National Institutes of Health, Public Health Service

Blood group antigens are membrane-specific molecules on the surface of erythrocytes. The I antigen is a normal human adult antigen which is not expressed in the fetus. The appearance of these new species of molecules on tho surface of red blood eel Is takes place at about the same time as the changeover from the fetal to adult hemoglobin inside the cells.

A comparison and chemical events inside the ing molecular mechanisms. could take place in any of

correlation of the I antigen development to other bio­cel I may result in information relevant to the underly­The appearance of the adult hemoglobin and the I antigen the fol I owl ng orders.

According to one hypothesis, there are tv10 independently differentiating cell lines: one line making fetal hemoglobin predominantly, present in the fetus, and the other line making adult hemoglobin. In this case the I antigen might be exclusively expressed on the eel Is containing adult hemoglobin or may gradually appear en adult eel Is exclusively. Alternatively there could be a gradual appear­ance of the antigen on both cell lines. According to another hypothesis only one stem eel I I ine differentiates to produce hemoglobin. Ce! Is would gradually change from fetal to adult hemoglobin in an analogous way to enzyme induction. The ex­pression of the I antigen could take place prior to, simultaneously with, or fol­lowing the synthesis of adult hemoglobin. Lastly, it is also possible that the I anti gen expression is contra II ed by other deve I opmenta I events and therefore is

154

independent from the control mechanisms of erythropoiesis.

The underlying molecular events are very different in each case. Jn the first case the eel I would become one or the other type and thus would require a molecular mechanism for committed, nonmodulated, chromosomal control so as to main­tain separate cell lines. In the other case, modulated chromosomal regulation would be necessary to gradually turn some genes off and to slowly activate the genes for the I antigen and adult hemoglobin. Quantitative experimental studies of the degree of correlation of this surface marker with the type of hemoglobin within eel Is could distinguish between these mechanisms.

Erythrocytes from infants of different ages have been fractionated accord­ing to their I agglutinabi lity. The hemoglobin types in these cells have been quan­titated using ion exchange chromatography and alkali denaturation. In addition, single eel Is were studied with the aid of microspectrophotometry. Cel Is were re­acted with purified anti-I antibodies and then fixed to a quartz slide. These cells are extracted with a low pH buffer which specifically removes adult hemoglobin. By measuring the optical absorption of a single eel I before and after extraction it is possible to classify the inner content of the cell.

The results indicate that the rate of expression of the I antigen is inde­pendent from the rate of appearance of adult hemoglobin. This could be best ex­plained by two independent modulatory regulations, whereby the enzymes involved in the biosynthesis of membrane-specific molecular entities are regulated independent­ly from the control of the hemoglobin subunit biosynthesis.

The biosynthesis of ABO blood group antigens have been wel I studied. Somo earlier observ~tion Indicated that the I antigen may have structural relations to the ABO system. Cel Is (from postnatal infants) fractionated with respect to their I agglutinabi lity have also been studied with 1251 labeled anti-A antibodies in order to compare the rate of A antigen appearance with the rate of I antigen ex­pression. The expression of the A cntigen is control led by the enzyme N-acetyl-

•galactosamine transferase. The synthesis of this enzyme is under the regulation of the erythroid eel I I ines, therefore a correlation between the I and tho A 'anti gens wou Id give evidence for a s i mi I ar regu I ati on of the I anti gen expression. The results with the labeled anti-A antibodies indicate such a mechanism.

Erythrocyte membranes were fractionated and the ABO and the I antigen activities were located in several fractions. Al I the molecular tractions contain­ing I antigen also contained ABO blood group activity, but not al I the fractions with ABO antigens had I activities. These observations indicate that there might be two different molecular carriers for these antigens with ABO and I present simultaneously on only one of the carriers.

Reference:

Dreyer,

London, I. M. et al. ( 1967) Devel. Biol. Suppl. I: 227.

PlBLICATIONS

w. J., Laico, M. T., Ruoslahti }. E. I., and Papermaster, D. S. ( 1970) The fundamental polypeptide subunits of biological membranes: A large pep­tide or "miniprotein." Fed. Proc. 29: 606 (abstract).

I.•.· .

ii I'. ~g

i I

155

Dreyer, W. J. ( 1969) A proposed new and general chromosomal control oochanism for commitment ot specific CGI I I ines during development. Symp. on Develop­mental As;iects ot Antibody Formation, Prague, June 1969. Sterzl (ed.), Academic Pross, N.Y. (in pr~s).

Gray, W.R. and Smith, J. F. (1970) R;opid sequence analysis of sm<:lll pe;itidos. Anni. Biochem. 33: 36.

Gray, W.R. and del Val le, U. E. < 1970) Application ot mass spectrometry to protein chem is try. I • Method tor amino-term i na I sequence ana I ys is ot prate ins. BiochGmistry 9: 2134.

Gray, W. f<., Wojcik, L. H., and Futrel I, J. H. ( 1970) A;ipl ication ot chemical ionization mass s;iectroscopy to tho amino terminal sequence analysis ot proteins. American Society tor Mass S;iectrometry, June 1970.

Laico, Marion T., Ruoslahti, E. I., Papermuster, U.S., and Uroyer, W. J. (1970) Isolation ot the fundamental polypeptide subunits of biological mem­branes. Proc. l~at. Acad. Sci. <in press).

156

BIOLOGICAL SYSTEMS ANALYSIS

NEUROPHYSIOLOGY

PSYCHOBIOLOGY

DROSOPHILA GENETICS

Booth Computing Center

Kerckhoff -Alles

(Third Floor)

~-----

I 1.".-.. ·.:.•.'.• •. :.·.-.· ..• -•. •· ; .E••·

Professor: Derek H. Fender

* * Graduate Students: Antony W. Goodwin , Robert N. Kavanagh , Cary Lu * -... , *

Laboratory Ai des: A I ton Story , Francis Wal yak l

145. THE HUMAN OCULOf-OTOR SYSTEM

I nvestl gators:

Support:

* Derek H. Fender, Antony W. Goodwin

·National Institutes of Health, Public Health Service

157

Target tracking and fixation experiments are being used to examine the control mechanisms of human eye movements. Linear systems techniques are not being used because of their I imitations and unrealistic description of the known physiology; instead, non-linear techniques are used with the prime objective of understanding the mechanisms of visual processing, and secondary objective of pro­posing a realistic model. Targets of carefully chosen color, intensity, shape and background Intensity make it possible to determine to sc:me extent what processing of the stimuli for fixation and target fol lowing ls occurring at the retlnal level, and what at higher levels.

146. VISUAL EVOKED POTENTIALS IN MAN

Investigators: * Derek H. Fender, Robert N. Kavanagh

Support: National Institutes of Health, Public Health Service

The potential field arising from the human visual evoked response has been measured with considerable resolution through the use of a 50-channel record­ing system. These surface potentials, when displayed in movie form, show that dur­ing certain intervals of the normal visual evoked response the equipotential con­tours are indicative of some simple equivalent electromagnetic generators.

Models of the head as a homogeneous sphere or as concentric spherical shells are being studied to relate the surface measurements to underlying cortical activity. Techniques for locatlng and characterizing equivalent dipole generators have been produced and are being applied to the visual evoked response data.

Reference:

Fender, D. H. and Kavanagh, R. N. ( 1970) Functional activity of popula­tions of neurons in the human visual cortex. Association for Research In Ophthalmology, Spring Meeting, Sarasota, Florida.

147. STEREOPSIS AS A FUNCTION OF COLOR AND LUMINANCE

Investigators: Derek H. Fender, Cary Lu

Support: National Institutes of Health, Pub I ic Health Service

This study of the interaction of color and luminance with stereopsis

* Division of Engineering and Applied Science, California Institute of Technology.

158

uti I izes primari Jy the random-dot stereo patterns which are free of tho normal sec­ondary cues to depth perception. By using a 4-channel optical system, these pat­terns can be viewed as squares of one color and luminance against squares of another color and luminance rather than black and white.

It is found that unambiguous stereopsis requires a luminance difference between the squares; patterns with uniform luminance but strong differences in color (monochromatic I ight is used) do not permit stereopsis. Furthermore, retinal ri­valry in color over the entire pattern does not prnclude the possibility of stere­opsis, providing the luminance contrast is great enough. Conversely, rivalry in luminance with identical colors prevents stereopsls at al I contrast ratios.

Use of stereo perspective drawings of geometrical objects with all their attendant secondary cues permits a percept of a 3-dimensional object over a wide range of rivalrous luminances and colors where random-dot stereo patterns cannot be perceived in depth. This suggests that tho classical demonstration of stereo fusion of one image with white lines against a black background and another image with black lines on white, is a special case and cannot be generalized to the nor­mal performance of the visual system.

PUBLICATIONS

Lehmann, D. and Fender, D. H. ( 1969) Averaged visual evoked potentials in humans: Mechanism of dichoptic Interaction studied in a subject with a split chiasma. Electroenceph. cl in. Neurophysiol. 27: 142-145.

Lehmann, D., Kavanagh, R. N., and Fender, D. H. C 1969) Field studies of averaged visually evoked. EEG potentials in a patient with a split chiasm. Elec­troenceph. clln. Neurophysiol. 26: 193-199.

St. Cyr, G. J. and Fender, D. H. (1969) The interplay of drifts and flicks in binoc-• ular fixation. Vision Res. 9: 245-265.

St.>Cyr, G. J. and Fender, D. H. ( 1969) Noni inearities of tho human oculomotor system: gain. Vision Res. 9: 1235-1246.

st. Cyr, G. J. and Fender, D. H. (1969) Nonlinearities of tho human oculomotor system: time delays. Vision Res. 9: 1491-1503.

I

l I

l<esearch l\ssoci ate: Ken-I chi Naka

• Senior f{csoarch Fol low: Patrick VI. Nye

\ t<oseorch Fe I I ovJ: Nob uyosh i Matsumoto

159

Our aim is a comprehensive understanding of the functional relations ex­isting among tho various types of neurons in the vertebrate retina which ultimately can lead to a model of the vertebrate retina. This can only be accomplished by a close I iaison between the biological experimentalist (who provides the datal and tho theoretician (who binds the results into a unified structure), each refining his opproach by mutuol interaction. As the ti rst stop toward this aim, experiments are being carried out to gather information on the dynamic behavior of neurons in the retina.

148. THE FUNCTION OF S-POTENTIAL IN THE VISUAL PROCESS

I nvosti gator: Kon-I chi Naka

National Institutes of Health, Public Health Service

Though the properties of the S-potential (responses from tho row of hori­zontal eel Isl have now been well established, the function ot tho potential in the visual process has yet to be explained. The retina of the channel catfish has pro­vided a chance to examine this problem as tho catfish retina is rather simply orga­nized functionally and probably morphologically too.

Our results can be summurized by the tol IONing crude scheme. There is a box in the external plexitorm layer, probably the bipolar coll, which compuros tho input from the receptors and inputs trom the hori zonta I ce 11 s. It the input from tho horizontal eel I is larger than that from tho receptor, the comparator box sends a signal to produce a sustained discharge. This signal reflects tho waveform of tho S-potential 11hen it is passed through a low-pass ti ltcr. V/hen tho signal from tho receptor is larger than that from the horizontal eel I, tho resulting re­sponse is transient. There are several combinutions of the two inputs tor this scheme. For al I of those possibi llties, the actual results agree with tho predic­tion. Tho site of this interaction between the receptor signal and the horizontal eel I signal is probably tho cone podlcle where tho three components of this scheme make synaptic contacts.

References:

Naka, K.-1. ( 1969) Biophys. J. 9: 845-859.

Naka, K.-1. and Nye, P. W. (1970) J. Neurophysiol. (in press).

* Division ot Engineering and Applied Science, California Institute of Technology.

160

149. ANALYSIS OF S-POTENTIALS FROM THE CHANNEL CATFISH RETINA

Investigators:

Support:

* Ken-lchi Naka, Patrick W. Nye

National Institutes of Health, Public Health Service

Under moderately dark-adapted conditions the catfish (lctalurus punctatus) $-potential is generated by a signal originating from a cone pigment with an absorp­tion maximum at 625 nm. The absorption curve closely fits that of cyanopsin. The voltage-log intensity curve (curve of V plotted against log I) can be precisely fitted to a logistic function. Thus the variables of the catfish S-potential can be visualized in terms of a solid figure having three axes representing wavelength, log intensity, and voltage. The plane formed by wavelength-log intensity is the log spectral sensitivity of the pigment involved. The voltage-log intensity plane is the logistic function displaced acccrding to the spectral sensitivity curve. The wavelength-voltage plane represents the height of the S-potentials obtained by spectral flashes of equal energy. Dark adaptation shifts the logistic function (obtained by flashes of a given wavelength) toward dimmer I ight without any change in the parameters of the function including the d.c. level.

References:

Naka, K.-1. (1969) Biophys. J. 9: 845-859.

Charlton, J. S. and Naka, K.-1. ( 1970) Vision Res. (in press) .

•

* Division of Engineering and Applied Science, Cal lfornia Institute of Technology.

i lliL 161 '1!>···

~

Professor: Anthonie Van Harreveld

Senior Research Fel ICltl: Eva Fitkova

Research Fel ICltls: Nachum Datny, Janett Trubatch

Graduate Student: Lee-Ming Kow

Research Assistants: Ruth E. Estey, Josephine Pagano

Much of the work during the last years was involved with the distribution ot 1vater and electrolytes in the central nervous system, and with the mechanisms which change this distribution. Furthermore, Investigation ot the mechanisms which result in hypertone and spastlcity was continued.

150. CONCOMITANTS OF SPREADING DEPRESSION AND ASPHYXIATION OF CENTRAL NERVOUS TISSUE

I nvestl gators: A. Van Harreveld, Eva Fitkova

Support: National Institutes of Health, Public Health Service •

The similarity ot the ccncomitants of spreading depression and asphyxia­tion ot the cerebral cortex suggests that they are caused by a common mechanism. Both spreading depression and asphyxiation are acccmpanied by a slowly developing surface negativity ot the cortex and by a marked increase.In tissue impedance. In both Instances extracellular chloride and water accumulate Into certain cellular elements. A loss of extracellular material can be shCltln in suitable electron mi­crographs.

It has been proposed that the underlying mechanism ot these changes Is an increase In sodium permeabi I tty ot the plasma membrane ot the eel lular structures involved. The disturbance of the double Donnan equilibrium then can account tor the transport of extrace 11 u I ar sodium ch lorl de and water into these structures. Since the current used in impedance measurements is carried mainly by extracellular electrolytes this also explains the impedance increases. The resulting depolariza­tion ot the tissue elements accounts tor the slCltl potential change.

It was postulated that the Increase in ion permeabi I ity ot the plasma membrane is caused by a release ot glutamate from the lntracel lular compartment into the extracellular space. The chicken retina exhibits spreading depression when stimulated with direct current or with a potassium chloride solution. After being charged with 14c-glutamate this tissue releases the label under these cir­cumstances. The retina also releases the 14c-glutamate when stimulated with un­labeled glutamate. This suggests the existence ot a process with positive feed­back. When a stimulus tor spreading depression releases some glutamate in the extracellular space this will cause an increased glutamate permeability and an additional release of the amino acid until a maximum is reached. This mechanism may be at the basis ot spreading depression and explains the al I-or-none character of this phenomenon.

To account for a glutamate release during asphyxiation of the cerebral cortex it has been postulated that some glutamate is released constantly from the intracellular compartment but that this is actively transported back into the cells and fibers of the tissue. Asphyxiation, after the energy reserves are used up,

162

makes the active transport impossible, leading to an accumulation of glutamate in the extracellular space which at a certain point would set in rrotion the mechanism with positive feedback leading to a maximal release of the amino acid.

It has been attempted to produce in tho isolated retina the asphyxia! changes resembling spreading depression studied in detal I in the cerebral cortex. In the superfused preparation this has been found impossible. Neither Oz-lack nor the application of metabolic inhibitors caused the transparency change which is characteristic for spreading depression in the retina, or a release of glutamatG. It was postulated that the small amount of glutamate released continuously is re­moved even without the benefit of active transport by diffusion into the superfu­sion fluid. The concentration would at no time reach a high enough level to set the release with positive feedback in rrotion. When the retina is superfused with a glutamate solutlon of insufficient ooncentration to cause spreading depression, diffusion may be impaired and it can be postulated that under these circumstances only the assistance of active transport can prevent the glutamate release with pos­itive feedback. ln such a preparation a metabolic inhibitor might cause a marked glutamate release. Evidence has Indeed been obtained that under such circumstances the application of dinitrophenol increases the glutamate release.

151. EFFECT OF GLUTAMATE ON CORTICAL TISSUE

Investigators: A. Van Harreveld, Eva Fifkova

Support: Natlonal Science Foundation

It was postulated that glutamate released in the extracellular space causes the profound changes ln central nervous tissue which are the concomitants of asphyxiation and spreading depression. Glutamate was deposited eloctrophoret­i ca I I y from a mi crop i pette in the cerebra I cortex of rats . Those res u I ts wore

•studied with tho I lght and electron microscope.

Glass micropipettes usually fi lied with 150 mM sodium glutamate wore in­troduced about 0.5 rrrn deep in the cortex. Currents from 5 to 0.02 µA wore passed through the pipette for times varying between 5 minutes and I hour. The brain was later fixed by glutaraldehyde perfusion. The light microscope showed in cortex fixed shortly after the perfusion a spherical area of grossly swollen structures, sharply delineated from the surrounding tissue. The cells in this area were shrunken. When fixed 24 hours after the glutamate injection asimilar situation was found with some regression of the swelling. When the fixation was postponed tor 2 or 3 days the tissue elements were sti 11 enlarged. A number of rather darkly stained col Is had appeared. When fixed after I or 2 weeks, the tissue elolll3nts were only slightly enlarged. Some dark eel Is wore sti Ir present.

The electron micrographs of cortices fixed shortly after the glutamate injection showed that many of the swollen structures are dendrites. However, gila and presynaptic terminals are also often grossly swollen. The cells in the gluta­mate lesion are electron-opaque. In electron micrographs of preparations fixed after 2 to 3 days tho swelling had regressed. Glial· elements surrounded the dark neuronal structures.

The magnitude of the glutamate spot was found to be proportional to the cube root of the current in a series of experiments in which the pipettes wore fl I led with glutamate of the same concentration and the current was ~assed for the same length of time (I hour).

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These experiments show the profound effect ot glutamate on central ner­vous tissue and cire in keeping with the postulate that ttie releose of glutamate is Q primory I ink in the chain of events resulting in tho concomitants of asphyxiation and sprouding depression.

1~2. GLUTAMATE RELEASE DURll<G GLUTARALDEHYDE FIXATION

Investigators: A. Van Harreveld, Eva Fifkova

11ational Institutes of Health, Public Health Service

Tho perfusion ot the cerebral cortex with one of the common fixatives tor electron microscopy, glutaraldohyde, is accompanied by some of the concomitunts of us~hyxiution and spreading depression. There is a simi lur increase in tissue imped­unco, an accumulation of chloride into the apical dendrites ot the cerebral cortex and in suitable electron micrographs a loss at extracellular material was demon­strated. It can be postulated that these changes aro produced by tho re lease ot glutamate which is believed to cause the concomitants ot asphyxiation and spread­ing depression.

The superfusod retina charged with 14

c-labolod glutamate was used to in­vestigate this ~ossibi lity. One minute ~ortions, first of the physiological solu­tion, later of the fixative flowing over the retina, were collected and analyz8'd for amino acids. Changing from the salt solution to tho fixative (2.5% glutaral­dehyde) usually caused an increase in transparency of the retina which is charac­teristic tor spreading depression. During the supertusion with a physiological salt solution some glutamine but very I ittle glutamate is released from the retina. During the glutaraldehyde perfusion two relatively large nCM substances appeared which were neither glutamine nor glutamate but seemed to be a compound ot these amino acids and glutaraldehyde. They do not shov1 the ninhydrin reaction. A further identi ti cation is attempted. The results so tar suggest that during glutaraldehyde superfusion glutamate is released which then forms~ compound with tho fixotive.

153. THE EFFECT OF l'ONOCULAR DEPRIVATION ON AXOSOMATIC CONTACTS OF THE VISUAL CORTEX

I nvosti gator: Eva Fitkova

Support: National Science Foundation

Uni lateral visual deprivation (of 2-month duration) causes significant chongos in the volume and eel I density in contra lateral visual centers and utfocts the dendritic spine_s ot corticul neurons. Detectable changes huvc been obsorvod attor 10-days deprivation and the maximum 1·1as reached attar 30 days. No recovery from tho deprivation effect was observed utter reopening of the originally closed eye.

In electron micrographs ot the visual cortex connected with the deprived eyo a change in density and sizo of synapses wos found. These changes were mostly located in cortical layers 11 and 111; layer IV and the upper part ot layer V being less uftected. Since axodendritic synupses were examined exclusively the oim ot the ~resent investigation was to study eventual changes ot axosomatic synapses.

Tho material was derived from 6 rats in which the Iott eye had been sutured at 14 days ot ago. 2 months later tho brain was perfused with

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glutaraldehyde and embedded in Maraglas. Three normal rats perfused at the age of 10 weeks were used for controls. From cortl cal I ayers I I, 111, IV and from part of layer V thin sections for electron microscopy were cut.

The criterion used for distinguishing synapses were the presence of synaptic vesicles in the axonic terminal and the typical membrane thickening. Only 2-3 synaptic contacts were found per cell, not enough to count their density. The length of synaptic apposition of 20-30 synapses was measured in each of tho layers. Right and left visual cortices were compared. A total of 1,565 synapses were mea­sured in experimental animals and 742 in controls.

In al I experiments but one the synapses connected with the I id-sutured eye were signiflcantly smaller than those on the normal side; the average differ­ence being 23%. In the controls no consistent differences were found between the right and left side. On the deprived side a large percentage of the synapses have a diameter of 0.1 to 0,2 µ,whereas on the side connected with the open eye synap­ses of 0.3 to 0.4 µprevail. In the control group there were no consistent differ­ences in the size classes between the right and left visual cortices.

The possibi I ity was considered that the changed axosomatic syna;:ises pre­vai I in certain cytoarchitectonlc layers. The whole region of layers 11, 111, IV and part of V was by alternating thick and thin sections divided in 8 levels which were 80 µapart. The mean size of the synapses of al I the experimental and of all the control animals was calculated for each of the levels. Significantly smaller contacts were found in the cortex connected with the deprived eye at al I levels. The changes do not seem to be concentrated in any of the cytoarch i tectonic layers. No significant changes were found In the controls.

Contacts with flattened as wel I as with round vesicles were found in the nxosomatic population with a prevalence of the first ty;:ie (about 85%). The possi­bility was considered that both types of synaptic contacts are not affected in the same way by visual deprivation. The synapses with round and oval vesicles were t"erefore measured separately. The size of the contacts with round vesicles was more affected by the deprivation than the size of the contacts with flattened ves­icles, in layers I I and IV. This difference in the size reduction of round and oval vesicles is statistically significant. In layer 111 there was no difference between the two types. The percentage of contacts containing round vesicles was significantly higher on the deprived side in layers I I and IV indicating a larger reduction of the flattened vesicle terminals or a change from flattened to round vesicle endings.

154. THE EFFECT OF VISUAL DEPRIVATION ON SYNAPTIC CONTACTS OF THE RETINA

Investigator: Eva Fi fkova

Support: National Science Foundation

Monocular visual deprivation causes changes in the synaptic contacts of the visual cortex as was described in the preceding abstract. It was assumed that the visual cortex is not the only visual,structure where deprivation would cause changes in the ultrastructure. The retina was examined because of its uniform laminar arrangement which makes it possible to choose homologous layers for com­parison. The inner plexiform layer of the retina, where the axons of bipolar and amacrlne eel Is make contact with each other and with the dendrites of gang I ion cells, seems to offer a favorable object tor the study of the effect of deprivation

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on the synaptic organization.

The lids of II albino rats (14 days old) were sutured on the right side and animals kept for 8 weeks. The eyes were then fixed by glutaraldehyde perfusion In urethane narcosis and the eye bulbs were carefully removed. The occipital pole was dissected and transferred to 2% osmic acid in the phosphate buffer for 2 hours, By that time the tissue was hard enough to permit the isolation of a strip (2-3 nrn wide) of the sclera with the retina attached. From a block next to the optic tract thin sections for the electron microscope examination were prepared. Electron mi­crographs of the Inner plexiform layer are being studied.

155. INPUT TO tlOTONEURONS DURING SPASTIC STATES

I nvestl gators: Janett Trubatch, A. Van Harreveld

Support: National Institutes of Health, Public Health Service

In the lnvestlgatlons of spastlclty during the last decade emphasis has been placed on the peripheral structures involved in the myotatic reflex which is in general involved In this phenomenon. The purpose of the present experiments is to examine both the peripheral and central activation of motoneurons In preparations exhibiting 4 different kinds of rigidity and to distinguish between the effec~s of suprasplnal, lntraspinal and peripheral influences.

The reflex nature of intercol I icular decerebrate rigidity was ti rst dem­onstrated by Sherrington. In such preparations supraspinal mechanisms activate the gamma efferent motoneurons which cause a contraction of tke intrafusal fibers in the muscle spindles, ellcltlng trains of Impulses in the la fibers which in turn monosynaptically activate the alpha motoneurons resulting in a muscular contrac­tion. In preparations decerebrated anemlcal ly, deafferentiation does not abolish the rigidity and therefore the suprasplnal output seems sufficient to fire the alpha motoneurons without the peripheral input through the la fibers. Jn addition, two types of rigidity result from asphyxiation of the spinal cord; "secondary" tone appears within a few hours of asphyxiation of 30-60 minutes and lasts up to a couple of days, whl le "late" tone begins 10 days to 2 weeks after 30 to 35 minutes asphyxiation and lasts the remainder of the animal's I ife. Both of these asphyxia I tones have been shown to be reflex tones, dependent on an input from the la fibers. In secondary tone a temporary functl onal recovery of i nterneurons seems to be in­vol ved which enhances ganrna activity. Late tone, however, is not a gamma tone, but is believed to be due to an increased excltabl lity of the remaining motoneurons, while spindle contracture may provide an enhanced Input through the la fibers.

Previous experiments showed that the stretch reflex is elicited in mid­brain decerebrate preparations and in animals with secondary tone with only moder~ ately high la frequencies. In spinal animals, however, reflex activity could only be produced during very rapid stretch. If al I that is needed for rigidity is a dis­charge in the la fibers of a sufficiently high and stable frequency, then it should be possible to replace the gamma innervation by a sufficient stretch of the entire muscle. Since this is impossible, the gamma fiber activation does not seem suffi­cient to fully explain the rigidity of midbrain decerebratlon. An additional In­flow of Impulses either from the supraspinal structures (decerebrate rigidity) or from spinal interneurons (secondary tone) seems to be needed to produce rigidity.

To verity the existence of such an additional Input the potentials of femoral motoneurons are recorded with an intracellular technique. An extensor muscle must be used since decerebrate rigidity is mainly an extensor tone. These

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neurons are found in the region of the spinal cord where focal potentials elicited by antidromic stimulation of the femoral nerve can be recorded. The origin of the excitatory and inhibitory postsynaptic potentials recorded from these cells can be determined by el iminatlng first the peripheral input by severing the femoral nerve and then the supraspinal input, in the two decerebrate preparations, by spinaliza­tion. The effects of the modification of decerebrate rigidity by changing the position of the heud (reflexes of Magnus and deKleyn) wi 11 also be examined.

The excitatory activity in the motor cells is expected to decrease mark­edly u;ion deafferentiation in al I but the anemic decerebrate preparations. Spin<Jl­ization should decrease the remaining activity in both intercol I icular and anemi­cally decerebrated preparations. Deafferentiation in preparations with secondary tone may not eliminate the background activity which could be derived from an ab­normal interneuronal activation. The individual postsynaptic potentials during late tone are expected to be larger than those during secondary tone due to the enhanced excitability of the motoneurons.

156. TRANSM I SS I DN DF HI G-1 FREQUENCY TRAINS DF I MP ULSES

l nvesti gator: Janett Trubatch

Support: National Institutes of Health, Public Health Service

This investigation was instigated by the finding that upon application of procaine to a muscle nerve the disappearance of the stretch reflex seemed attrib­utable to the decreased abi I ity of nerve fibers from the stretch receptors (la) to transmit high frequency trains of impulses. Recording from the appropriate dorsal and ventral roots of the cat's spinal cord while stimulating the sciatic nerve showed a decrease in the height of the later action potentials of a train of im­pulses with respect to the ti rst, as the impressed frequency was increased from 50/second to 500/second. Procaine abolished or severely reduced the later spikes in the high frequency trains before the first spike or an entire low frequency train was affected.

The reason for these effects cannot be inferred from the behavior of the entire nerve, containing thousands of fibers, since the effects of reduction of am­plitude due to relative refractoriness cannot be distinguished from a dropping-out of a percentage of the fibers. With a train of impulses it is also possible that there exist additive effects of the stimuli. In a procainized region an impulse arriving during the refractory period of the previous spike may increase the refrac­toriness of the nerve for the fol lowing one. Due to the al I-or-nothing relationship, ha.;ever, the main effect outside the procainized region is a reduction in the firing rate of the fibers, unti I they are completely blocked. Therefore information about the actual effects of high frequency stimulation can best be obtained from a single fiber.

The sciatic nerve of the frog is suitable for this investigation since a single fiber can be dissected free over a smal I region and the remaining fibers cut. The resulting preparation is placed in a moist chamber, the dissected region in a smal I pool of Ringer's solution. The entire nerve is then stimulated at the proximal end while recording from the"-distal end. The single fiber remains vi ab le for hours.

It is planned to observe the effect of progressively increasing tho ap­plied frequency on the action potential of the single fiber. An equal diminution of the second and al I subsequent action potentials and then a dropping-out of

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alternate spikes as the frequency is increased would imply that each spike Is af­fected only by the one previous to itself. The occurrence of additive effects would show themselves in any variation of spike amplitude after the second spike or in an irregular pattern of impulses. The ~ffects of procaine on a train of Impulses can then be investigated by replacing the pool of Ringer's surrounding the single fiber by the drug. Pre I iminary results indicate that additive effects are minimal, procaine does not effect the amplitude of the spike outside the procalnlzed region, and the all-or-nothing law is valid within the procalnized region.

Si nee It has been suggested that removl ng the connect! ve tissue and other axons from the vicinity of a single fiber drastlcal ly affects Its mi lieu and there­fore Its properties, it wl II be attempted to repeat these experiments on a single fiber impaled by a glass mlcroelectrode.

157. ELECTRON MICROSCOPY OF INSECT GANGLIA

Investigators: Nachum Dafny, A. Van Harreveld

Support: National Science Foundation

Gangl la of the leech (Mooreobdel la fervldal were Investigated previously using the technique of freeze substitution. In this material the nerve eel I bodies were surrounded by mu I tip le g 11 a lame! I ae separated by s I its f i I led wl th extra-cel I u lar material. In the connectives the nerve fibers and gila elements were separated by siml Jar silts. There was in general more extracellular space in the micrographs of the leech central nervous system prepared by freeze substitution than In those of material fixed with conventional methods.'

In the present Investigation freeze substitution was applied to the gan­glion chain of roaches CBI ate I la germanlca, Periplanata amerlcanal. Pre I iminary results revealed less extracellular space than observed In the leech.

158. THE EFFECT OF CALCIUM ON THE IMPEDANCE OF CENTRAL NERVOUS TISSUE

Investigators: A. Van Harreveld, Nachum Dafny

Support: National Science Foundation

Adey and his coworkers Injected a near-Isotonic calcium chloride solution in the ventricle system of rats and observed a marked decrease of the electrical impedance of the surrounding tissue. Since the tissue resistance, as mentioned above, Is related to the amount of extracellular electrolytes, the resistance decrease might indicate an increase of extracellular material. If this Is the explanation It would be possible to demonstrate such an Increase in electron mlcro­graphs of the brain tissue prepared by freeze substitution. However, the struc­tures studied by Adey et al. are not suitable for freeze substitution. It has therefore been attempted to show the Impedance drop .due to calcium application in the cerebral cortex, which can be freeze substituted.

A hanogeneous current field ( 1000 cycle AC) was estab I !shed in a tissue slab consisting of the rabbit's cerebral cortex and the subcortical white matter. Through this region a probe was passed which measured the potential at various depths in this current field. The slope of the current-depth relationship is a measure of the specific resistance of the tissue. In a normal cortex this slope is

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smaller in the superficial about 2 mm than in the deepest few tenths of an mm of the slab. This is due to the difference in specific resistance in gray and white matter. After treatment of the cortex with an isotonic calcium solution for I hour the graph of the depth-potential relationship consisted of 3 parts; a superficial part (about 0.5 11111) of relatively low resistance, the next part (about 1.5 11111) of higher resistance, and the deepest part consisting of the white matter of still higher resistance. Calcium chloride in relatively high concentrations thus seerrs to cause a drop of resistance in the cortex also. The application of a histochem­ical method for calcium showed that this ion penetrates about 0.5 mm into the cortex after bathing the cortex for an hour with an isotonic CaClz solution.

Electron micrographs prepared by freeze substitution of cerebral cortex treated with the CaClz solution showed a much enlarged extracellular space as com­pared with the normal cortex. This space was present especially between the pro­f! les of smal I tissue elements (nonmyel inated axons). The extracellular space was in many electron micrographs tilled with a tine granular, rather electron-opaque mater! al.

These observations support the postulate that large changes in tissue impedance are in general caused by changes in the amount ot extracellular material.

This work was done in collaboration with Dr. Fahmy I. Khattab, Zoology Department, Ain Shams University, Cairo, Egypt.

Reference:

Wang, H. H. and Adey, W. R. ( 1969) Exptl. Neurol. 25: 70-84.

159. CHANGES IN INTRACELLULAR ELECTROLYTES OF CHICKEN RETINA DURING SPREADING DEPRESSION

Investigator:

Support:

Lee-Mi ng Kow

McCal lum Fund Genera I Fund

From the results of experiments on the changes of cortical impedance during spreading depression and of histochemical studies, it has been suggested that the initial step of this phenomenon is the entrance of extracellular sodium ions into the eel Is. This suggestion has not been proved directly. The present investigation attempts to provide more direct evidence by substituting Na+ with u+ in the bathing solution. It is known that extracellular u+ behaves very similarly to extracellular Na+, but once Inside the neuron it can not be pumped out by the "Na-pump" but only leaks out very slowly. Therefore, if the entrace of the Na+ be the initial step of spreading depression, then with al I Na+ replaced by u+ the cells with spreading depression will trap u+ in their intracellular compart­ment.

In a single experiment both retinas ot the· same chick were used which initially were not removed from the bisected eye. One ot them, serving as control, was bathed in a physiological solution'eontaining in addition to u+, Mg++, which prevents the occurrence ot spreading depress I on. The other was immersed in a s i m­i lar solution in which the Mg++ had been replaced by an equal molar amount ot cho-1 ine, al lowing spreading depression to occur. After some time both retinas, one' exhibiting spreading depression, the other not, were separated from the pigment epithelium. The isolated retinas were then washed with isotonic sucrose solution

f I

r

I

I

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for the same period of time to remove extracellular electrolytes. After the wash­ing, the excess fluid vias removed and the retinas weited, digested and properly diluted for further analysis. The contents of Nil+, K and u+, now only confined to the intracellular compartment, were·'Onalyzed with the flame spectrophotometer.

The Na+ contents for both groups are the same and very low. These sodi­um i ans may represent the bound Na+, for they cannot be rep I aced by u+, nor are they affected by spreading depression. The u+ content of the retl nas with spread­ing depression Is significantly higher than that of the retinas without. The dif­ference is approximately 30 meq/kg intracellular water. The situation for the K+ content is reversed, but the difference is smaller, approximately 15 meq/kg intra­cellular water. Thes·e results indicate that during spreading depression an appre­ciable amount of Lt+ gets Into the cells, replacing Na+ and part of K+ which are extruded into extracellular space. The sum of the Na+, K+ and Li+ content for both groups is also significantly different, with the spreading depression group larger than that of the controls. It we assume that the content of electrolytes other than Na+, K+ and Lt+ Is very small and can be neglected, and that the lntracel lular fluid is isotonic with the fluid in which the retina is bathed, then the intracel­lular compartment of the retina would swel I during spreading depression to a vol­ume 108.5% of the controls.

Since Na+ behaves slmilarly to LI+, these results strongly suggest that during spreading depression sodium moves from the extracellular space into intra­cellular compartment together with c1- and water. Part of the entering Lt+ may also replace K+ causing its extrusion into extracellular space.

PUBLICATIONS

Ahmed, N. and Van Harreveld, A. (1969) The iodide space in rabbit brain. J. Physiol. 204: 31.

Fifkova, E. and Van Harreveld, A. (1970) Effect of glutamate on the corpus striatum of the chicken. Fed. Proc. 29: 264.

Fifkova, E. ( 1970) The effect of monocular deprivation on the synaptic contacts of the visual cortex. J. Neuroblol. I: 285.

Trubatch, J. and Van Harreveld, A. (1970) Effect of procaine on asphyxia I rigidity. Exptl. Neural. 27: 76.

Van Harreveld, A., Khattab, F. I., and Steiner, J. (1969) Extracellular space in the central nervous system of the Leech, Mooreobdella fervida. J. Neuro­b i o I • I : 23.

Van Harreveld, A. and Fifkova, E. (1969) Glutamate release from the retina during spreading depression. 2nd lnternat. Meeting lnternat. Soc. Neurochem., R. Paoletti, R. Fumagal Ii and C. Gal Ii (eds.), Milano, Italy, p. 208.

Vun Harreveld, A. and Steiner, J. (1970) Extracellular space in frozen and ethanol substituted central nervous tissue. Anat. Rec. 166: 117.

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Van Harreveld, A. (1970) A mechanism for fluid shifts specific for the central nervous system. In: H. T. Wycis Ced,), Current Research in Neurosciences. To;iical Prob I. Psychiat. Neurol. 10: 62.

Van Harreveld, A. and Niechaj, A. ( 1970) A ;iossibly monosyna;itic component of the dorsal root ;iotentlal. Brain Res. 19: 105 .

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Professor: C. A. G. Wiersma

Research Fe! lows: Thomas A. De VI Ieger, Keijl Yanagisawa

Graduate Student: William A. Hill

Electronic Special !st: Kenneth L. Hol I is

Research Assistant: Joan L. Roach

The work on_single unit responses from neurons In Arthropods has proved to be a terti le field tor obtaining insight into the ways a central nervous system deals with Integration. It Is now clear that here, as In many other forms, Includ­ing mammals, specific reactivity ot individual neurons Is an Important aspect. By a study ot the "behavior" of a neuron to changing circumstances, such as specific, modi tying and hormonal Influences, and comparing these with concurrent behavioral changes in the animal, a link can be laid between these two, a necessary step to explain behavior by neurophyslologlcal events. Because ot our present awareness ot such factors as length ot day and night on responsl veness, the quarters where the animals are kept have been thoroughly rebui It. Exposure to light Is strictly regulated and temperature kept constant within a narrow range. There ls also 1)ro­vlsion tor rearing crayfish In complete darkness, which appears to have Interesting influences on their optic system (Biology 1969, No. 109). •

160. BINOCULAR INTERACTION IN VISUAL RESPONSES OF THE CRAYFISH

I nvestlgator: Keijl Yanaglsawa

Support: National Science Foundation

As reported previously, the response ot a sustaining fiber ot the crayfish optic nerve is Inhibited by the light on the symmetrlcal area ot the opposite eye <Biology 1969). It has now been found that this Inhibition Is apparently especlally caused by the initial burst ot the contralateral sustaining fibers and decays rather rapid I y, I ts I atency is longer than that ot the onset of nerve I mpu I ses in the contralateral sustaining fiber. This Inhibitory Influence ot the contralateral eye is also present when light Is presented to the other areas of that eye. Two spots of Ii ght on the contra I atera I eye show a summatl on of the I nh I b I ti on. I ncreas Ing the intensity of light on an area of the opposite eye also results In an Increased inhibitory effect. When the Inhibition Is strong enough, the effect lasts as long as the inhibitory area Is lighted. These properties of contralateral Inhibition are quite slml lar to the small light spot Inhibition of the homolateral eye (Wiersma and Yamaguchi, 1967), but the contra lateral ettect Is much weaker.

These experiments support the hypothesis concerning the functional sig­ni ticance ot the heterolateral optic fibers In the decapod Crustacea: ti be rs ot a given type coming to the optic gang I la wi 11 Influence partlcularly those reactions which are related to the spec! tic stimulus parameters they sense (Wiersma et al., 1964).

References:

Wiersma, C. A. G., Bush, B. M. H., and Waterman, T. H. ( 1964) J. Cel I. Comp, Physiol. 64: 309-326.

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Wiersma, C. A. G. and Yamaguchi, T. ( 1967) J. Exptl. Bio:. 47: 409-431.

161. VISUAL INTEGRATION OF LOBSTERS

Investigators:

Support:

Kelji Yanagisawa, C. A. G. Wiersma

National Institutes of Health, Public Health Service National Science Foundation

The experiments on single unit analysis of optic nerve fibers in the Pacific coast rock lobster, Panulirus interruptis, have been continued. To date we have established some I 10 units, each found at least 5 times, and more than 100 more fibers with fewer findings. Al I of the expected sustaining fibers, namely 23, are now es tab I ished, and no others have ever been encountered. Of the 23 jittery moverrent fibers, 22 have been established, and the other one found. In the medium movement fiber class, which fire with I ittle adaptation on approaching objects, 15 have been established, and only 2 never seen. Fast movement fibers are difficult to determine, which explains why only 8 have been established and 4 never found. Dimming fibers appear indeed to exist in the lobster as they do in crayfish and crab (Biology 1970, No. 164), but they have not been found sufficiently isolated to determlne their sensory flelds and so none have yet been estab llshed. Another rea­son is that their discharges, as well as those of the sustaining fibers, arc often very noisy in the lobster in comparison with other species. We have also obtained good evidence for the presence of heterolateral simple visual fibers, especially in the jittery movement fiber class, which appear to run, as also noted in other species, near their homolateral partners. Surprising is the dearth of findings of heterolateral sustaining fibers. The heterolateral fibers of these classes seem to be just branches of the corresponding homolateral fibers as in the crayfish.

A new class of fibers, discovered after their more obvious presence in another species of rock lobster (Biology 1970, No. 163), respond to a moving light through their visual field, and not to constant Illumination or to quick I ight sweeps. Their properties appear thus to be complementary to those of the jittery npvement fibers, which "see" smal I moving dark spots only. At present 4 fibers of this class have been established. They again appear to have the same visual field boundaries as the other simple visual fiber classes.

162. COMPLEXLY INTEGRATING UNITS OF THE OPTIC NERVE IN THE ROCK LOBSTER

Investigators:

Support:

Keljl Yanagisawa, C. A. G. Wiersma

National Institutes of Health, Public Health Service National Science Foundation

An assortment of fibers, of which a nunber have become recently estab­lished, convey Information regarding sensory input of several modalities from fur­ther or nearer body parts to the optic ganglia. Complexly integrating units in which centrifugal information ls Integrated with optical events are to be expected. With such an increase in complexity, it becomes more and more difficult to define al I the properties of such fibers. Rela'tlvely simple in this respect are the space­constant fibers, of which a member was found in each class of the simple visual units. Their fields, which appear to be without exception the upper eye half in the normal body position, through Inhibition by statocyst input of the lower half, change in such a manner that all ommatidia looking downward are inhibited from

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contributing to the input.

However, for unidirectional movement fibers, which have also been studied further, additional factors come into.,play. The ones perceiving horizontal motions appear to be only slightly influenced by gravity. Fibers perceiving vertical mo­tions appear to be divided into 2 classes. Those seeing up-to-down movements have fields limited to the lower eye half, and show no space constancy at all. But for those seeing upward motions in the normal position in the upper eye half only (of which there are at least 3, one for the front, one for the middle and one for the back of the eyel, inverting the animal moves the field to the lower eye half, but never sideways. Withln thls area movement from any direction to the highest 01T111a­tidia causes a discharge, so that their unidirectionality is then lost.

Another interesting unit has recently been determined, and being a larger fiber can be much more easily found than the previous set. It is a multimodal fiber Integrating mechanoreceptlve Information from the anterior appendages with visual Input from both eyes, from the anterior halves only. Usually, when one or the other eye ts covered, the input from the homolateral eye proves to be much stronger than from the other side. In both instances the fiber then reacts much I ike a jittery movement fiber, showing speedy habituation; but when both eyes are uncovered, habituation is slow, and the response to a given stlmulus much stronger (more spikes l than with one eye covered. · ·

Quite recently we have obtained evidence for some type of form vision in certain fibers. These fibers show in addition the necessity of some type of "attention" mechanism to trigger the first spikes. These fibers are triggered by movement of a dark object in the field and wll I often fire repetitively and for longer than the stimulus lasts once they have been "warmed up," but only when the object remains visib 18. Turning off i 1 luminatlon even fo

0

r a moment wi 11 stop the discharge. Further experiments with such fibers may make it possible to define more cl8arly the mechanisms lnvolv8d in this type of attention.

163. UNITS IN THE OPTIC NERVE OF A SOUTH AMERICAN RJCK LOBSTER

I nv8sti gators: C. A. G. Wiersma, Keljl Yanagisawa

Support: National Science Foundation Nati ona I Institutes of Health, Pub Ii c Hea I th Service

When in the spring of 1969 the Pacific coast rock lobster 1;ent off the market because of the Santa Barbara oi I slick, a South American species, Panul irus graci lis, became COITlllercial ly available for a short period. This gave us the op­portunity to study simi laritles and differences between two not very closely related species. The slml larities were very evident. Though only a total of 8 animals could be investigated, 31 units identical In behavior with units in the local spe­cies could be established, which means that they were found at least 5 times. Another 26 fibers known from the local species were encountered fewer times. Thus a total of 57 out of a possible 95 were shown to be the same in both species. In a few instances fibers were found which were not known tor the local species. The majority of these have since been shown to be present in the local species. This illustrates a feature previously encountered in species comparison in crayfish, namely that certain units, though present in both, are much more often encountered in one. The most likely cause is that such fibers are bigger in one species than in the other. It is of special interest that I ight movement fibers, referred to above (Biology 1970, No. 161l, were especially easily found in P. graci lis and be­c<ime only subsequently established in P. interruptus.

174

164. VISUAL RESPONSES FF\l'.JM THE OPTIC NERVE OF A CRAB

Investigator: C. A. G. Wiersma

Support: National Science Foundation Medische Facultelt Rotterdam

In previous work it was strongly indicated that crabs have a more sophis­ticated sot of optical fibers than crayfish. One main difficulty for comparison was that tho crab experiments were performed before tho remarkab Io proporti es of the so-called jittery movement fibers were discovered. Since Carcinus is a good, widely distributed laboratory species, the types of fibers obtainable with our methods were detennined. Results were severely I imited by the fact that responses were largely confined to large fibers. Presumably because of tho absence of such fibers in other classes, no new types were discovered. It was shown however that in Carcinus too, some of the jittery movement fibers are among the largest fibers present. CI asses found with certa l nty we re sustaining, dimming, jittery movement, medium approach movement fibers, and space constant members in al I these classes. Since crab eyes are more mobile than either crayfish or lobster ones, tho latter observation was of particular interest.

References:

•

' 165.

Watcnnan, T. H. and Wiersma, C. A.G. (1963) J. Cel I. Comp. Physiol. 61: 1-16.

Watennan, T. H., Wiersma, C. A. G., and Bush, B. M. H. ( 1964) J. Cel I. Com~. Physiol. 63: 135-155.

Wiersma, C. A.G., Bush, B. M. H., and Watennan, T. H. (1964) J. Cell. Comp. Physiol. 64: 309-326.

Wiersma, C. A.G. (1970) Proc. Kon. Ned. Akad. Wet., C 73: 25-34 •

THE INFLUENCE OF STATE OF EXCITEMENT OF THE ANIMAL ON DISCHARGES IN THE OPTOMOTOR FIBERS OF A CRAB

Investigator: C. A. G. Wiersma

Support: National Science Foundation Medi sche F acu l te it Ratte rdam

In the crayfish pronounced changes in firing rate of optomotor fibers are present when the animal's behavioral state ch<1nges. In general many more impulses are present when there is excitement. This overlies the firing frequencies caused by postural or optic reactions. In the crab very similar frequency changes were observed, though the system is much more complex because of the presence of addi­tional motor neurons and eye muscles. One notable exception to the rule of in­creased firing rate with increased excitement was found for tho motor fiber which tonical ly withdraws tho eyecup to the protected position in the groove of tho carapace. This fiber shONod pronounced inhibition during excitement. Functionally this would make the animal better aware of f'ts environment during an excited state, as the visual field is severely I imlted when the cup is withdrawn. It was further found that during the summer months tonic discharges caused by postural changes showed very pronounced habituation with time - within a few minutes the frequencies fell by half or more; whereas in spring animals, such habituation >1as practically

175

absent. This may be related to the fact that in summer the animal I ivos in brack­ish water and in winter in deeper seawater.

This 1wrk was done in col laboNtion with Dr. L. Fiore at tho Department of Physiology, Medical Faculty, Rotterdam.

166. UNIDIRECTIONAL OPTOMOTOR FIBERS IN CARCINUS

Investigator:

Support:

C. A. G. Wiersma

National Science Foundation Medische Faculteit Rotterdam

In the crayfish and rock lobster the positional adjustments of the eye­cups, and the discharges in the motor fibers causing these, can be largely ex­plained by the influence of tho action of gravity on the statocysts. The influ­ences are mediated by tonic discharges in smaller nnd larger fibers, tho latter showing some directional preference. It was found that in the crab Carcinus addi­tional very large fibers are present which respond only phasical ly during very quick turns in one dirnction. Such fibers were found for al I directions for which tonic fibers were found, and often accompanied these in the appropriate sets. The most interesting were the ones associated with the optokinetic movement fibers., As in the crayfish, the latter do not fire when the animal is turned in the dark, or in o surround without visual clues. The large phaslc fibers, hONovor, responded as wel I in the dark as in the 1 lght. A responsiveness to this type of acceleration is most likely not due to the statocyst as such, but to loose hairs in the stato­cyst cavity, whose physiological reactivity has previously been described by Cohen ( 1955) for the lobster. The most interesting result was the discovery of a fiber type intermediate in reactivity between the optokinetic and rotation fibers. These wi 11 respond to rotating stripes. At low rotation speeds there wi 11 be no effect from rotation of the animal in the dark, though a good response is obtained in the I ight for a striped background. However, at speeds of a few degrees per second, they will start to fire in the dark and this rate will increase the faster the ro­teition. Thus ut speeds where the "slow" optoklnetic fibers no longer res;>0nd be­cause of tho "blurred image," these fibers 1dll still respond by virtue of their statocyst input. Their presence in Carcinus and absence in crayfish und rock lobster may well be connected with the much quicker movements performed by crabs, which would make eye position adjustments necessary to compensate for the fast turns.

This work was done in collaboration with Dr. L. Fiore at tho Department of Physiology, Medical Faculty, Rotterdam.

Reference:

Cohen, 1:1. J. ( 1955) J. Physiol. 130: 9-34.

16 7. OPTO~:OTOR FI BERS IN THE RCCK LOBSTER

I nvcsti gutors:

Support:

C. A. G. Wiersma, Keiji Yanagisawa

National Institutes of Health, Public Health Service National Science Foundation

Uuring the studies concerning the fibers in the optic nerve, optorrotor

176

fibers are regularly encountered. Most often, though with occasional exceptions, these fibers are clearly located not in the optic nerve itself, but outside of it. As In the crayfish, only 3 sets of fibers appear to be present. These are opto­kinetic fibers for horizontal movements of objects in the surround, or turning the anlmal in the horizontal plane, eye-up and eye-down fibers stimulated by rotating the animal on its length axis, and head-down and -up fibers stimulated by turning the animal on its horizontal axis. Of these the head-up fibers appear to be more difficult to find. There is no evidence of the existence of unidirectional motor fibers as were found in the crab, nor are there fibers for 45° positions. These fibers, though reacting to visual stimuli like those of the crayfish, appear to do so more weakly and less constantly. For the optokinetic reactions the homolateral eye is by far the stronger influence, much more so than in the crayfish. A fourth type of fiber appears homologous with the crayfish eyecup withdrawal fibers; the mechanical effect Is, h011ever, rather weak and consists of a downward-forward move­ment of the eye. This brings the eye, when the antenna Is kept backward, behind a big spine on Its first joint, thus providing protection. These fibers have shown clear evidence of being Inhibited by the excited state, in contrast to al I the aforementioned sets.

168. COORDINATICN AND ACTIVATION OF SWIMMERET MOVEMENTS IN THE CRAYFISH

Investigator:

Support:

Thomas A. De Vlieger

Free University Amsterdam National Science Foundation

Previous experiments <Hughes and Wiersma, 1960; Ikeda and Wiersma, 1964; Wiersma and Ikeda, 1964) have shOl/n that motor Impulses normally leading to swim­meret movements can be recorded in the isolated abdominal cord of the crayfish. These impulses appear as bursts in the first roots of the abdominal ganglia, either £pontaneous or elicited by electrical stimulation of special corrrnand fibers. In both cases the fifth ganglion always has the lead and successive bursts can be ob­served in the anterior gang! la after delays of 100-200 msec per gang I ion.

Confirming the previous results, with the aid of suction electrodes, it was found that the fourth or the third ganglion has the lead after cutting the cord between the fifth and fourth or between the fourth and third ganglion respectively, while the Initiation of bursts in the first and second ganglia is entirely depen­dent on the posterior ganglia. This happens to both spontaneous and corrrnand fiber­activated bursts. An underlying neuronal network is indicated which coordinates the bursts in the fifth, fourth and third ganglia and activates them in the second and first ganglia. The fibers of this network, if present, must be located close to the command fibers In the cord as it was not possible to cut them separately.

In certain conditions, bursts could become very long both with respect to remoter and to promoter elements, which then were very clearly distinguishable.

Besides the aforementioned methods of activation of the bursts, it ap­peared that bursting could also be elicited by stimulation of the ventral hairs of the head of the animal by means of a stceam of air bubbles. This bursting could be inhibited by hair stimulation of the dorsal and lateral carapace. The inhibi­tory stimulation habituated very quickly. It remains to be investigated hOll these effects are mediated through the exciting and inhibiting command interneurons.

177

He fe rences :

Hughes, G. M. and Wiersma, C. A. G. ( 1960) J. Exptl. Biol. 37: 657. -, Ikeda, K. and Wiersma, C. A.G. (1964) Comp. Biochem. Physlol. 12: 107.

Wiersma, C. A. G. and Ikeda, K. ( 1964) Comp. Biochem. Physiol. 12: 509.

169. DISCHARGE PATTERN OF JOINT ~'OVEMENT FIBERS IN THE CRAB

Investigator: C. A. G. \~lersma

Support: National Science Foundation Medi sche Facu I te it Rotterdam

In the original investigation of the properties of the so-cal led move­ment fibers in the crab Carcinus (Wiersma and Boettiger, 1959), it was observed that these fibers showed a remarkable discharge pattern in the spikes caused by joint motions. Instead of a gradually increasing firing rate after the threshold of their reactivity was reached, the spikes appeared in an irregular fashion until a saturation frequency of about 50 per second was reached. This aspect was now in­vestigated by repeatedly recording discharges during movements between threshoid and saturation, a rather narrow band of velocltles. It was found indeed that the time intervals present were multiples of the one at saturation up to at least 10-fold intervals. But a dependence on the experimental circumstances was also found, pointing to the presence of a resonance factor caused by these conditions, which determines the saturation frequency itself. Therefore, th~ functional significance of th is remarkable property remal ns uncertain.

This v/Ork was done in collaboration with Ors. F. van der Mark and L. Fiore at the Department of Physlology, Medical Faculty, Rotterdam.

References:

Wiersma, C. A. G. and Boettiger, E. G. ( 1959) J. Exptl. Biol. 36: 102-112.

Wiersma, C. A.G., van der Mark, F., and Fiore, L. (1970) Comp. Biochem. Physiol. 34: 833-840.

170. OBSERVATIONS ON THE CRAYFISH SINUS GLAl'D

Investigators: Joan L. Roach, C. A.G. Wiersma

Support: National Science Foundation National Institutes of Health, Public Health Service

The crustacean sinus gland is a neurohemal· organ located in the eyestalk. It is considered to be a storage site for at least five hormones which are released into internal blood sinuses when needed. Bunt and Ashby (1967) have described five types of granules in the axonal terminations of the crayfish, Procambarus clarki, which are believed to represent these different hormones. The mechanism of hor­monal release in another s;iecies of crayfish has been sh0tm to be by prelimlnary fragmentation and subsequent release (Shivers, 1969). An alternate method, extru­sion of the complete granule core, has been described for a land crab by Weitzman

178

( 1969). Figure I is an electron micrograph of axon terminations adjacent to a blood sinus, with portions of two blood cells, in P. clarki. Throe granules are seen abutting the terminal membrane. A complete granule core is in the process of being released; structures are attached to the membrane partially surrounding the granule and smal I clear vesicles appear nearby. Two free granules are also present, one between the basement membrane of the blood sinus, and the other in the blood sinus itself, An "active site" for granule release, described by Shivers ( 1969) as an accumulation of very small dense granules next to a thickened patch of mem­brane with smal I clear vesicles nearby, ls also seen.

Ce/ I inclusions appearing in the blood cells in Fig. I may be of the type described by Shivers ( 1969) as "crystal line mitochondria" in typo 2 neuroscrctory ce 11 s of the X-organ. The inset shONs an en I argod view of the "customary" mi tochon­dri on and neighboring "crystal/ins" types. Ths lattsr havs not been sesn in axon terminations in the sinus gland, at least some of which are presumably from the typs 2 neurosecretory ce 11 s, a I though mitochondria are present.

The significance and distribution of the "crystal line mitochondria" are unknown. It is hoped that further study with environmental stimuli assumed to affect sinus gland hormones will elucidate the release mechanisms involved, as well as providing insight into the unusual cell inclusions. Correlation of a specific granule type with a specific hormone may also be possible under these conditions,

References :

Bunt, A. H. and Ashby, E. B. ( 1967) Gen. Comp. Endocrinol. 9: 334-342.

Shivsrs, R. R. ( 1969) Z. Ze/ lforsch. 97: 38-44.

Weitzman, M. (/969) Z. Zel lforsch. 94: 147-154.

• PUBLICATIONS

l\rOchiga, H. and Wiersma, C. A.G. ( 1969) Circadian rhythm of responsiveness in crayfish visual units. J. Neurobiol. I: 71-85.

Ardchiga, H. and Wisrsma, c. A.G. (/969) The effect of motor activity on the reactivity of single visual units in the crayfish. J. Neurobiol. I: 53-69.

Wiersma, C. A.G. (1969) Regulative mechanisms for the discharges of specific intorneurons. In: M.A. B. Brazier (ed.), The lnterneuron, pp. 113-129.

Wiersma, c. A. G, (/970) Neuronal components of the optic nerve of the crab, Cnrcinus maenas. Proc. Kon. Nsd. Aknd. Wet., C 73: 25-34.

179

Electron micrograph of the crayfish sinus gland. be, blood eel I; g, granule abutting terminal rrembrane; gr, granule being released; as, active site; fg, free granule; m, mitochondrion; cm, crystal I ine mitochondrion. Mag. 31,900. Inset mag. 55, 250.

180

Professor: Fe Ii x Strumwasser

Senior Research Fellow: Robert W. Meech

Research Fellows: Stephen Arch, Arnold Eskin, Bertram Peretz, Mary A. Weir, David L. Wi Ison

Graduate Students: Jeffrey L. Ram, Barry S. Rothman

Engineering Test Assistant: James J. Gi I liam

Research Engineer: Floyd Schlechte

Research Assistant: Renate Alvarez

Laboratory Assistants: Suzy Bower, Shelly A. Rempel

171. IN VITRO ENTRAINMENT OF THE CIRCADIAN RHYTHM OF OPTIC NERVE IMPULSES FROM EYES OF APLYS I A

I nvosti gator:

Support:

Arnold Eskin

National Institutes of Health, Public He<ilth Service National Aeronautics and Space Administration

Ci rcndian rhythms of optic nerve impulses have been recorded from iso­lated eyes of Aplys-ia in filtered seawater, under conditions of darkness, for 2 days nnd in organ culture medium for 3 days (Jacklet, 1969a). Tho period of the rhythm was shown to be 24 hours or less when tho eyes wore maintained in seawater and about 27 hours when the eyes were maintained in culture medium. Jacklet (f969b) also found that one eye could be phase shifted in vivo relative to tho other oyo

•and that ono Ii ght exposure in vitro cou Id phase shift an oyo. Tho experiments to be described were performed so that characteristics of the rhythr:i of tho eye during

'in vivo and in vitro entrainment could be compared. In addition, the possibility of extending the life of the eye in vitro was investigated.

Aplysin were entrained by light/dark (LO) 12:12 pretreatment light cycles before being placed into the experimental conditions. The light cycle for the con­trol groups was the same as the pretreatment cycle. The I ight cycle for tho experi­mental groups was phase delayed II hours relative to the control cycle. For the in vivo entrainment experiments, one group of animals was placed under the control I ight regime nnd one group under tho experimental (phase shifted) regime. After the first dny under the experimental conditions and each.successive day, an experi­mental and a control animal were sacrificed and one eye was removed from each ani­mal. Optic nerve impulses were then recorded tor 24 hours from each eye maintained in ti ltered seawater and under constant dark conditions. For tho in vitro entrain­ment experiments, eyes we re removed from the an i ma Is and a th read was attached to each nerve for hand Ii ng purposes. The eyes were then rinsed in steri le seawater and placed into a smal I plastic petri dish with 12 ml of culture medium (Strumwasser and Bahr, 1966). One eye was then placed under the experimental I ight regime and the other eye, from the same animal, wh-ich served as a control, was placed under the control I ight regime. After each day of isolation, two eyes from tho same ani­mal were romeved from the entrainment conditions and their optic nerve impulses recorded while the eyes were in ti ltered seawater and under constant dark conditions.

After one day of entrainment, the eyes that were exposed to the

181

experimental cycle in vivo were almost completely phase shifted. The eyes exposed to the light cycle in vitro did not appear to be phase shifted as much as the eyes exposed in vivo after I day of entrainment but this difference may not be signifi­cant. After 2 days of entrainment tl\e phase shifts of both the in vivo and in vitro exposed eyes were essenti a I ly complete. Though the phase shifting character­istics of the in vivo and in vitro treated eyes were slmi Jar, the phase of the rhythm of the in vitro treated eyes was delayed about 4 hours relative to the in vivo treated eyes. This result indicates that the period of the rhythm of the eye in organ culture medium may be longer than the period of the rhythm of eyes in vivo.

The isolated eyes were fol lowed for 8 days of entrai nmcnt. Eyes which had been isolated for this time expressed rhythms and were sti 11 entrained. The amp Ii tu de of the rhythm of the In vitro treated eyes was about 60% of the amp Ii tude of the in vivo entrained eyes for the first 4 days of isolation. From the 4th to the 9th day of isolation, the amplitude of the rhythm of the Isolated eyes de­creased by 50%.

The results discussed above have established that the nature of the mech­anism underlying the rhythm of optic nerve impulses may be explored in two comple­mentary ways. Since Aplysia eyes can be entrained by light cycle in vitro, the mechanism by which environmental light information ls transferred to the osc:;illator responsible for the rhythm of optic nerve impulses is within the eye. Thus,' it is possible to Investigate the nature of this mechanism by Investigating the eftects of drugs applied to the isolated eye In the presence of a light cycle. Also the results discussed above have established that Isolated eyes wl I I last for at least 9 days in culture media. Therefore drugs may also be applied to the Isolated eye while it is free-running In constant conditions in order to investigate the nature of the mechanism directly responsible for the rhythm. To perform these types of experiments, techniques are currently being developed for the continuous monitoring of the optic nerve activity from an individual eye in vitro.

References:

Jack let, J, W. ( l969a) Science 164: 562-563.

Jack let, J. W. ( 1969b) Amer. Zool. 9: 1103,

Strumwasser, F. and Bahr, R. (1966) Fed. Proc. 25: 512.

172. THE EFFECT OF ACTINOMYCIN DON THE CIRCADIAN RHYTHM OF THE EYE OF APLYSIA CALIFORNICA

Investigator: Barry S. Rothman

Support: National Institutes of Health, Public Health Service

It has been demonstrated, by recording optic nerve impulses, that the eyes of the sea hare, Aplysia californica, maintain an endogenous circadian rhythm in vitro (Jacklet, 1969). In the present experiments, animals were trained on a schedule of 12 hours of I ight fol lowed by 12 hours of dark for at least 5 days. Eyes from such Aplysia were dissected out during the light portion of the cycle and monitored in the dark by means of a suction electrode on the optic nerve. From this recording it was found that two eyes from the same an I ma I remain in phase (+

or - I hour) at least until the end of the next projected light cycle. Al I control eyes sho1ved onset of endogenous activity toward the onset of the projected I ight period; and offset of activity toward the end of this projected I ight period.

182

Actinomycin D, a well known inhibitor of DNA-dependent RNA synthesis, has been used in previous investigations of circadian rhythms. It was administered to one of each pair of eyes in extracellular concentrations varying from 16 to 0.25 µg/ml dissolved in the usual perfusion medium of ti ltered seawater. The drug was af\;ays given at the end of the projected 1 lght cycle, a few hours after dissection. The effect of the higher doses (16, 8 µg/mll was to completely inhibit tho appeur­ance of the next period of projected I ight-time activity. Lower doses al lowed the manifestation of this expected cycle, but markedly affected its duration. At I µg/ml of actinomycin D the eye maintained its activity cycle for 24 hours rather than 12 hours. At 0.25 µg/ml this cycle was extended to only 16 hours. This lengthening of the projected I ight-time activity is clearly different from effects observed in lower organisms (Karakashian and Hastings, 1964; Sweeny et al., 1967), and suggests the possibility of a different clock mechanism in Aplysia.

Studies have been started to determine the level of RNA synthesis within the eye at different actinomycin D concentrations by measuring the uptake and in­corporation of 3t-f uridine.

References:

Jacklet, J. W. ( 1969) Science 164: 562-564.

Karakashian, M. W. and Hastings, J. W. ( 1964) J. Gen. Physiol. 47: I.

Sweeny, B. M. et a I • ( 1967) J. Gen. Phys i ol . 50: 647.

Strumwasser, F. (1965) In: J. Aschoff (ed.), Circadian Clocks, pp. 442-462.

173. MOLECULAR WEIGHT DISTRIBUTION OF PROTEINS SYNTHESIZED IN SINGLE, IDENTIFIED NEURONS OF APLYSIA

1nvesti gator:

S.Upport:

David L. Wi Ison

Helen Hay Whitney Foundation National Institutes of Health, Public Health Service National Aeronautics and Space Administration American Heart Association

The abdominal ganglion of Aplysia callfornica, the sea hare, contains a number of large (up to 1/2 1m1 diameter), identifiable nerve cells. Others huve shown that the neurons differ greatly from each other in terms of morphology, elec­trical activity, and neurosecretory function, but I !ttle work has centered on Aplysia nerve eel I biochemistry.

I have been studying amino acid incorporation in the single neurons. Isolated abdominal ganglia were incubated (4 to 20 hours) in a defined medium con­taining 3t-f-leucine. Single nerve eel I somas vtere removed, and their proteins ex­tracted and analyzed by SDS-polyacrylamide gel elect·rophoresis. Techniques were developed for working with miniature gels, Including an easy, efficient method for I iquid scintillation counting of radioactive gel s I ices. I found that a number of proteins arc synthesized in abundance in ~the sing I e neurons, and that different neurons shovt reproducibly different gel labeling patterns.

These experiments were designed to form a foundation for determining whether correlations exist between nervous _function und protein synthesis.

183

Experiments are under way to detennine the effect on protein synthesis of I) block­ing ganglion impulses with tetrodotoxln, and 2) electrical stimulation of the neu­rons (synaptic and antldromic). Information from such experiments could prove use­ful in understanding such complex pro~esses as learning and memory.

References:

Wi Ison, D. L. (1970) J. Gen. Physlol. Ctn press>.

Ward, s., Wi Ison, D. L., and GI II lam, J. J. (1970) Methods for fraction­ation and sclnti llation counting of radioisotope labeled poly­

·acrylamide gels. Anal. Biochem. (submitted for publication).

174. ATPase ACTIVITY IN APLYSIA NERVOUS TISSUE

Investigator: Jeffrey L. Ram

Support: National Science Foundation National Institutes of Health, Public Health Service

The parabolic burster neuron of the sea hare, Aplysl a, has endogenous "slow wave" act Iv ity which produces bursts of spikes when the wave reaches a par­ticular threshold. Studies by Strumwasser and Kim (1969) have shown that the slow wave can be observed Independently of the spiking activity in the presence of tetro­dotoxin and ca++-free seawater. They have also shown that the slow wave is ouabain­blocked and CNa++c1->-linked. Since the slow wave appears to be produced by an electrogenic pump, and since Ion pumps are believed to operate by means of ion-1 inked ATPases, I have undertaken a search to discover whether Aplysla nervous tis­sue has a (N~++c1->-I inked ATPase. These studies are ultimately aimed at studying this ATPase in single eel Is. Previous studies by David Carpenter (NIH, personal communication) on pooled ganglia from Aplysia had indicated that a ouabaln-sensitive ATPase did exist.

Measurements of ATPase activity were done on crude homogenates of the paired pedal-pleural ganglia. Three methods of measuring ATPase activity were tried: colorimetric, automatic titration, and 32p. Ouabain, a potent inhibitor of ion pump ATPase, produced Inhibition of some ATPase activity only for the titra­tion method and the 32p method, but not for the colorimetric method. Tho pH de­pendence of the total ATPase activity as measured colorlmetrical ly was similar to the pH dependence of the ouabain-insenslttve ATPase measured with 32p. Since the main difference between the experiments done colorlmetrical ly and those done by the other methods is that the tissue concentration Is higher In the former, the sug­gestion is made that an endogenous Inhibitor of ouabain-sensltlve ATPaso is present in the Aplysia nervous tissue and is effective at the higher tissue concentrations used in the colorimetric method.

Km curves measured with the 32P method for both the ouabaln-sonsitlve ATPase and the ouabain-insensltlve ATPase give at least two Km's for each, a high one about 10-4 M and a lower one of about 10-5 M. The dual Km behavior is In agreement with a few other studies in the literature; Neufield and Levy (1969) have studied in some detail a Na+-ltnked ATPase which they say accounts for the lower Km· The suggestion is made here that this enzyme is actually a (Na++c1-)-linked enzyme cc1- dependence was not looked at) slmi lar to the one being searched for in Aplysia.

184

References:

Strull1'/asser, F. and Kim, M. ( 1969) The Physiologist 12: 367.

Noufiold, A.H. and Levy, H. M. A. (1969) J. Biol. Chem. 244: 6493-6497.

Lowry, O. H. and Lopez, J. A. (1946) J. Biol. Chem. 162: 421-428.

Cummins, J. and Hyden, H. (1962) Biochim. Biophys. Acta 60: 271-283.

175. SEASONAL REPRODUCTION IN APLYSIA: BAG CELL PERFUSION

Investigator:

Su;iport:

Stephen Arch

National Institutes of Health, Public Health Service National Aeronautics and Space Administration

Tho bag eel I clusters adjoining the parieto-visceral gang I ion CPVG) of Aplysia fulfill most of the morphologic criteria for neurosecretory cells. In the last few years evidence has accumulated Indicating that these cells ;iroduce a poly­peptide hormone which induces egg-laying <Toevs and Brackenbury, 1969). Hm1ever since egg-laying ls seasonal (Strumwasser et al., 1969) it seems clear that either the synthesis or release of the hormone and/or the receptivity of its target tissue changes during the year.

As part of a series of studies concerned with the regulation of seasonal reproduction in Apl~sia I have begun investigating tho physiology of hormone re­lease from the bag eel I clusters. The experimental protocol consists of isolating a PVG and incubating it for 16 hours in a defined medium which has been supple­mented with 3H-leucine. After incubation the ganglion is rinsed for 6 hours in fresh medium supplemented with 1000-fold excess of cold leucine (medium Rl. The

•rinse Is fol lowed by 4 hours of perfusion with fresh medium R at a rate of I cc/6 minutes. Forty I-cc fractions are collected during the perfusion. By aliquoting U.2 cc from each fraction onto glass fiber filter discs it is possible to determine the total radioactivity of the perfusate as well as the total TCA-precipitable radioactivity and sti II have 0.8 cc of perfusate for additional analysis (Lawry's, gel electrophoresis, bioassay, etc.).

So far only one treatment has been employed to release neurosecretory products from the ganglion. This treatment has consisted of introducing a 6-24 minute pulse of high potassium medium (medium Kl during perfusion. The results of medium K pulses are clear. There is a significant rise in the radioactivity of the crude perfusate during stimulation. Furthermore, the maj_or portion of this rise is contributed by acid-insoluble material. The I lkelihood that this acid-insoluble material is a neurosecretion ls strengthened by the observation that if 3 pulses of medium Kare given during perfusion, the amount of acid-insoluble material re­leased decreases sharply from the first to the third pulse. This suggests that the neurosecretory stores are being depleted by the repeated pu I ses.

Currently I am attempting to characterize the material released by medium K. Further experiments are p I anned to ,investigate the effects of various neurotrans­mitters, as wel I as electrical stimulation, on tho release of neurosecretions from the PVG.

References:

Toevs, L. and Brackenbury, R. (1969) Comp. Biochem. Physlol. 29: 207-216.

185

Strum.vasser, F., Jacklet, J., and Alvarez, R. ( 1969) Comp. Blochem. Phys­lol. 29: 197-206.

176. HABITUATION AND DISHABITUATION IN THE ABSENCE OF A CENTRAL NERVOUS SYSTEM

I nvestl gator: * Bertram Peretz

National Institutes of Health, Public Health Service National Aeronautics and Space Administration

Habituation and dlshabltuatlon have been observed In a semi-Intact Aplysla preparation In which the central nervous system is removed. The amplitude of withdrawal responses In the gill decreases In proportion to the rate of water drops applied, 1/0.5 to 1/2.5 minutes at 15°C. The effects of habituation last for at least 2 hours. A d!shabituated response is ellc!ted by stopping water drops or electrically stlmulat!ng the gill. Further, the gill was found, by histology\ to contain nerve cell bodies, and habituation and dlshabltuatlon appear to be properties of these peripheral neurons.

Reference:

Peretz, B. ( 1970) Science (In press>.

177. INTRACELLULAR CALCIUM INJECTION CAUSES INCREASED POTASSIUM CONDUCTANCE IN APLYSIA NERVE CELLS

Investigator: Robe rt W . Meech

Support: Los Angeles County Heart Association American Heart Association National Institutes of Health, Public Health Service

Small quantities of calcium salt were injected Into Aplysla neurons. The membrane potential was hyperpolarlzed because of a specific Increase in potassium conductance. The hyperpolarlzatlon was reversibly blocked by tetraethylammonium chloride. Injection of potassium citrate caused membrane depolarization. Entry of calcium may activate the Increased potassium conductance during the action po­tential and/or may participate In the so-cal led inhibition of long duration (ILDA).

Reference:

*

Meech, R. W. (1970) Intracellular calcium ·Injection causes Increased potassium conductance in Aplysla nerve cells. Science (sub­mitted for publication).

Department of Physiology and Biophysics, University of Kentucky, Lexington.

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178. SYNAPTIC INFLUENCES ON IDENTIFIED NEURONS IN AN ABERRANT PARIETO-VISCERAL GANGLION OF APLYSIA

* ** Investigators: Jon W. Jacklet, Bertram Peretz , Felix Strurr111asser

Support: Natlonal Institutes of Health, Public Health Service

One aberrant parieto-visceral (abdominal) ganglion in Aplysia californica was found in which the right hemigangllon was separated from the left hemiganglion by a commissure approximately l cm long. Simultaneous records were made from Rl5 (intracellular) and the convnissure In the isolated right hemigangl ion; from LI I (in­tracel lularl and the genital and pericardlal nerves in the isolated left hemigan-gl ion; in addition, intracellular records were made from Rl4. These and other eel Is were found in their respective hemlganglia in the position they usually occupy in the normal ganglion. The recordings have al lowed us to indicate the relative in­fluence or lack of influence of LIO and lnterneuron II on Rl5 and LI I in their re­spective hemiganglia and the position of lnterneuron II as being in the right hemi­ganglion. Rl5 was shewn to send an axon Into the inter-ganglionic commissure. Rl5 1s bursting activity was modulated by an ILD-E input from lnterneuron II in the right hemiganglion.

Reference:

Jacklet, J. W., Peretz, B., and Strumwasser, F. (1970) Z. vergl. Phys­iologie 66: 318-325.

PUBLICATIONS

Jacklet, J. w., Peretz, B., and Strumwasser, F. (1970) Synaptic influences on iden­tified neurons in an aberrant parieto-visceral gang I ion of Aplysia. Zeit. f. vergl. Physiol. 66: 318-325 •

• Meech, R. W. and Strumwasser, F. (1970) Intracellular calcium injection activates potassium conductance in Aplysia nerve eel Is. Fed. Proc. 29: 834.

Meech, R. W. (1970) lntracel lular calcium injection causes increased potassium conductance In Aplysia nerve eel Is. Science (submitted for pub I ication).

Peretz, B. (1969a) Central neuron inltlation of periodic gi I I movements. Science 166: 1167-1172.

Peretz, B. (1970) Habituation and dlshabltuation in the absence of a central nervous system. Science 169: 379-381.

Strumwasser, F. and Kim, M. ( 1969) Experimental studies of a neuron with an endog­enous oscillator and a quantitative model of its mechanism. The Phys­iologist 12: 367.

Strumwasser, F. (1970) The cellular basis of behavior In Aplysia. In: J. V. Brady (ed.), Problems, Practices and Positions in Neuropsychiatric Research. J. Psychiat. Res. (Jn press). ,,

* Department of Biological Sciences, State University of New York at Albany.

**Department of Physiology and Biophysics, University of Kentucky, Lexington.

vlard, S., Wilson, D. L., and Gilliam J. J. (1970) Methods for fractionation and scintillation counting of radioisotope labeled polyacrylamide gels. Anal. Biochem. (submitted tor publicatlon).

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Wi Ison, D. L. (1970) Molecular weight distribution of proteins synthesized in single, identified neurons of Aplysia. J. Gen. Physiol. (submitted tor pub Ii cation).

•

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Professor: Roger W. Sperry

Visiting Associate: Harbans L. Arora

Senior Research Fellows: Evelyn Lee-Teng, Colwyn B. Trevarthen

Research Fellows: Linda Fagan, Stanley Jaffee, Santosh Kumar, Myonggeun Yoon

Graduate Students: Larry I. Benowitz, Harold W. Gordon, Cary Lu, Ronald L. Meyer, Je rre Levy , Robe rt D. Nebes

Electronic Specialist: Peter Jonkhoff

Research Assistants: Sarah M. Ingersol I, Ruth T. Johnson, Charles D. Kreuter, Lois E. MacBird, J. Geoffrey Magnus

Research Aides: Eef Goedemans, Dahlia Zaidel

Laboratory Assistants: Donna Berker, Cathy Greene, Melissa A. Honack

Work in psychobiology has encompassed a wide spectrum of activities aimed at understanding the higher functions of the brain, ranging from studies on the functional effect of I ight on the developing visual system In salamander embryos to theoretical speculations on the nature of psycho-neural interaction in the cerebral hemispheres ot man. Special emphasis has been focussed on a continuing series of fol low-up studies on human neurosurgical patients of Vogel and Bogen with complete and partial sections of the corpus callosum, aimed at problems concerning differ­ential specialization of mental faculties in the right and left hemispheres and related phenomena of perception.

Continuing animal experiments have been centered around visual learning in.one-day-old chicks and on functional compensation achieved in brain-operated newborn kittens. A new line of experiments has been started involving electrophys­iohoglcal mapping of regenerated optic fiber connections in the midbrain optic lobe of I owe r i nve rteb rates .

179. RIGHT HEMISA-IERIC DOMINANCE FOR THE PERCEPTION OF PART­WHOLE RELATIONSHIPS IN HUMAN COMMISSUROTOMY PATIENTS

Investigator: Robert D. Nebes

Support: National Institutes of Health, Public Health Service

Although the dominance of the human left hemisphere for language is well documented, the role played by the right hemisphere in perception, especially of visuo-spatial relations, is not clear. The presence in human commissurotomy patients of two essentially normal but disconnected hemispheres al lows direct com­parison of the two sides of the brain of a single Individual on a given task. The present experiment was designed to investigate the ability of each hemisphere to handle part-whole relationships. The task required the subject to estimate the

' size of a circle from examination of an are of that circle. In order to test each hemisphere separately, examination of either the arc, or the circles, or both was restricted to touch by one hand at a time. The arcs and circles used us choices were made from raised plexlglas rings mounted on cards.

I 89

Results shCl.'I the left hand, and thus the right hemisphere, to be far su­perior to the right hand in these tests. The scores for the right hand rarely rose above chance, demonstrating the Ineptness of the left hemisphere on this sort of perceptual task. The test is present_ly being given to several patients with uni­lateral brain damage, as well as to a'ser!es of normal subjects, in order to better define the capabi I !ties of the two sides of the brain for visual !zing the whole from a part.

180. LATERALIZATION OF PERCEPTUAL UNIFICATION IN COMMISSURCTOMY PAT! ENTS

Investigator: Robert D. Nebes

Support: National Institutes of Health, Public Health Service

Further investigation into the ab! !tty of each cerebral hemisphere to perceive a whole figure from Its parts involved shCl.'ling commissurotomy patients a series of geometrical shapes, each of which had been cut into several pieces and the pieces drawn apart. The subject's task was to mentally slide the parts to­gether and then to find the resultant shape among three alternatives. The choices, plexiglas forms, were hidden behind a screen and were avai !able for tactual exam­ination by only one hand at a time. Of the two Incorrect choices for each figure, one was in shape of one of the pieces, whl le the other had the same general ~ize and angularity as the correct choice.

The performance of the left hand (right hemisphere) on this task was sig­nificantly superior to that of the right hand. The right hand usually picked the choice shaped I ike one of the parts rather than the unit~d figure. Together with the results of the previous experiment (Biology 1970, No. 179) this demonstrates the superiority of the human right hemisphere for perception of the whole from in­complete or fragmented input.

181. THE PERIODICITY OF EARLIEST EYE MOVEMENTS

Investigator: Colwyn B. Trevarthen

Support: National Institutes of Health, Public Health Service

Newborn infants, who have been born without effects of sedatives or anes­thetics, are frequently highly alert Immediately after delivery. Their eyes are open if the light level is not too high, and they make conjugate saccadic displace­ments of gaze, to left and right, and less frequently up and down. These appear I ike eye movements of an adult scanning a large picture or a scene for visual in­formation, but the infant's eye movements are only occasionally influenced by structure in the visual array.

A ful I-term Infant, born without artificial aids or drugs was observed in the ti rst 10 days of Ii fe. Fi !ms were made of eye movements from the third day and at 9 days polygraphic records were made of horizontal eye and head rotations using techniques devised for infant research. lvhen the baby was in a state of arousal known as "quiet alert" barrages of eye saccades (jumps of fixation) were observed. The recordings show that these movements are close in speed and ampli­tude to adult exploratory eye movements. The eyes rotate together from 5° to 30° taking npproximately 50 mi II !seconds to do so. The remarkable synchrony of muscu­lar contractions to displace and stop the eye which is suspended between antagonist

190

muscle-pairs in the orbit, is present at birth,

The period of oculomotor displacement was obtained by measuring the inter­vals between 300 saccades tor the 9-day old while the baby was "quiet alert." The frequency histogram shows two clear peaks, the longer at 0.3 seconds, the other at 0.6 seconds. Comparison with a histogram obtained from 5 Russian adults by Yarbus l 1967l shows that one-third of a second is a basic attentional interval in human vision when scanning a large picture. The main cadence of saccadic oculomotor ori­entation of gaze is therefore innate and evident at birth.

The baby's rhythmic eye movements continued when al I I ights were extin­guished in the room. The period is clearly independent of structure in the visual array. However, a change, and progressive decline of eye movements in the dark Indicates that a I ight array may influence the infant's visual exploration and the influence irey be stored tor a short period. The 9-day old infant was recorded tracking a lighted ping-pong ball moving slowly across the visual field.

Films have been made of a second infant during the first three days ot Ii te. These cont i rm the above ti ndi ngs.

Re te rences :

Trevarthen, C. and Tursky, B. ll969l Behav. Res. Meth. and Instr. I: 291-293.

Yarbus, A. L. (1967) In: Eye Movements and Vision. Plenum Press, New York.

182. COORDINATION OF EYE ANO HANO IN HUMAN INFANTS

Investigators: Colwyn B. Trevarthen, Sarah M. Ingersoll •

National Institutes of Health, Public Health Service

I ntants 3 months o Id rarely reach tor and grasp a nearby object which they are fixating visually. By 20 weeks ot age most infants immediately reach tor and grasp a smal I tami liar object suspended close enough in front of them. We have studied the beginning of voluntary reaching in infants with the aid of movie films. The babies were supported with head and llrrbs tree, but torso stationary, in a special chair, and were ti lmed in a system of mirrors which provided two simul­taneous camera angles, one from In front, one from vertically above. A smal I toy was placed in different positions at eye height within reaching distance. Seven infants were observed covering the period from 10 to 22 weeks of age.

Al I subjects made responses of wrist and hand, or of arms, directed to the object of visual attention. These responses were regulated according to the distance of the object, giving clear evidence that the baby was detecting the loca­tion of the object in space and following its displacement. However, before 16 to 18 weeks the infants do not combine coordinated gras?ing of the hands with aimed extension of the arm. The attainment of well-regulated reach and grasp at 18 to 22 weeks was accompanied by the ability to guide the hand to take effective hold of the object when it was brought close to the object (visual groping), We have named the early components "pre-reach! ng" to indicate their preadaptive character. Their assembly in the fully coordinated reach and grasp pattern appears to involve both accelerated growth changes in the nervous system, and acquisition of refined structure as a result ot experience with the sensory consequences of reaching and

191

gras~ing m::>vements.

It has been claimed that babies learn to guide the hand to an object by shitting gaze between object and han&, linking them in visual space (Piaget, 1952). Ive have tested this by attaching blinkers to the infant on the outside edge ot one or both eyes, to obscure the hand from view when it is resting at the side against the infant's body. We have never observed the baby search tor his hand, nor is the accuracy of the earliest reaches affected in any way by thus cutting the hand off from view while the object is fixated by the baby. Hand regard, which does occur, is part of another kind ot attention concerned, not with locating tho object, but with the exploration ot patterns ot wrist and finger movement and the! r sensory consequences. It is part of the mechanism by which refined manipulatory ski 11 is attained at a later period (after 20 weeks).

We conclude that the attainment ot aimed reaching coordinated with grasp in the fifth month ot lite by most infants is the result of an inherent pattern of sensory-neuro-motor growth. There is evidence that this grC111th, which must involve the specification of highly complex coordinating structures defining a space of visual perception coincident with a space for voluntary action, is faci I itated by practice, or is plastic. Serious deprivation can retard this development (White, Castle and Held, 1964). It would appear, hC111ever, that plasticity has a secondary role to play in determining the normal order of events and time of appearance in infancy of the earliest voluntary reach and grasp to an object perceived vistially.

This study of voluntary reaching in infants employed films made by James Alt at the Center for Cognitive Studies, Harvard University. The project was inspired by Professor Jerome Bruner's investigations into the biological founda­tions of human intelligence at the Center. We have conttnued analysis of the films at Caltech in collaboration with James Alt.

References:

Piaget, J. (1952) In: The Origins of Intelligence in Children. lnter­nati ona I University Press, New York.

Woite, B., Castle, P., and Held, R. (1964) Chi Id Development 35: 349.

183. DETECTION AND DISCRIMINATION OF VISUAL STIMULI ON BOTH SIDES BY SUBJECTS WITH DISCONNECTED HEMISPHERES

Investigator: Colwyn B. Trevarthen

Support: National Institutes of Health, Public Health Service

Patients with the cerebral hemispheres surgically disconnected offer an opportunity to test the autonomy of mental processes going on in the two hemi­spheres, or alternatively to examine their dependency upon integrative steps in­volving the undivided brain-stem mechanisms. We have measured the speed and accu­racy ot responses by such people when they are performing two simple visual search tasks simultaneously, with stimuli presented both left and right of a fixation eoint. They were asked to simply raise an index finger on the same side as the stimulus vihen the criteria of a correct stimulus were satisfied. Each index finger is mainly controlled from the opposite half of the brain, so this form of reseonse gives us tw0 hopefully independent mechanisms of response, one for e<Jch hemisphere.

There were two kinds of tests. In one the subject was asked to detect

192

a black square <:ippearing for 0.15 seconds unpredictably to Jett or right ot a fixa­tion point. Sometimes no square appeared and sometimes there were two squares appearing simultaneously. In the other test letters were presented in the same way, but now the subject was required to discriminate a target Jetter (Z) from 8 other letters to which he was not to respond. Simple detection ot the appearance ot a black square on left or right imposes no hardship for the commissurotomy subject. Simultaneous responses to the appearance ot two stimuli were made in a normal manner. Abnormalities ot response latency or form could be explained by the epi Jeptic con­dition ot the subjects before commissurotomy.

The double discrimination test, hOtlever, revealed marked deficiencies ot vigi Janee. The operated subjects are unable to concentrate attention simultaneously upon two complex perception processes and Initiate simultaneous responses as a normal subject may do. The responses to simultaneous presentation ot two Z's sh0\'1 rivalry of psychological processes, one finger rising in response before the other, or both rising together, with a delay ot 0.15 to 0.2 seconds after the usual time for a response to a single Z appearing to Jett or right (0.5 seconds). This con-t I ict ot response does not occur with normal subjects which are readily ab le to distribute their attention so that response to two Z's is as rapid as response to one.

Such phenomena, reflected In the accuracy of finger responses as wel I as in the! r latency or speed, show clearly that commissurotomy reduces readiness to perceive or respond. It causes disequilibrium of visual attention for simultuneous stimuli when the subject Is fixating a point between the stimuli. When attention is on one side there is a brief Joss of attention to the other side.

Two epileptic subjects with partial section ot the hemispheric commis­sures (posterior one-third ot the corpus cal Josum intact) had slowed responses, but they did not exhibit the disturbances of perceptual readiness seen in those subjects with complete commissurotomy. We therefore believe that the slowing responses ob­se.rved when two stimuli are presented reflect a Jack of communication between the cerebral mechanisms of visual perception, rather than a defect in motor coordina­tion.

An acallosal subject (born lacking the major interhemispheric commissure> showed minor discoordination of responses and attentional defect, but the responses of th is subject are closer to normal than they are to those of the surgically oper­ated subjects vsith brain completely bisected.

This work was done in collaboration with Dr. Marcel Kinsbourne of the Department of Pediatrics, Duke University Medical School.

184. EYE MOVEMENTS JN COMMISSUR'.ITOMY SUBJECTS

Investigator: Colwyn B. Trevarthen

Support: National Institutes of Health, Public Health Service

Visual attention is served by eye movements. The most frequent are selective jumps of fixation (saccadesl whtch displace the fovea of each eye relative to the I ight focussed on the retina. We are normally unaware of the saccades made at up to 3 ;ier second whl le we scan the visual field for detai I. While we read, the eyes jump slightly rrore frequently as they scan the line.

When inspecting visual arrays human subjects with the cerebral hemispheres

193

surgically disconnected move the eyes in normally conjugate manner to left and right. In general the eye movements belie the fact that each hemisphere receives detal led visual information from only~,one-half of visual space at any instant. Clearly the perception of the visual world depends upon a store of impressions ob­tained in many fixations.

By exposing directive stimuli for 1/8 second (too I lttle time for the brain to initiate a fixation eye movement) to the left or the right of the fixa­tion point, we have demonstrated with commlssurotomy subjects that stimuli entering by either side of the retina may direct an eye movement to the left or the right. A stimulus in the right half of the retina is projected to the left hemisphere, and vice versa. An arrow pointing left or right, or a word "left" or "right" was pre­sented at 3° to one or the other side of the fovea to inform the subject which of two longer lasting stimuli, placed at 6° to the side, were to be Inspected when they appeared one second later.

In most trials, the subject made a brief fixation at the place where the first brief stimulus had been exposed after it had disappeared, looked in this di­rection at some remembered Image of It, then made the appropriate larger step to look at and read one of the pal r of lateral stlmul l. The tact that the subject could aim the eyes to left or right to obey complex visual Information given"to either hemisphere alone, confirms the hypothesis that each cerebral hemisphere is equipped to perceive in both halves of the visual field at any instant. •

Inspection eye-movements to where the directive stimulus had been ex­posed shCMed a consistent asymmetry, suggesting that the left cerebral hemisphere took approximately 1/5 second longer to perceive the information in the stimulus. This asyrrmetry may be related to Inherent differences !n'the visual perception processes of the hemispheres or to serial orienting impulses in left and rights ides ot the brain.

When comparing paired stlmul I in motion at left and right or the orien­tations of pairs of lines crossing the two halves of the visual field, commissu­rotomy subjects show a pronounced tendency to make rapid, Involuntary glances to the right from the midpoint of the display. This would relocate the two stimuli of the display in the left half of the visual field of each eye. It is possible that this orientational bias may reflect a prepotence of the right hemisphere for making judgments of relative motion or relative orientation in the visual field. Conversely, concentration on verbal report of letters flashed to left and right of the fixation point results tn eye movements to the left.

Commissurotomy subjects complain ot abnormal fatigue in reading since their operation. Recordings made of their eye movements while reading indicate that they fa! I to scan efficiently along the I ine with the regular rapid lett-to­rlght steps made by normal readers. Comm!ssurotomy subjects make many more steps, and frequently go back over the line. The causes of this difficulty are being examined. Apparently both hemispheres are involved in the normal process of reading. This skll I would appear to be one depending upon intact neural connec­tions between the hemispheres.

194

185. FINGER-TAPPING TEST FOR DISTRIBUTION OF ATTENTION IN BRAIN-BISECTED PATIENTS

Investigators: Colwyn B. Trevarthen, Charles D. Kreuter

Support: National Institutes of Health, Public Health Service

A finger-tapping test has been used to examine the distribution of inter­hemispheric attention during performance of verbal tasks in 3 cerebral commissur­otomy patients. Finger movement patterns and base rates were determined with the left and right Index fingers tapping independently, simultaneously and alternately. Tapping behavior was then observed during verbal loading (the examiner repeats a simple sentence), during storage (the patient rehearses the sentence si lentlyl, and while the patient repeats the sentence aloud. Simultaneous tapping performance was evaluated during mathematical problem solving and alphabet recitation.

Al I patients were right-handed and all shO<Jed significantly higher tap­ping rates with the right finger alone. Normals, however, showed no frequency dif­ference between left and right hands. Simultaneous tapping rates for all patients were never greater than the maximum frequency of the left (slow) hand and were consequently much slower than for normals. The patients experienced difficulty in digital coordination during simultaneous and alternate finger tapping and much of the responding was asynchronous.

Verbal loadlng, storage and recitation tasks produced slowing of tapping rates for each hand with maximal slowing or interruption during recitation. Math­ematical problem solvlng whl le tapping with both hands was accompanied by inhibi­tion of either the left or the right and less frequently of both hands. Simple problem solving (add 3+1) showed no effect on responding, while more complex prob­lems (add 3-3/4+2-3/8) produced suppression, usually in only the right hand.

• One patient was asked to recite the alphabet omitting alternate letters, while finger-tapping with both hands. There was virtually no change in the left hand response, but the right hand stopped tapping. During one 5-second interval the patient tapped 18 times with the left finger and only twice with the right. The right finger started to tap only during periods when the patient was not attending to the task, i.e., when letters were not skipped properly. Both hands stopped tapping when the patient became completely lost.

Hemispheric control of finger-tapping can originate in either the dominant (left) hemisphere or in the minor one or in both together. Undoubtedly some of the mechanism for production of regular tapping is in sub-hemispheric levels of the brain. Test results indicate that performance of simple verbal tasks involving minimal cortical processing wi 11 not affect response in either hand. Hemispheric processing for a complex task, however, wi 11 compete and interfere with coordinated activity directed by that hemisphere, leaving activity directed by the other hemi­sphere unaffected. Confusion caused by a difficult task may stop cerebral control of both hands.

This work was done in collaboration with Marcel Kinsbourne of the Depart­ment of Pediatrics, Duke University Medi~al School.

195

186. COMPLETION OF VISUAL PERCEPTS BY OISCONNECTED HEMISPHERES

Investigator: Co I wyn B. Trevarthen

Sup port: National Institutes of Health, Pub I ic Health Service

A German neurologist named Poppelreuter, working after the ti rst World War with men suffering gunshot wounds in the head, first drew attention to the tact that people with brain injury may sometimes have holes in their visual fields, but not be aware of it. Something entirely in the blind region is not seen, but other­wise perception extends into, or completes vision in this part. Objects lying across the blind area-are seen to continue through it. A comparable perceptual completion causes the blind spot we al I have for each eye to be hard to locate. Most of us are unaware of this 5° hole in the visual receptor system of each eye,

Perception tests we have performed shew that patients with the cerebral hemispheres surgically disconnected have two complementary halt-fields which, at least near the vertical meridian, are blind-but-perceiving in this sense. The left hemisphere has tul I visual reception to the right of the fixation point only, with a very sharp cut-oft at the vertical meridian. But the other ha! f of the field is not blind for this hemisphere. Our tests show that percepts of words 1 and figures given at the mldline are completed into the left field by the left hemi­sphere. A complementary, or opposite, rightward completion phenomenon is show~ tor the right hemisphere when the commissurotomy subject is asked to make drawings with the left hand of what is presented at the midi ine. Thus we have proved that while each hemisphere receives detailed visual input for the contra lateral ha! f of space only, it perceives in both halves. The subject sees complex percepts extending on both sides of the meridian with each half of his brain, an<J may have two differing percepts coincident at the place he is fixating.

Perception by each hemisphere in the ipsi lateral half-space is affected by information crossing through primitive visual pathways which are not divided by the surgery. These latter processes offer a variable loose coupling between the perceptions of the two cerebral mechanisms of visual awareness.

Two subjects with partial section of the corpus cal losum (posterior, visual section intact) did not exhibit visual completion. Likewise, a young lady born with no corpus cal losum (a congenital agenesls case) perceived the stimuli normally and did not complete the partial figures or words exposed at the midi ine.

This work >ms done in collaboration with Dr. Marcel Kinsbourne of the Deportment of Pediatrics, Duke University Medical School.

fleference:

Trevarthen, C. ( 1970) Brain, Behav. and Evol. (in press).

187. PERCEPTION OF CHIMERAS 8Y COMMISSUROTOMY PATIENTS: FACES, NONSENSE SHAPES, OBJECTS AND PATTERNS

I nvcsti gators: Jerre Levy, Colwyn 8. Trevarthen, koger W. Sperry

~1ational Institutes of Health, Public Health Service Frank P. Hixon Fund

Four commissurotomy ;:iutients wore tested \tith u variety of stimulus

196

materials in order to determine hemispheric dominance for Gestalt perception. Ear­lier work (Levy-Agresti and Sperry, 1968; Levy and Sperry, 1969; Sperry and Levy, 1969) had suggested that the mute right hemisphere of these patients was superior to the speaking left hemisphere for a task requiring the transformation of a t\'lo­dimensional "opened-up" picture into its three-dimensional percept.

In the present study visual chimeras consisting of t\'lo half-stimuli (for example, t\'lo half-faces) joined together at the midline and presented tachistoscop­ical ly for 0 .15 seconds, were shown to the subjects as they fixated on a point cen­tered in the stimulus. Under these conditions the subject's right hemisphere re­ceived only the left half-stimulus and his left hemisphere received the right half­stimuius. As Trevarthen and Klnsbourne had shown earlier (see Report No. 186, Biology 1970), a half-stimulus which extends to the mldline of vision may be per­ceived by a hemisphere of these patients as a whole stimulus, that is, they may complete the stimulus across midline. Each hemisphere, therefore, under the present conditions, saw a whole and complete stimulus, but the stimulus perceived was dif­ferent for each half-brain.

After seeing the chimeric stimulus the subjects were asked to point, with either their left or right hands, to the one of a group of whole stimuli which was shown to them in free vision. They chose the one which matched what they had seen in the tachistoscope. For faces, nonsense shapes, line drawings of objects, and simple line patterns, the choice made In the vast majority of cases was in favor of that stimulus which was seen by the right hemisphere. In other words, the eyes oriented toward, and the hand pointed to, that stimulus which had been presented in the left-half visual field. When the subjects were asked for the same test stimuli to name what they had seen, thus forcing a read-out through the left hemi­sphere, the named stimulus was that which had been presented in the right-half field. However, under these conditions, the number of errors increased dramatical­ly.

These data show that, not only is the right so-cal led "non-dominant" hemi­sf1here superior to the left hemisphere in the recognition of visual gestalts, but it is also strongly dominant in taking control of eye and hand orientation in or~er to respond to what it has seen. Records of spoken exp I anati ons made by the subjects throughout testing also showed that when the left hemisphere did recognize a stimulus it did so, not by apprehending a gestalt, but rather by noting and labeling detailed features of the stimulus. We conclude that the deficit in gestalt perception suffered by the left hemisphere is just as clear and strong a defect as the classically recognized I inguistic deficit of the right hemisphere.

References:

Levy-Agresti, J. and Sperry, R. W. (1968) Proc. Nat. Acad. Sci. 61: 1151.

Levy, J. and Sperry, R. W. (1969) Lateral specialization and cerebral dominance in oommissurotomy patients. Paper read at the Inter­national Congress of Psychology, London.

Sperry, R. W. and Levy, J. ( 1969) Excerpta Medi ca International Congress Series No. 193. The Netherlands: Reidel, Dordrecht, p. 176.

188. RECOGNITION OF AUDITORY AND VISUAL IMAGES BY COMM! SSUROTOMY PATIENTS

Investigators:

Support:

Jerre Levy, Colwyn"il. Trevarthen, Roger W. Sperry

National Institutes of Health, Publlc Health Service Frank P. Hixon Fund

197

In the preceding study it was established that commissurotomy patients show a strong and consistent right hemisphere superiority and dominance for the recognition of complex visual gestalts. Using the same experimental design and some of the same stimuli, we found that a different mental set requiring the recog­nition of verbal-symbolic identity between the stimulus and choice objects shifted hemispheric dominance to the left hemisphere. In addition It was found that even ostensibly verbal material ls recognized by the right hemisphere If the instruc­tions required only a matching of visual appearance.

Presented with the same object chimeras as In the preceding study (Biology 1970, No. 187). The subjects were now instructed to point, not to the object seen in the tachistoscopic exposure, but instead to one of three different objects the name of which rhymed with that of the object they had seen. Care was taken ttiat neither the experimenter nor the subject spoke the name of any stimulus or choice object aloud. Under these conditions, even when using the left hand, the subjfcts pointed to the choice object which rhymed with the right field stimulus object, i.e., the object seen by the left rather than the right - in contrast to the results for direct visual matching.

When we presented non-chimeric intact whole stlmul i entirely in the left visual field, most subjects performed at chance level. One subject was exceptional in showing some indication under these conditions of being able to perform rhyming object matches, consistent with other evidence that this particular patient pos­sesses some right hemisphere speech. The present results strongly suggest that the right hemisphere's expressive language deficit is Intrinsic to the organization of the right hemisphere involving the coordination of speech with auditory images, and is not primarily dependent on extrinsic factors such as motor control. The "rhyming objects" test appears to be an extremely sens I ti ve measure of l ntrl ns i c ex;i ress i ve ability, Involving as It does the recognition of complex spatial patterns, thus favoring the right hemisphere and not requiring any motorlc linguistic output.

We were interested in the question of whether verbal material per se would always elicit a left hemisphere dominance. 6 chimeric pairs of symmetric 4-letter words, divided and recombined as in previously described tests, were presented so that right and left halves of the word were seen by left and right hemis;iheros respectively. Subjects were simply required to point to the word they thought had been exposed. In spite of the tact that the stimull were words, it was the left field which was responded to, suggesting that purely visual or spatial as;iects, rather than the sound or meaning of the word were prepotent in determining the response. It appears that if the instructional set does not specifically require anything other than visual recognition, then it is only this aspect of a stimulus which is used first.

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189. BRAIN MECHANISMS FOR PERCEPTION AND NAMING OF COLORS

Investigators:

Support:

Colwyn B. Trevarthen, Jerre Levy, Roger W. Sperry

National Institutes of Health, Pub I ic Health Service Frank P. Hixon Fund

Two kinds of detect in color perception have been reported to follow I oca I i zed brain injury. Patients may not be ab I e to name co I ors correct I y wh i J e showing in appropriate nonverbal tests that they are able to discriminate colors well. Alternatively, there may be no detect in saying what is seen or naming colors, but color discrimination itself is affected. We have investigated color naming and color discrimination In 6 commissurotomy subjects. Al I 6 are able to discriminate colors normally in free vision, and to give accurate verbal report of what they see in the right visual field. When color stimuli are confined to the left visual field smal I changes in brightness or hue are detected and brightness thresholds for isolated bands of the spectrum are normal, but verbal report of tho hue is poor. Nevertheless, tests revea I that what the subjects say about the I eft field stimuli does reflect recognition of both brightness values and some attribute of hue. Color is discriminated from gray, but when a wide range of brightnesses of gray and a variety of colors are given together, bright patches of light of any color may be confused with yel ICl.'I or orange, while darker patches are reported as blue or red. Significant scores for the approximate discrimination of hue as wel I as brightness have been obtained while the eyes remain fixated in the center of a white card and when verbal or manual signals capable of transmitting information between the hemispheres were eliminated. It is possible that detection of left field colors in the right hemisphere may generate specific autonomic responses leaking information to the left hemisphere by which spoken responses are normally generated. There is evidence that the presence of color is exciting or alerting. However, the latency of the responses obtained favors the view that some brain­stem visual processes are Involved in perceptions of color and that these processes

• may transfer information about the left field to the left hemisphere. Jn certain instances there was evidence of right hemisphere speech. In particular, the changes in double responses, where the first syllable of a color name was uttered and then speech arrested so that a change In response could be made, were almost invariably in the direction of an error. A correct response was cut short and an incorrect response substituted tor it. The first utterance may have been generated by the right hemisphere which perceived the color accurately, the second more arbitrary response is I ikely to coroo from the left hemisphere.

Using the'spl!t or ch!roor!c stimulus technique described above we have tested hemisphere preference for direct color recognition and naming of color. But­terfly-shaped stimu Ii were presented tach i stoscopical ly (0 .15 seconds) with wings symrootrically spread on either side of a black body centered on the fixation point. The wings were bright red, orange or green and al I 6 bi color combinations were pre­sented. The subjects were first asked to respond by pointing with right or left hand to butterflies with both wings red, orange or green; subsequently they were given the same spl It stimuli and asked to report verbally which of the colored butterflies they had seen. 2 of 6 commissurotomy subjects employed the right hemi­sphere for nonverbal matching (pointing) and changed to left hemisphere preference for naming the colors. The remaining 4 subjects were biased in favor of the left hemisphere even when making simple viStJal recognition and pointing. Apparently the tendency to name colors and use verbal mediation for these easily discriminated hues is strong. In some cases the subjects reported that they could see that the two wings of the stimulus were differently colored. They did not name the left wing correctly, but said they imroodiately recognized an imbalance of hue or bright­ness.

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190. HEMISPHERIC MECHANISMS FOR PERCEPTION OF SPATIAL ORIENTATION AND MOTION

Investigators:

Sup port:

Colwyn 8. Trevarth"Eln, Jerre Levy, Roger W. S?erry

National Institutes of Health, Public Health Service Frank P. Hixon Fund

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Chimeric stimuli were made In which arrowheads, clock hands, and oblique­ly inclined, hollow cylinders were represented in conflicting left-right pairs. These split-orientation directional stimuli were presented tachlstoscopically as described above (Biology 1970, No. 187), and the subject was asked to point to a matching stimulus among a ful I array of the individual stimuli.

Commissurotomy subjects shooed strong preferences to respond from the right cerebral hemisphere, as they did with other visual gestalts in the test described above. One subject, who had already shown a strong preference to res;iond to material he was naming with his left hemisphere, gave verbal responses in this test for the right hemisphere stimul I. The findings add further evidence that per­ception of spatial orientation is a function for which the right hemisphere is in­nately better equipped, a result which is In agreement with observations that, sub­jects with right hemisphere lesions as wel I as commissurotomy patients are ?oor at map reading and other spatial tasks, and that they become lost easily. •

In a second test, a right and left dot were made to appear to move simul­taneously but in different directions on either side of the fixation point. The sub joct was asked to report the mot I on perceived. This test al so exposed a strong preference for response according to the percept of the dght cerebral hemisphere, even with verbal response. Some subjects shooed striking disability in describing the orientations or directions of motion of the stimuli presented, indicating that the left hemisphere, in control of speech, is relatively poor at this kind ot dis­crimination.

In the above tests evidence was obtained for partial perception ot both halves ot the stimuli in unison. Some commissurotomy subjects responded as it blending the percepts of the two sides. This is further evidence tor the existence ot primitive anbient visual perception processes associated with centers in the undivided brain stem.

191. PREDICTABILITY OF A CROSS-MODALITY TEST

Investigators: Santosh Kumar, Jerre Levy

Support: National Institutes of Health, Public Health Service

A visuo-tacti le cross-modality test WilS developed by Levy ( 1969) to measure spatial visualization ability ot the commissurotomy patients who underwent surgery tor control ot epl lepsy. The cross-modal lty test mainly consisted of visu­alizing a three-dimensional object as represented in a two-dimensional diagram. In the present study a th ree-d I mens i ona I object (wooden b I ock) was presented to 34 college freshmen tactually in such a way that they were unable to see it, and the two-dimensional diagram was presented visually in the form ot a multiple-choice test. The subjects used right or left hand according to whichever was dominant. The average performance ot the two kinds of subjects shooed insignificant differ­ence as determined by the Fisher's t of 0.04. The rel iabl I ity coefficient of the cross-modality test was found to be .94.

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The cross-modality test had significant coefficient of correlation, at .01 a level, with DAT-space relations, evaluation of semantic relations CEMR) and cognition of figural transformation (CFT). The cross-modality test had an insig­nificant coefficient of correlation of .15 with cognition of figural units CCFU). EMR, CFT and CFU are the factors of the structure of intellect, hypothesized and identified by Guilford <1967).

Only DAT-space relations entered the multiple regression equation as fol­lows: Cross-modality test score= 32.15 + 0.61 DAT-space relations, The multiple R of 0.69 with only DAT-space relations In the multiple regression analysis was sig­nificant at .01 a level. The multiple R was 0.73 when al I 4 independent variables were also included in the multiple regression equation. An increase of 0,04 mul­tiple R was negligible.

Whereas the significant correlation coefficients between the cross-modality test on the one hand, and DAT-space relations and CFT on the other, indicate high predictabi I ity of the vlsuo-tactl le cross-modality approach, the significant coeffi­cient of correlation between the cross-modality test and EMR cal I for further anal­ysis of the visualization processes Involved in answering the verbal relations test.

References:

Guilford, J.P. (1967> In: The Nature of Intelligence. McGraw-Hill, New York.

Levy, J. (1969) Information processing and higher psychological functions in the disconnected hemispheres of human commissurotomy patients, Ph.D. Thesis, Cal lfornla Institute of Technology, Pasadena, Cali­fornia.

192. EFFECT OF RIGHT- OR LEFT-HANDEDNESS ON PERCEPTUAL AND REASON I NG AB I LIT I ES

l~vestigator: Santosh Kumar

Support: National Institutes of Health, Public Health Service

A significant perceptual deficit has been reported by Levy (1969) in sinis­trals, correlated with their greater bi laterality in language function, but no sig­nificant difference was found for verbal ability as measured by Wechsler Adult Jntel-1 lgence Scale. A measure calling for analytic reasoning ability might be expected to differentiate statistically between right- and left-handers. The present in­vestigation used a verbal reasoning test from the differential aptitude test <DAT> battery. It was also expected that the synthesizing tendency of the right hemi­sphere would be more appropriately measured by the 1970 revision of the visuo-tacti le cross-modality test described In the preceding study (Report No. 191, Biology 1970).

An experimental design was prepared to study the perceptual ability of right- and left-handed subjects (undergraduate and graduate students at Caltech). Each subject used both right and left hand. Half of right-handers used right hand first and left hand second, and vice ve~;i for the remaining right-handers. The same design was used with the left-handers. Al I subjects took the verbal reason­ing test, and answered the items with their preferred hands.

The data so far collected reveal that in the perceptual ability test, the average performance of left hands of the right-handers was superior to that of right

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hunds of the samE:'l subjects, statistically significant at .05 a level. The average performance of tho Iott hands of the left-handers was inferior to their right hands, stutistical ly significant at .01 o. level. Moreover, the average score of tho left hands of tho right-handers was greater than that of loft hands of tho left-handers. Tho uvorage score of the right hands or the right-handers was inferior to that of tho right hand of the left-handers. The average performance of the right-handers in the verbal reasoning test was superior to that of left-handers. The study is in progress, and ulthough the results agree with the hypothesis of Levy ( 1969), the conclusion wi I I await the completion of data collection and analysis.

l<eference:

Levy, J. ( 1969) Information processing and higher psychological functions in the disconnected hemispheres of human commissurotomy ?Uticnts. Ph.D. Thesis, California Institute of Technology, Pasadena, Cali­fornia.

193. 1970 REVISION OF SPATIAL ABILITY TEST

I nvosti gator: Santosh Kumar

National Institutes of Health, Pub I ic Health Service •

The previous edition of the spatial ability test consisted of 39 wooden blocks (items) and 13 response curds each containing 3 diagrams. Thus tho first 3 wooden blocks wore represented in the first card, the next 3 blocks were repre­sented in the second card, and so on. It was found that familiarity with the card acquired during the test was affecting the judgment of the subjects, and accordingly a corrective revision of the test was attempted. The length of the test wus reduced from 39 to 34 items. A new set of response cards were made, each corresponding to a wooden block, and each consisting of 3 diagrams out of which only one was u cor­rect response. The blocks are presented tactually, the cards visually. From the previous edition of the test, the items which were more I ike form perception were pooled together. Some new items wore made to construct a sub-test of 10 items tor the form perception. The 1970 revision of tho spatial ability test is being used in current studies of the present investigator >lith the normal subjects and commis­surotomy putionts.

194. IMPAIRMENT OF SINGING AFTER UNILATERAL ll~JECTION OF SOLJ I UM AMYT AL

Investigator: Haro Id W. Gordon

National Institutes of Health, Public Health Service

A recent sori es of patients of P. J. Voge I who were to undergo major bruin surgery was subjected to the Wada technique for determination of hemispheric speech lateralization (Wada, 1949). The clinical procedure consists of intracarotid sodium amytal ingestion which results in hemiplegia of the contra lateral side, while pur­posefu I acts continue with the side ipsi lateral to the injection. The ability of the patient to speak in the few minutes fol l01;ing injection is contingent upon the amount of speech contro I in the depressed hemisphere.

The purpose of this study is to assess the patients' ability to sing once speech has been established. Previous reports have indicated musicnl-type abi I !ties

202

reside in the non-dominant (non-speech> cerebral hemisphere (Mi Iner, 1962; Kimura, 1964). Therefore, if speaking were unimpaired in these patients after amytal inges­tion, singing should be markedly disturbed. The results confirm the hypothesis. After amyta I depression of the right hemisphere in right-handed patients, speech function was relatively Intact while singing was almost totally amelodic. At the same time, however, it appeared that the sense of rhythm was preserved. In 2 of the subjects amytal injections were made to the expected dominant (speech> side on a different day. Certain anomalies prevented cl ear-cut resu I ts, but in genera J, speech was markedly Impaired but singing less so. Thus, support is given tor music functions of the non-dominant hemisphere; yet as pointed out in a recent study with dichotic chords, the specific functions of nonverbal sounds or music have yet to be clarified (Biology 1970, No. 195; Gordon, 1970).

This work was done in collaboration with Joseph E. Bogen, M.D., Ross-Loos Medical Group, Los Angeles, California.

References:

•

Kimura, D. ( 1964) Quart. J. Psychol. 16: 355-358.

Mi Iner, B. (1962) Lateral ity effects in audition. In: V. B. Mountcastle (ed.), lnterhemispheric Relations and Cerebral Dominance. Johns Hopkins Univ. Press, Baltimore.

Gordon, H. W. (1970) Hemispheric asymmetries in the perception of musical chords (submitted for publication to Cortex).

Gordon, H. W. and Bogen, J. E. ( 1970) (in preparation>.

Wada, J, (1949) lgaku to Seibutsugaku (Medicine and Biology) (Japanese) 14; 221-222 •

195. MUSICAL ABILITIES OF THE CEREBRAL HEMISPHERES

Investigator: Haro Id W. Gordon

Support: National Institutes of Health, Public Health Service

Digits, melodies, and chords were presented in dichotic listening tasks to 20 amateur college musicians. Each of the types of stimul I were presented in separate tests in an ear rivalry situation. In the chords test for example, 2 chords were presented, I to each ear, simultaneously for short duration. The task was to select these chords from a multiple choice of 4 played after the stimulus pair. Each of the 3 tests was administered in asimilar manner. The left ear showed a significant superiority of the right In recognizing chords (p<0.02). A further analysis of the results reveals a significant ability for the left ear to perform the task ( p<O .O I) as compared to on I y chance I eve I performances tor tho right ear (p>0,05). This was in marked contrast for the melodies test where both oars could obtain an above-chance score (p< 0.01), yet the means did not differ signif­icantly from each other (p>O.I). Previous studies have shown a loft ear preference in melody recognition (Kimura, 1964) as h{'s been shown here for chords. The present melodics were particularly rhythmic In quality and it is concluded that the non­temporal, melodic qualities of music are the cues that determine lateralization. The left ear superiority in the present chords test and previous melodies tests reflects a right hemisphere dominance since the contralateral ear-to-cortex path-ways are more efficient in conflict situations introduced by the dichotlc stimuli

203

(Kimura, 1967). The chordal qualities found to be lateralized in the present study add to the I ist of nonverb~I functions tor which the nonspeaking, minor hemisphere is superior.

Ro fe roncos:

Kimura, D. ( 1964) Quart. J. Exptl. Psycho!. 16: 355-358.

Kimura, D. ( 1967) Cortex 3: 168-178.

Gordon, H. W. (1970) Hemispheric asymmetries in the perception of musical chords (submitted tor publication to Cortex).

196. DEVELOPMENT OF VISUAL BEHAVIOH IN KITTENS

Investigator: Linda Fagan

National Institutes of Health, Pub I le Health Service

The development of visual responses was studied in over 150 normal l~b­oratory-bred kittens as a basis for concurrent experimental studies on maturation and developmental plasticity in the visual system. Ages for eye opening (eo) qnd the onset of responses to specific visual stimuli, e.g., optokinetic (ok), depth perception by avoidance of a visual cliff (vcl, visual reaching (vr), tracking of small objects (tr), and spatial placing (pp), were recorded. The range and average ages are summarized in Fig. I.

As may be noted in Fig. I, responses to visual stimuli develop gradually over the 5 weeks afterbirth, fol lowing a consistent pattern. The onset of each response most probably corresponds with maturation of the neural elements (e.g., retina, midbrain, cortex) Involved. Attempts to correlate these behaviored data with histological and electrophysiological studies in the literature are in progress.

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197. DEVELOPMENT OF COMPENSATORY v I SUAL FUNCTIONS IN KI TT ms

Investigators: Linda F<igan, Roger W. Sperry

Support: Natlonal Institutes of Health, Public Heelth Service

The effects of early brain surgery on the visual behavior of kittens after their eyes havo opened is being analyzed. To what extent does the developing brain compensate for oarly brain damage - and is such plasticity greater than what might bo found after similar lesions in adult controls'/ Tho surgGry dGstroys spG­cific visual centers or pathways (e.g., visual cortex, midbrain, optic tract). Subsequent tests of movement detection, brightness and pattern discrimination, and depth perception determine whether visual responses develop normally, are retarded, or are completely lost.

Pre Ii mi nary resu Its suggest: (I) "Cortical" vision for pattern discrimination remains uncompensated

whon cortical areas 17, 18, and 19 are removed in the neonatal kitten (before eyes open) and vision involving depth perception shows little or no compensation.

(2) Vision involvlng movement detection and brightness discrimination <midbrain and tectum are intact in al I experiments so tar) showa I ittle or no loss in the tests employed.

(3) When one cortex remains, retarded development is found leading to approximately normal visual behavior, even with associated lesions in the optic pathway to the intact cortex.

198. RETINOTECTAL CONNECTIONS FOLLOWING RE~iDVAL OF HALF­TECTUM IN THE GOLDFISH

I Mvesti gators:

S lipport:

Harb ans L. Arora, Roger W. Sperry

Nati ona I Institutes of Hea I th, Pub Ii c Hoa I th Service Frank P. Hixon Fund

The optic system of the fish, with its ability to regenerate and to re­cover fol lowing various types of lesions and surgical interruptions, continues to offer promising possibilities for study of thG way in which the patterning of nerve connections may be determined in regeneration and also in development. The current investigation ls concerned with the fate of regenerating optic fibers when the area of tho tectum in which they normally terminate is completely removed.

Fi be rs from the nasa I ha I f of the retina, represent l ng the tempera I a roa of the visual field, normally terminate in the caudal or posterior half of tho tectum, while thosG from the temporal retina terminate in the rostral tectum. We have rerroved the caudal halt of the tectum in a number of specimens of adult gold­fish and find no evidence of subsequent regeneration of tectal cells, nor does the residua I tectum show any increase in size th rough out the recovery po ri od. In the period immedintely after surgery the visual field of the operated eye of the fish is restricted to the nasal area only and -removal of the tem;>oral half of the retina brings about total blindness. After a period of 90 to 120 days from the original operation, the fish recovers the ability to see within the temporal field, indicating that the fibers from tho corresponding portion of the retina have estab­lished functional connections with the remaining half-tectum, confirming an earlier physiological study of Gaze and Sharma ( 1970). Since the portion of the tectum

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regularly innervated by those fibers is no longer present, the connections must take pl;ce within the residual area that normally corresponds to the nasal field and was originully innervated by only the temporal half of the retinu. Although the pres­once of visual response in the temporal field can be demonstrated, its quality re­mains to be dotormined. Histological ~tudies are also being performed in an effort to determine the pathways fol lowed by the regenerating fibers cind their terminations as they enter the residua I tectum.

Reteronce:

Guzo, fl. M. and Sharma, S. C. (1970) Exptl. Brain Ros. 10: 171-181.

199. SPlC IF IC I TY ANll PLASTIC I TY IN NEURAL RECONNECT IONS BETWEEN THE EYE AND THE BRAIN OF ADULT GOLllF I SH

t nvesti gutors; Myonggeun Yocn, Harbans L. Arora, Roger W. Sperry

Support: National Institutes of Hen Ith, Pub I ic Health Service Frnnk P. Hixon Fund

Reti notecta I projections were mapped from norma I and operated adu I tcgol d­f i sh by electrophysiological methods. The maps of retinotectal projections ob­tained from operated animals whose intact tecta were reinnervated by regenera~ing optic nerves ure similar to those obtnined from normal goldfish. This result agrees with the previous results of Attardi and Sperry ( 1963) and Jacobson and Gaze (1965), implying that there exists u specific mechanism(s) for functional connec­tions between the retina and the optic tectum not only during embryonic development but also during regeneration at an adult stage. •

In another group of experimental animals the caudal half of the tectum was ablated and the contralateral optic nerve was crushed at the same time. In the case of one operated animal of this group, tested tor its retinotectal projection 4~ dnys after the operation, the positions of the center of receptive field tor 4 units, recorded from tectal positions near the posterior end of the remaining ros­tral ha! f-tectum, were displaced from the vertical median towards the temporal vis­ual field by 50°"'70°. These receptive field positions would have corresponded to the posterior end of (ablated) caudal tectum if the animal had had an intact whole tectum. This preliminary observation is not inconsistent with a recent result of Gaze and Sharma ( 1970) who report that the whole visual field is projected upon the rostral ha! f-tectum of nn operated goldfish in a contracted form. Further analysis of the observed plasticity is in ?regress.

References:

Attardi, lJ. G. and Sperry, R. W. ( 1963) Exptl. Nourol. 7: 46-64.

Jacobson, M. and Gaze, H. M. ( 1965) Exptl. Neurol. 13: 418-430.

Gaze, I<. M. and Sharma, S. C. <1970) Exptl. Brain f{es. 10: 171-181.

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200. VISUAL RESPONSES OF OPTIC TECTAL UNITS IN THE GOLDFISH

Investigators: Myonggeun Yoon, Ronald L. Meyer

Support: National Institutes of Health, Public Health Service National Defense Education Act

Action potentials were recorded from multi-units in the optic tectum of adult goldfish. Three types of responses, 11on, 11 11off, 11 and 11on-off 11 were observed (Jacobson and Gaze, 1964). Some units showed directionally selective response to a moving stimulus <Cronly-Di I Ion, 1964). The size of roceptive fields was estimated with a series of black test spots (with luminance 2.3 l~g10 ft. Lamberts) of various sizes (1°"-20° in diameter) against a white background (1.4 loglO ft. Lamberts) by determining subjectively a "threshold size" of the test stimulus above which the response magnitude did not increase any further as the area of test stimulus in­creased. The determined size of receptive fields, for which the effect of stray light from a test light spot was completely eliminated, ranged from 5° to 10° in diameter.

When a preparation is in good condition, most tectal units show spontane­ous discharges at a mean rate of I0"-30 spikes/second. The mean rate usually de­creased as the background luminance increased. The spontaneous discharges are ex­tremely sensitive to anoxia. When the perfusion of water to the animal 1s gi I I through the mouth was interrupted, the spontaneous discharges disappeared within a minute, whereas transient responses could be evoked by (stronger) I ight stimuli up to 10 minutes after the interruption of water perfusion. When the perfusion wus resumed, the spontaneous discharges reappeared within I or 2 minutes and gradually recovered to the previuus mean rate at the same background luminance.

References:

Jacobson, M. and Gaze, R. M. ( 1964) Quart. J. Exptl. Physiol. 49: 199-209.

Cronly-Dillon, J. R. (1964) Nature203: 214-215.

20 I . THE PROCESS I NG OF VISUAL INFORMATION ASSOCIATED WITH LEARN I NG IN CH I CKS

Investigator: Larry I • Be now i tz

Support: National Institutes of Health, Public Health Service

Interocular transfer of an avoidance learning task was used to determine the pathways involved In the processing of visual information in chicks. Training was done with only one eye open. Chicks wero trained to avoid a rretal I ic lure at which they are normally predisposed to peck, and then tested with either the same lure, or I of 2 dissimi lur lures. In one group of subjects, the same eye used in training was open for testing, while in another group the other eye was used. /1 difference was found according to whether the sarre or contra lateral eye was used. using the same eye for testing and trainl..ng, chicks avoided the lure used in train­ing but pecked at the dissimilar lures. Using the opposite eye for testing, chicks avoided the dissimilar as wel I as the same I ure to a greater extent than had the group using the same eye. This suggests that the discrimination learning in tho hemisphere contralateral to the trained eye was more precise than that in tho ipsi­lateral hemisphere. Combining these results with ablation studies, which show

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local izable areas responsible for spec! fie memory functions, we are attempting to understand the processing of visual information and its association with learning.

202. ABSENCE OF A~NESIC EFFECT WITH CURRENT ADMINISTERED TO CHICKS REEXPOSED TO THE TRAINING SITUATION

Investigator: Evelyn Lee-Teng

Support: National Institutes of Health, Public Health Service

Transcranial currents administered within 30 seconds after a training tri­al have been shown to cause retention deficit of the training experience in newly hatched chicks; the shorter the training-to-current interval, the poorer the reten­tion. These results have been interpreted in terms of disruption by current of memory trace formation. Sane recent studies with other animals suggest, however, that current Impairs any memory active prior to its administration, instead of only memory of events experienced for the first time. To check this possibility, day­old chicks were trained In one trial to suppress their predilection to peck at a small shiny bead. I hour after the training trial, the bead was re-presented to each chick, followed inmediately by transcranial current. Re-presentation of the bead served to activate memory of the training experience first introduced 60 minutes previously. No amnes I c effect was observed when retention was tested I day later. Together with earlier demonstrations of complete amnesia by current given immediately after the original training trial, the present result indicates that, at least in the chicks, current does not affect indiscriminately any memory active before its delivery, rather it only disrupts memory of new /earning.

Re te rences :

Lee-Teng, E. and Sherman, S. M. ( 1966) Proc. Nat. Acad. Sci. 56: 926-93/.

Hisanin, J. R., Mi I /er, R.R., and Lewis, D. J. ( 1968) Science 160: 554-555.

Lee-Teng, E. ( 1970) Proc. 78th Ann. Conv. Amer. Psycho/. Assoc., 225-226.

203. RETROGRADE AMNESIA GRADIENTS BY SUBCONVULS/VE AND HIGH CONVULSIVE TRANSCRAN/AL CURRENTS IN CHICKS

Investigator: Evelyn Lee-Teng

Support: National Institutes of Health, Public Health Service

Either a subccnvulsive or a high convulsive current was delivered trans­crania//y at various intervals after a learning trial to independent groups of day­old chicks. The effect of current on retention was measured I day later. Results indicate a current-sensitive memory consolidation period of about 30 seconds that cannot be grossly prolonged by a 10-fold increase in current intensity and a simul­taneous 2-fold increase In current duration. On the other hand, at comparable t ri a I -to-current i nterva Is with In this brief period, more retention deficit was caused by the higher current. Beyond this critical period, both currents sti II effected some retention deficit, but it did not seem to vary within the trial-to­current interval from I minute to 24 hours. Overt muscular convulsion was unnec­essary for the retrograde amnesia effect. The subconvulsive current may be a more pure agent for consolidation studies because the high, convulsive current may affect

208

test performance in ways other than disrupting the consolldatlon processes.

References:

Gherkin, A. (1969) Proc. Nat. Acad. Sci. 63: 1094-1101.

Lee-Teng, E. ( 1970) Proc. Nat. Acad. Sci. 65: 857-864.

204. TWO SEPARABLE PHASES OF BEHAVIORALLY DEM:JNSTRABLE MEt-ORY FOR ONE-TRIAL LEARNING IN CHICKS

Investigators: Evelyn Lee-Teng, J. Geoffrey Magnus

Support: National Institutes of Health, Public Health Service

Earlier studies with post-training administration of transcranial current in chicks indicate that memory trace responsible for retention at I day is gradually formed. If so, some other phase(s) of memory must exist to account for retention before its formation. By varying the training~to-current interval and the train­ing-to-test interval, we found that immediate post-training administration of cur­rent left a low level of retention at 30 minutes which disappeared within 2 hours. When current was delayed for I minute after the training trial, substantial reten­tion was observed at 30 minutes which increased to asymptotic level at I hour. These results may indicate the existence of 2 phases of measurable memory. Phase I is irrrnune or only partially susceptible to current disruption and lasts for at least 30 minutes. Phase 2 is Induced within seconds after training, but its trace formation µs a long process involving several stages; only the beginning stage is curront-disruptable, but this phase of memory is not expressed until the entire process is comp I ete.

Re te rences :

Lee-Teng, E. (1970) Proc. Nat. Acad. Sci. 65: 857-864.

Lee-Teng, E., Magnus, J. G., Kanner, M., and Hochman, H. (1970) lnternat. J. Neurosc i ences I : (in press) .

PUBLICATIONS

Lee-Teng, E. and Sherman, S. M. ( 1969) Effect of forebrain lesions on acquisition and retention of one-trial learning in chicks. Proc. 77th Annual Con­vention, APA, pp. 2-3-204.

Lee-Teng, E. ( 1970) Retrograde amnesia gradients by sub.convulsive and high convul­sive transcranial currents in chicks. Proc. Nat. Acad. Sci. 65: 857-864.

Lee-Teng, E. (1970) Absence of amnesic effect with current administered to chicks reexposed to the training situation. Proc. 78th Annual Convention, APA, pp. 225-226.

Lee-Teng, E., Magnus, J. G., Kanner, M., and Hochman, H. ( 1970) Two separable phases of behaviorally manifest memory tor one-trial learning in chicks, lnternat. J. Neurosciences (in press).

2()9

LGvy, J. <1969) Possible busis tor the evolution of lateral specialization of the human brain. Nature 224: 614-615.

S;>erry, R. W. (1969) A modified concept of consciousness. Psychological Review 76: 532-536. '•

Sperry, R. W., Gazzaniga, M. S., and Bogen, J.E. (1969) lnterhemispheric relation­ships: The neocortical commissures; syndromes of hemisphere disconnection. In: P. J. Vinken and G. W. Bruyn (eds.), Handbook of Clinical Neurology, Vol. 4, Chap. 14. North-Ho! land Pub I ishlng Co., Amsterdam.

Weaver, T. A. and Magnus, J, G. (1969) Effect of unconditioned stimulus I inked subconvulsive current in chicks. Psychon. Sci. 16: 265-266.

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210

Professor: Edl'lard B. Lol'lis

Research Fol low: Kimiko Asano

Graduate Students: Michael B. Klayman, Gary C. Scheidt

Research Assistants: Kay S. Groisen, Margaret M. Phi I lips, Ameliu ;,1. Smit

Laboratory Aides: Linda M. Hollywood, Judith E. Rush, Kitty L. Van Buoron, Curo Ii no Vermaes, I reno E. Von Hartmann

Near tho middle of the right arm of tho third chromosome of lJrosophi la are a group of remarkable mutants which profoundly alter the body segmentation pat­tern in sharp, well-defined ways, such as causing extra pairs of wings or logs to apeoar on the fly. We ilro concentrating on finding out hCl.'I this region of the chromosome is intogrutod into the rest of tho genome. One of tho major lines of attack is to use chemical mutagens to identify as many as possible of tho gene functions which cun exist in this "bithorax" region of the third chromosome. Gary Scheidt, a graduate student, is engaged in studies of this kind.

Some of the mutant effects arise only because of changes in position brought about by chromosomal rearrangorrents. Michael Klayman, another gruduuto student is undertuking an analysis of il special cluss of rearrangements with break­uge points in tho vicinity of one of tho major regulatory genes of tho bithorux region. A number of regulatory-like genes have been identified within tho bithorax region and in surrounding regions. The study of such a class of genes, whose func­tion appears to be to turn on and off other genes at specific times and places, should tell us much about how genes arc able to program the growth and development of an organ ism.

205 .• ANALYSIS OF REGULATORY GENES 11~ DRJSOPH I LA

* E. B. Lewis, Y.-w. Fong

U.S. Atomic Energy Commission National Science Foundation

The way in which genes bring about the orderly growth and development of an organism is a central problem in biology and ono which is largely unexplored. We are attempting to determine to what extent developmental pathways in a higher organism are under the same typo of genetic control as are the biosynthetic path­ways in a microorganism. In tho latter case, tho switching on and off of tho structural genes which control the enzymatic steps in such pathways is accomplished, as Jucob and Monad first showed, by a special class of regulator genes. Mutants of such regulutor genes result in tho turning on or off of \'/hole groups of genes involved in one or more bi osynthet i c pathways. Mutants of regu I ator genes which control tho switching on or off of genes control I ing developmental pathways should load to bizarre developmental effects such as the transformation of one organ or part of the body into unothcr, homologous one. Just such mutants are known in a number of higher organisms. In Drcsophi la Q striking example is tho cuse of mutonts which cause tho fly to develop 4 wings instead of 2. At least 2 mutants ore noodod to accomplish this change and the genes involved are located close together in the right urm of tho third chromosome of Drosophila. Those mutants known as bithorux

* Undergraduate, Cul ifornia Institute of Technology.

211

(bxl and postbithorax (pbxl evidently represent mutated forms of genes which are normally concerned with suppressing wing-development on the third thoracic segment. As a result, that segment bears only a rudimentary pair of appendages, the halteres, in place of wings. The normal bx gen~, bx+, and the normal pbx gene, pbx+, are regulatory in the sense that they evidently prevent whole blocks of wing-forming genes from being turned on in the third thoracic segment. There presumably are still other regulator genes which prevent the bx+ and pbx+ genes from being turned on in the second or wing-bearing segment. Otherwise the f I y wou Id be wing less.

We now have evidence for the existence of this latter type of regulator gene in the form of two types of mutants, one (Rg-pbxl which acts as a pCN1erful re­pressor of pbx+, wh i. le the other ( Rg-bxl acts as a feeble repressor of bx+. We have been concentrating our studies on the more dramatic mutant, Rg-pbx. It causes the fly to express to a variable degree wing-like halteres, on either or both sides. The analysis is complicated because the mutant, which arose after mutagenizing with ethyl methane sulfonate, is associated with a smal I inversion. Nevertheless it has been possible to shCN1 that the Rg-pbx is located at least 50 bands to the left of the bx and pbx genes, which are tightly clustered In a region near the middle of the right arm of the third chromosane. That the Rg-pbx mutant exerts its effect by repressing the pbx+ gene ls made likely by gene dosage studies which shCN1 that the more doses of the pbx+ gene, the more nearly normal the development of the third thoracic segment.

Since the Rg-pbx gene is relatively close to the pbx+ gene, a systematic attempt has been made to separate these two genes by Inducing chromosomal rear­rangements. To accomplish this, a series of translocations between the Y and third chromosomes was first synthesized by Yee-Wing Fong. From a cytological analysis of these rearrangements, one translocatlon <Y-3lP9 has be~n found which causes pbx+ to be shifted to the Y chromosome while the Rg-pbx mutant remains in its original position relative to the centromere of the third chromosome. In spite of the physi­cal separation of pbx+ from Rg-pbx in this trans location, the phenotype of the Rg­pbx mutant ls not noticeably altered. However, a quantitative study of the pheno­type has not yet been completed, so that a position effect of Rg-pbx on pbx+ is not ruled out. This study has provided the starting point for developing smal I chromo­somal fragments carrying the Rg-pbx mutant. Experiments involving X-irradiation of the <Y-3JP9 translocation are underway to produce such tragoonts, which will then be used to study the effect of varying the number of doses of Rg-pbx in the presence of a constant number of pbx+ genes. This approach should provide an ad­ditional test of the validity of the model that Rg-pbx acts as a repressor of the pbx+ gene.

Further studies have been made of the Rg-bx mutant which acts as if it were a weak and variable repressor of the function of the bx+ gene. It has. been found that this effect is dominant and evidently is the result of a deficiency for a region (888-C) of the sal I vary gland chromosomes that is approximately 50 bands to the left of the bx region (in 89E). This places the locus of Rg-bx close to one of the breakage points of the Rg-pbx i overs ion and suggests that Rg-bx and Rg-pbx may be in proximity the way bx+ and pbx+ are. Experl ments are under way to develop a double mutant between Rg-bx and Rg-pbx. This involves irradiation of a chromo­some carrying Rg-pbx to produce a deficiency for the Rg-bx region. Such deficien­cies should be detectable because the existing Rg-bx deficiency includes the locus of a visible eye color mutant, red.

212

206. GENE FUNCTIONS IN A SMALL REGION OF A DF\IJSOPHILA CHROMOSOME

Investigator: Gary C. Sche I dt

Support: National Science Foundation

It is ordinarily not possible to saturate a portion of the genetic map of Drosophila with mutations In any systematic way, since in a typical mutagenesis ex­periment mutations are randomly induced and are spread about the whole map. However, smal I regions of the map can be intensively studied by utilizing known deficiencies and selecting for mutations included inside the limits of these deficiencies. Such a method is currently being used to study a region In the right arm of the third chromosome which includes the blthorax mutants. The deficient region is thought to contain perhaps two dozen genes, but only one gene in addition to the bithorax series is now known. The experimental procedure involves mutagenesis of suitably marked stocks with the chemical mutagen ethyl methane sulfonate. Matings are car­ried out in such a way that in the F2 generation, flies heterozygous for the defi­ciency and the treated chromosome are recognizable, and any mutations induced in the region are identified in these flies. Other flies in this generation are used to establish stocks of the mutants.

Preliminary experiments have shCW1n that the selection procedure outlined does in fact allCWI recognition of mutations confined to this region. About 20 le­thals have already been isolated, and complementation tests between these lethals are being carried out currently. New mutant screening experiments are also planned. It is hoped that the mutants isolated may give some indication of the diversity of genetic function in smal'I regions of chromosomes of higher organisms; in particu­lar, it would be interesting to recover new clusters of mutants I ike the bi thorax series.

207: A NEW CASE OF COMPLEMENTARY LETHALITY IN DF\IJSOPHILA

I nveSti gator: Gary C. Scheidt

Support: National Science Foundation

Sturtevant C 1956J has described a complementary lethal system involving the genes prune, pn, and Killer of prune, K-pn. All £.!2_;K-pn flies die in the second larval instar for unknCW1n reasons; otherwise, K-pn has no detectable pheno­type.

In recent crosses a new al Isle of K-pn, designated K2-pn, has been found. Detailed studies have not yet been performed;-bllt K2-pn resemiiTSSl<-pn in that al I alleles of pn so far tested are ki lied. In addition, no recombination between K-pn and K2-pn has been observed in I imlted tests. However, K2-pn differs from K-pn in two ways. First, pn;K2-pn flies apparently die at the end of the third larval instar rather than atthe end of the second instar. Second, a variable and extreme­ly low percentage of pn;K2-pn flies survive to adulthood. This result has been ob­tained with two alleles of prune and so is probably a characteristic of K2-pn rather than of the prune al Isles. No escapers hav~-ever been reported for K-pn-:--lnitial observations indicate that at least some pn;K2pn third instar larvae live as long as 3 weeks or more without pupating, and thaT"'"'S""ome larvae are eventually able to pupate and give rise to the escapers. Further experiments are planned to clarify the exact time of lethality of £!!_;K2-pn flies and the genetics of the surviving flies.

I

I

213

Reference;

Sturtevant, A.H. (1956) Genetics 4i: i 18,

PUBLI CAT i ON

Abrahamson, S. and Lewis, E. B. C 1970) The detection of mutations in Drosophila melanogaster. In: A. Hoi laender Ced.), Environmental Chemical Mutagens. Plenum Publishing Corp, Cin press).

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214

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BIOPHYSICAL CHEMISTRY

CELL BIOLOGY

Church - Alles

(Second Basement)

Professor: Jerome Vinograd

Senior Research Fellows: Lajos Pi k6, John A. Petruska

1l Research Fellows: Wi I liam R. Bauer, Irving H. Brown, David A. Clayton, Robert Eason, Horace B. Gray, Jr.*, Richard L. Hallberg, John M. Jordan*, Harumi U. Kasamatsu, Bernard M. J. Rilvet, Mark G. Rush, Maurice Schmlr", Henry T. Tai, Alex J. van der Eb

Graduate Students: Wesley M. Brown, P. John Flory, Jr., Charles Allen Smith, C1arn J. B. Tibbetts, Wi 11 iam B. Upholt*

Associate Biologist: Patrick F. Koen

Associate Biochemist: Robert Watson

Research Assistants: Jean Edens, Marlyn R. Tep I itz

Research Aides: Cheryl A. Everton, Denzl I le T. Stephens

Loboratory Aides: Julie van den Kolk, Wanda L. Owens

208. COMPLEX MITOCHCA'IDRIAL DNA IN LEUKEMIC LYMPHOCYTES •

Investigators: David A. Clayton, Marlyn R. Teplitz

S up;iort: National Institutes of Health, Public Health Service

215

The occurrence of circular dimer mitochondrial DNA CrrilNA) in human leuke­mic myelocytes has been established (Clayton and Vinograd, 1969). This study has been extended to include human leukemias of the lymphatic system. Preliminary re­sults indicate that mitochondria from white eel Is of 2 patients with chronic lym­;ihatic leukemia do not contain circular dimer rrilNA. A survey of the mDNA from 6 ;iatients with acute Jymphocytlc leukemia showed that 3 of the patients had circular dimer rrDNA ranging in frequency from 2 to 20%. The other 3 patients had no circu­lar dimer rrilNA. These results Indicate that the correlation between the occurrence of circular dimer mDNA and lymphocytic leukemia is not the same as tor myelogenous I eukem ia. A larger survey is needed to c I ari ty the above res u I ts.

RefBrence:

Clayton, D. A. and Vinograd, J. (1969) Proc. Nat. Acad. Sci. 62: 1077.

209. CIRCULAR DIMER MITOCHONDRIAL DNA IN MALIGNANT MELANOCYTES

** Investigators: Jeanne M. Wallace , Marlyn R. Teplitz

Support: National Institutes of Health, Public Health Service

In order to study functional aspects of circular dimer mitochondrial DNA CmDNAl, it is desirable to have an established cell line which has a reasonable

* Division of Chemistry and Chemical Engineering, California Institute of Technology, ** NSF Summer Trainee.

216

proportion of its rrONA in the clrcular fonn. A survey of the rrDNA populations of 3 ma! ignant mouse eel I I ines, CL, GH-1, and melanoma clone M, was undertaken.

The rrONA from the CL and GH-1 cells was similar to that found in a vari­ety of nonnal tissues. In contrast, the melanoma I ine has the circular dimer species at a significant level of 3.3+1%. It should therefore be possible to uti-1 i ze this ce 11 I i ne in experiments des! gned to e I uci date the functi ona I ro 1 e of circular dimer rrONA.

210. COM"LEX MITOCHONDRIAL DNA IN CELLS IN CULTURE

Investigators:

Support:

* John M. Jordan , Al ex J. van der Eb, Jerome VI nograd

Netherlands Organization for the Advancement of Pure Research National Institutes of Health, Pub I le Health Service

We have undertaken a survey of the occurrence of complex forms in selected estab Ii shed eel I I Ines: BHK 21/13 from hamsters, 3T3 and 3Tl2 from mice, Wl38, intestine 907, and HeLa SI from humans. Except for Wl38, a diploid eel I, and 3T3, a contact-inhibited cell, the mitochondrial DNA (rrONAl in the above I Ines contained a 2- to 3-fold higher level of catenated fonns than in anlmal tissue. The rrONA from the 3T3 I ine and the Wl38 strain contained a nonnal level of catenated forms.

The rrONA in a SV40 transformed BHK 21/13 line contained a further 2-fold increase in the fraction of catenated molecules; the mDNA in the SV40 transfonned 3T3 I ine was even more complex - 20% catenated dimers and 4% circular dimers as re­ceived from Dr. G. Todar-0 (National Institutes of Health). Further subculture under control led growth conditions in this laboratory resulted In a progressive increase in circular dimers and a decrease in catenated dimer forms. The distribution reached an apparent steady value of 65 weight% of single and interlocked forms of circular dimers after approxlmately 40 generations. The time course suggests a selection for eel I types containing a high frequency of circular dimers under our growth conditions.

21 I. EFFECTS OF THE INTERCALATING DYE ETH I DI UM BROMIDE ON THE MITOCHONDRIAL DNA OF CELLS IN CULTURE

* Investigators: Charles Allen Smith, John M. Jordan , Jerome Vinograd

Support: Natlonal Science Foundation National Institutes of Health, Public Health Service

The intercalating dye ethldium bromide (EB) has been extensively used to isolate and study closed circular DNA. It has recently been reported that EB in­hibits the formation of mitochondria I RNA in HeLa eel Is. Studies are now in progress to examine the effects of EB on the properties of mitochondrial DNA CrrDNAl and on the growth characteristics of HeLa eel Is and the eel Is of a SV40 viral trans­formed mouse cell line.

At EB concentrations of about 0.1,µg/ml, HeLa eel Is can be maintained in a growing state for several generations, after which eel I division ceases. As the concentration of EB is raised, cell growth diminishes. There is some evidence that even at O. I g/m I , synthesis of new mDNA ceases.

*Division of Chemistry and Chemical Engineering, California Institute of Technology.

217

The closed circular DNA isolated from eel Is treated with EB is character­ized by an altered superhelix density, a0 • The absolute value of this quantity is increased by a factor of about 3. The sedimentation coefficient of this closed DNA is about 30% greater than the DNA frOfll cells grown in the absence of EB. Simi lilr results were obtained with the mouse eel I I lne. The mouse eel Is u?;>ear to be more sensitive to EB than HeLa ce I ls.

The results of experiments with eel Is grown for a long period in the pres­ence of radiooctive DNA precursors allow us to conclude that the superhelix density of the closed mDNA formed prior to ;iddition of EB ls increased. It has therefore been estublished that mlJNA in eel Is in a medium containing EB serves <is a substrate for both Qndonucleas~ and polynuclootlde ligase-like activities.

212. MICRJINJECTION OF MITOCHONDRIA INTO THE FERTILIZED MOUSE EGG

* I nvesti gutors: Lajos Pik6 , David A. Clayton, Jerome Vinograd

Sup port: National Institutes of Health, Public Health Service

Experiments on the transfer of mitochondria In molds indicate that 'mutant mitochondria are able to survive and function when microinjected into the cyt~plasm of u normal (wi Id type) strain. We have undertaken similar experiments in mammals, using the ferti Ii zed mouse egg as the recipient, and mitochondria from cultured tis­sue cells for microinjection. The donor mitochondria are selected on tho basis of distinct properties of their DNA, e.g., distinct buoyant density or the prevalence of one or another of the complex forms of mitochondrial DNA. In this system a smal I admixture of donor-type mitochondrial DNA could be detected by buoyant density cen­trifugation or electron microscopy.

So far the technical problems of microinjection have been successfully overcome. Approximately 3D to 50% of mouse eggs mlcroinjected at the pronuclear stage undergo subsequent cleavage in vitro. However, when the eggs microinjected with foreign mitochondria were reimplanted into 2 recipient females <which had been mated previously with a vasectomized male to induce pseudopregnancyl, no offspring were obtained. Since the success of reimplantation is expected to be of a low rate, no conclusions can be drawn from these initial results. The fate of the foreign mitochondri<i is also being investigated by electron microscopy of fixed and sec­tioned eggs at various times after microinjectlon.

213. DISTRIBUTION OF CLOSED CIRCULAR MITOCHONDRIAL DNA IN THE AN I MAL KINGDOM

I nves ti gators : Wesley M. Brown, Jerome Vinograd

Support: National Institutes of Health, Public Health Service

The existence of closed circular mitochondrial DNA CrrDNAl with a contour length of approximately 5 µhas been demonstrated in vertebrates, echinoderms, and insects. It is not known how extensively this type of mDNA is distributed among the remaining animal phyla, and a systematic survey is currently in progress to determine this. CJ osed circular 5 µ mDNA from the Ca Ii torn i a musse I C My ti I us californianus) has been found and characterized, thus establishing its occurrence

* Veterans Administration Hospital, Sepulveda, California.

218

in the phylum Mollusca. Circular 5 µDNA from a sea anerrone (genus Anthopleura) preparation has been viewed in the electron microscope, but the occurrence in this instance may be due to contaminating parasites or commensals.

When the survey is complete, mDNA from representative species wi I I be used in cross-hybridization studies. It is hoped that the results of the survey and the hybridization studies will help to clarify the relationships arrong some of the animal phyla.

214. RELATEDNESS AMONG MITOCHONDRIAL DNAs IN THE ANIMAL KINGDOM

Investigators: Richard L. Hallberg, Wesley M. Brown, Jerome Yinograd

Support: National Institutes of Health, Public Health Service

Circular mitochondrial DNA CmDNA) of approximately uniform size occurs in animals as primitive as the Echiuroid worms. These mDNAs, which can be readily obtained in pure form, represent a single collection of genes. Evolutionary changes in these genes can be studied by examining the relatedness between mDNAs of differ­ent species. The complements of mDNA, read! ly separated from each other in alkaline buoyant CsCI density gradients, are used to form interspecies heteroduplex rrolecules. Such hoteroduplexes can be examined for relatedness by Kleinschmidt electron micros­copy, buoyant density centrifugation, hybridization competition with radioactive UNA, and by thermal melting behavior fol !Oiled by absorbance changes or by elution from hydroxy apatite c?I umns.

We have prepared separate heteroduplexes from the complements of mDNA from Xenopus laevis (toad), chicken, rrouse, and human cells. The buoyant densities of the heteroduplexes are between the mean buoyant densities of the separate strands an~ the mean buoyant densities of the two native DNAs in question. The fractional duplex content indicated by this procedure varies from 45% for the Xenopus/human heteroduplex to 65% for the rrouse/human duplex.

f\ reproducible map of duplex and denatured loop regions has been found in electron micrographs of the Xenopus/human heteroduplex. Approximately 50% of the I ength is in 12 duplex segments. Mouse/human heterodup I exes for-med under the same renaturing conditions and prepared for electron microscopy under the same denaturing conditions appears to consist entirely of duplex material. When the latter heteroduplexes are prepared for electron microscopy under more severe dena­turing conditions, single-stranded loops appear. This result indicates that the Xenopus/human map may depend upon the conditions used in preparing specimens for electron microscopy.

The absorbance melting profile of the rrouse/human heteroduplexes is essen­tially noncooperative. Melting, as indicated by hyperchromiclty, begins at approxi­mately 25°C and increases monotonically up to about 90°C. These heteroduplex mole­cules are therefore thought to contain smal I single-stranded loops or mismatched regions in the duplex that are not visible in the electron mlcrosc~pe. These studies are continuing with human mDNA and more closely related primate mDNAs.

,,_

215. THE MOOE OF REPLICATION OF MITOCHONDRIAL ONA IN HELA CELLS

Investigators: P. John Flory, Jr., Jerome Vinograd ,_

Support: National Institutes of Health, Public Health Service

219

The use of density labeling to study the replication of mitochondrial ONA (mONA) has continued with an examination of the kinetics of labeling. Col Is have been grown for varying lengths of time in the presence of 5-bromodeoxyuridine <~-BUOR) and the mDNA examined by buoyant density analysis. Fully labeled CHH) niJNA appears in smaJI amounts after less than half a cell generation. This obser­vation raises the possibility that a given mONA molecule may rep I icate more than once during a eel I generation, but synchronized cultures wi 11 be necessary to set­tle this point. In addition, unlabeled (LL) mONA persists after 2 cell generations, indicating that not every mDNA molecule replicates during each eel I generation.

These studies have also revealed the existence of 2 unexpected species of molecules which appear after loss than I eel I generation as 2 peaks between the LL and hybrid C 1/2 HHl peaks. Their average buoyant density corresponds to one­quarter heavy (1/4 HH), and tho separation of the 2 peaks is exactly one-half the separation of the 2 hybrid peaks. The peaks were isolated and examined in the electron microscope. Typically more than 40% by mass of the ONA is catenated dimers, 25-30% is higher catenanes, and the rest Is monomers. These values were cortobo­rated by analytical sedimentation velocity experiments. The obvious explanation is that the 1/4 HH DNA consists of catenated dimers made up of an LL monomer catenated to a l/2 HH monomer. Thus, only I of the 4.5 µsingle strands would be 5-BUDR labeled. There would be 2 species of this type, since the 2 strands of niJNA In HeLa eel Is have different numbers of thymidylate residues which are replaced by 5-BUDR. The higher catenanes which are found in this region would be molecules with less than half of the 5 µsingle strands labeled with 5 BUDR. Experiments are in progress to test this interpretation.

If this interpretation Is correct, the next problem is to explain how these forms arise. They could be formed either by some recombination process be­tween LL and HL monomers to give catenanes, or by a repl !cation process in which one of the submolecules of a catenane replicates and releases one of the daughter molecules from the topological bond.

216. A BUOYANT METHOD FOR THE DETERMINATION OF THE SUPERHELIX DENSITY OF CLOSED CIRCULAR ONA

* * Investigators: Horace B. Gray, Jr. , Wi Iii am B. Upholt, Jerome Vinograd

Support: Natl ona I Institutes of Hea I th, Pub Ii c Hoa I th Service

The suporhelix densities of several in vivo and in vitro closed circular DNAs have been calculated from the mole ratio of ethidium chloride bound per ONA nucleotide at the minimum of plots of sedimentation velocity vs. concentration of the dye. The required binding Isotherm for the ethidium-DNA complex was determined spectrophotometrically for calf thymus DNA, and the data treated according to the Scatchard independent binding site model in order to obtain the mole ratio of ethidium bound per nucleotide as a function of ethidium concentration. The separa­tion (relative to that for SV40 DNAs I and I I) between open and closed forms of each

* Division of Chemistry and Chemical Engineering, California Institute of Technology.

2W

DNA in buoyant CsCI gradients in the presence of ethidium bromide or an ethidium bromide analog was measured. Tho relative separation in the dye-CsCI gradient, when corrected for small effects due to differences in base composition and in dis­tance from tho axis of rotation, was found to vary linearly with tho suporholix density. This result has been predicted theoretically (Bauer and Vinograd, 1970). Tho measurement of tho separation between bands of closed and open circles in a dye­CsCI gradient thus makes possible tho determination of tho superholix density of any closed circular DNA from a single experiment.

Reference:

Bauer, W. and Vinograd, J. (1970) J. Mol. Biol. (in press>.

217. THE SEDIMENTATION VELOCITY BEHAVIOR OF CLOSED CIRCULAR SV40 ONA AS A FUNCTION OF SUPERHELIX DENSITY, IONIC STRENGTH, TEMPERATURE, AND COUNTERION

* * Investigators: Wi I liam B. Upholt , Horace 8. Gray, Jr. , Jerome Vinograd

Support: National Institutes of Health, Public Health Service

The sedimentation coefficients of 9 closed circular SV40 DNAs with dif­ferent suporholix densities have boon determined. Tho DNAs were closed in vitro with tho enzyme polynuclootido ligase in tho presence of variable amounts of the intercalating dye, othidium bromide (EB). Tho sedimentation coefficient in tho absence of EB initially increases in the A region with tho absolute value of the suporholix density, a0, and attains a local maximum at about -0.02 suporholical turns per 10 base pairs. It then decreases slightly in tho 8 region untl I at a 0 ~ -0.04 it again Increases monotonically in tho C region at least up to a0 • -0.08. Comparable curves for s vs. a have been obtained with tho individual DNAs when a was varied by addition of EB to tho sedimentation solvent. The appear­an't:o of tho enzymatically closed DNAs in tho electron microscope suggests that tho molecule is in tho form of an oxtondod linear suporhelix at tho BC junction and br~nching in tho suporholix may account for tho rise in the sedimentation coeffi­cient in the C region.

Tho dependence of a upon ionic strength, counterion, and temperature has also been investigated by tho observation of tho sedimentation behavior of closed circular SV40 DNAs of several different superhelix densities under various sedimen­tation conditions. Tho absolute superholix density Increases with increasing ionic strength and decreases with increasing temperature. It Is also dependent upon countorion with Cs DNA having a larger absolute number of suporholical turns than Na DNA. These changes aro duo to changes in the angle botwoon tho base pairs of the Watson-Crick ho! ix of the DNA.

* Division of Chemistry and Chemical Enginooring, Cal ifornla Institute of Technology.

218. THE EFFECT OF BOUND BASIC PROTEINS ON THE SUPERHELIX DENSITY OF CLOSED CIRCULAR SV40 DNA

* I nvos ti gators: WI I I lam B. Upholt ,, Jerome Vlnograd

Support: National Institutes of Health, Public Health Sorvico

221

Previous work has Indicated that changes of ionic strength and tempera­ture from in vivo conditions to the in vitro Investigation conditions are not suf­ficient to lntrod.uce the nuntier of superhellcal turns observed In closed circular DNAs. Another possible source of superhellcal turns Is bound protein at the time of closure of tho DNA by polynucleotlde I igaso In the cell. To Investigate this possibility, experiments are in progress to determine the effect of closing nicked circular SV40 viral DNA with polynuclootide I lgase in the presenco of varying amounts of bound basic protein. Tho protein is subsequently removed and tho su;icr­hel ix density of the resulting closed circular DNA is examined by the ethidium bro­mide-cesium chloride buoyant gradient method.

219. INVESTIGATIONS OF INTERCALATIVE DYE-DNA INTERACTIONS

* I nvostl gators: Maurice Schml r , Jerome VI nograd

Support: National Science Foundation National Institutes of Health, Public Health Service

Recent work In this laboratory has shown that propidium di iodide (3,B­diamlno-5-(N'-methylldiethylaminopropyl-6-phenylpondanthrifllnlum dllodidol enhances tho buoyant separation of closed and nicked circular DNAs by a factor of 1.7 to 1.8, compared with tho phenanthrldlne analog, othldlum bromide. Untl I recently, only impure samples of tho new dye have been aval lab lo, making quantitative studios of the behavior of clrcular DNA In tho presence of prop!dium dllodlde impossible. New lots of tho dye prepared to a very high purity have been fully characterized and tested with closed and nicked circular DNA derived from PM2 bacteriophage. Tho sources of tho new dye's efficacy are currently being Investigated. Binding studies using PM2 DNA wl II allow a meaningful comparison of the behavior of the closed cir­cular DNA in tho presence of propldlum di Iodide and eth!dlum bromide under condi­tions of sedimentation velocity in tho analytical ultracentrifuge. This information and the data derived from buoyant density studies wt 11 al low us to decide tho extent to which (IJ Intrinsic association constants between DNA and dye, (2) partial spe­cific volume of bound dye, or (3) unwinding angle of a bound dye molecule, is re­sponsible for tho enhanced separation.

220. VISCOMETRIC DYE TITRATIONS OF CLOSED CIRCULAR DNA

I nvestl gators:

Support:

* Bernard M. J. Rdvet, Maurl co Sch mi r

Centre de Recherches sur le Cancer North Atlantic Treaty Organization National Science Foundation National Institutes of Health, Public Health Service

In general tho Intrinsic viscosity of a macromolecule is more sensitively related to changes in the configuration of the macromolecule than is the

* Division of Chemistry and Chemical Engineering, Callfornla Institute of Technology.

222

sedimentation coefticiont. However, larger amounts of DNA aro roqui rod for tho measurements. Tho closed DNA from the bacteriophage PM2 has beon usod in a study of the intrinsic viscosity of its suporcol lod and relaxed forms in a rotating cyl­inder viscometer. It has also beon used In viscomctric dye titrations with ethidium bromido and propidium dilodido in a capillary viscometer.

At low shear gradients the intrinsic viscosity of closed PM2 DNA in 2.85 M CsCI at neutral pH is about one-half that of tho nicked form, a rosult that is siml lar to that found in an oarl ier study of RF (ijX DNA (Opschoor ot al., 1968). Approximately tho same ratio was obtained in the capi I lary viscometer. Tho vis­comotrlc dye titration exhibits the same type of depondonce on added dye as doos the sedimentation veloclty-dyo titration. However, since changes in configuration that decrease the sodimontation coefficient increase the viscosity, tho resulting titration curves are inversely related. As dyo is added the viscosity first in­croases to a local maximum, decreases to a minimum, and then rises sharply to a maximum corresponding to the fully relaxed closed molecule. It then fa! Is to a new local minimum as positive supercoi ling develops. The detal Is of the viscometric dye titration curve arc more clearly defined and subject to lower experimental error than the sedimentation volocity-dyc titration curve. The applicability of this procedure tor the elucidation of the structure of closed DNA and its Inter­action with added reagents ls being explored.

Reference:

Opschoor, A., Pouwels, P.H., Knijnonburg, C. M., and Aten, J.B. T. ( 1968) J. Mol. Biol. 37: 13.

221. SEDIMENTATION PROPERTIES OF PURIFIED CATENATED MITOCHONDRIAL DNA FRJM HELA CELLS

Investigators: Irving H. Brown, Jerome Vinograd •

Support: National Cancer Institute of Canada National Institutes of Health, Pub Ile Health Service

Although most of the clrcular mitochondrial DNA (mDNA) from HeLa cells is in tho form of 5 µ length dup I exes, about 15% of the DNA ls in "catEmated" forms, in which 2 (or morel circles are Interlocked. Pure forms of tho catenatod species, containing 2 clrcles (tho dimer) or 3 circles <the trimer), have been isolated by means of preparative velocity sedimentation in the presence of the dye ethidium bromide. The fluorescence produced by the binding of DNA to dye in the gradient was used to locate the position of the catenatcd species in tho gradient. The catcnatod species were Identified by examination with an electron microscope.

Catenated dimers In which both duplex circles have no single-strand breaks sedimented at 515 in the analytical ultracentrifuge, and catcnatcd trimers sod i mentcd at 635. By treatment with tho enzyme DNase, sing I a-strand breaks or "nicks" were introduced into tho dimer. This treatment reduced tho dimer sedimen­tation coefficient to 42S, then to 365. While tho 425 material has single-strand breaks in only one of tho interlocked circles, tho 36S material has single-strand breaks in both ci rclos and is sti 11 a cat<inated dimer.

The above results, with one exception, confirm the sedimentation proper­ties of the catenatcd species suggested by Hudson and Vinograd ( 1969). This work establishes 365 as the correct sedimentation coefficient for the dimer with singlo­strand breaks in both ci re I cs.

223

Reference:

Hudson, B. and Vinograd, J. ( 1969) Nature 221: 332. -,_

222. ALKALI-INDUCED STRAND BREAKAGE OF CIRCULAR MITOCHONDRIAL DNA

Investigator: Irving H. Brown

Support: National Cancer Institute of Canada National Institutes of Health, Public Health Service

DNA from mitochondria of HeLa and other cells can be isolated in tho form of covalently closed circular molecules. At high pH, mitochondrial DNA (mDNAl undergoes sing lo-strand b reakagc at a hi ghcr rate than c i rcu I ar vi ra I DNAs. Ho La eel I mDNA at pH 12.5 ls degraded at least 10 times as fast as SV40 viral DNA under tho same conditions. Tho lnstabi lity of mONA raises tho possibi llty that mDNA con­tains special chemical features which may be biologically significant.

In order to determine the extent of alkal inc breakage, DNA is reacted with alkali, then sedimented in CsCI solution containing formaldehyde. Tho.,tech­nique physically separates undamaged circles from those which have undergone at least one single-strand break due to alkaline treatment. Tho time of roact4>n in alkali is varied in order to determine the rate of breakage. Present studies are being carried out on HoLa col I mDNA. Studies later wi 11 measure breakage of mDNA from other sources, and wil I attempt to determine the reasons for tho sensitivity to alkali.

223. INTRACELLULAR CLOSED CIRCULAR FORMS OF SV40 VIRAL DNA IN AFRICAN GREEN MONKEY CELLS JN CULTURE

I nvosti gators:

Support:

Mark G. Rush, Robert Eason, Jerome Vlnograd

Damon Runyon Memorial Fund for Cancer Research, Inc. National Institutes of Health, Public Health Service

Circular DNA having twice tho molecular weight of viral DNA was isolated and purified by sedimentation velocity procedures from extracts of SV40-infectod BSC-1 cells. These extracts prepared by solectlve alkaline denaturation contain approximately 2% by number of dlmeric molecules. Approximately half of those are interlocked mcnomer-longth circles and half are circles of dimer length. Such dimers have not been observed in tho DNA from purified virus.

In tho early stages of Infection, prior to formation of complete virus, tho intracellular SV40 DNA is more than 80% in the form of closed circular duplex mclecu I es. Tho degroo of twisting of th Is DNA appears to be s Ii ght I y sma II er than that of DNA isolated from the virus. This result suggests that intracellular form is n i eked and roe I osed in tho cou rso of v I rus assemb I y.

224

224. REPLICATIVE INTERMEDIATES OF THE SINGLE-STRANDED DNA OF THE MINUTE VIRUS OF THE MOUSE

Investigators: Robe rt Eason, Mark G. Rush, Dona Id L. Robberson

Support: National Science Foundation Damon Runyon Momorl a I Fund for Cancer Research, Inc. National Institutes of Health, Public Health Service

Crawford (1966) has recently shown that tho nucleic acid of tho Minute virus of the mouse, a member of the picodnavlrus group, is a single-stranded DNA. The kinetics of the formation of viral Intermediates and mature virus have boon de­termined. During the Infective process tree intracellular single-stranded viral DNA could not be detected in labeling experiments with 3H-thymidine. In these samo experiments, 2 major sedimenting components having sedimentation coofficionts of approximately IOS and 14S wore observed. Tho 14S components appear to bo a I inoar duplex in tho oloctron microscope. On the basis of pulso-chaso experiments those i ntormed i ates appear to be precursors of vi ra I DNA.

f<o to re nee :

Crawford, L. V. ( !966) Vi rel ogy 29: 605.

PUBLICATIONS

Bauer, W. and Vinograd, J, (1969) A thermodynamic theory for interacting systems at oqui librlum in a buoyant density gradient: Tho reaction between a small molecular species and a buoyant macromolecule. Ann. N.Y. Acad, Sci. 164: 192-225.

Bauor, W. and Vi nograd, la ti ve dyes. J. Mol. Biol,

J. ( 1970) Interaction of closed circular DNA with intorca-11. The freo energy of suporhelix formation in SV40 DNA. 47: 419-435.

c'layton, D. A. and Vinograd, J, (1969) Complex mitochondrial DNA in leukemic and normal human myeloid eel Is. Proc. Nat. Aced. Sci. 62: 1077-1084.

Clayton, D. A., Davis, R. W., and Vinograd, J. ( 1970) Homology and structural rela­tionships between the dimeric and monomeric circular forms of mitochondrial DNA from human leukemic leukocytes. J. Mot. Biol. 47: 137-153.

Williams, A. E. and Vinograd, J. (1970) Tho buoyant behavior of RNA and DNA in cesium sulfate solutions containing dimethylsulfoxide. Biochim. Biophys. Acta (submitted for publication). ~

I l t I

I

225

Professor: Giuseppe Attardi

Research Fellows: Yosef Alon!, James M. England, Murial Lederman, Livia Plea Mattoccia, Sharon T. Vaughan -,

Graduate Students: Barbara F. Attard!, Wt II lam I. Murphy, Brian Storrle

Visiting Biologist: Solomon A. Kaplan

Associate Biologist: Laverne T. Wenzel

Research Assistants: Benneta Keeley, Deanna K. Ojala

Research Aide: Rosina K. T. Kinzel, Wanda L. Owens

Laboratory Aide: Christine D. Edmonds

The transcription of mitochondrial DNA Jn HeLa cells has been Investi­gated with rogard to the properties of the transcription products, the strand se­lection and extent of transcription, and the relationship of transcriptlon,to tho eel I cycle.

• The characteristics of the mitochondrial protein-synthesizing apparatus

in HoLa cells have been analyzed in an in vitro system and shCN1n to be strikingly di fferont from those of the ext ram l tochondrl a I apparatus.

Tho existence in HeLa col I mitochondria of speclflc 60S ribosomes, con­taining mitochondrial DNA-coded RNA species, has been demonstrated.

Tho investigations on the properties of membrane structural protein from HeLa eel Is and on the fractionation of Hela eel I metaphase chromosomes have been continued.

225. RAPIDLY LABELED HETEROGENEOUS RNA ANO DISCRETE RNA COMPONENTS FROM THE MITOCHONDRIAL FRACTION OF HELA CELLS

Investigators: Barbara F. Attard l, Giuseppe Attard i

Support: National Institutes of Health, Public Health ServlcG

Investigations concerning the characterization of tho RNA from thG mito­chondrial fraction of HeLa cells have been continued. Both rapidly labeled hetero­geneous RNA componGnts, varying ln sedimentation constant from 45 to 50S or more, and dlscrGte RNA species have bGen Identified in the sedimentation pattern of RNA from the mitochondrial fraction.

The labo Ii ng of the heterogeneous RNA, in short-term oxporlments, has been shown to be almost completely sensitive to othidium bromide, an intercalating dye which alters the tertiary structure of closed circular DNA, at a concentration which docs not appreciably affect nuclear RNA synthesis Cl µg/ml). This observa­tion has provided additional support for the previously reached conclusion that mitochondrial DNA is tho template of this RNA fraction (sec Biology 1969).

Tho discrete RNA species associated with the mitochondrial fraction con­sist of the following: Cal l6S, 12S, and 4S components, which for their kinetics

226

of labeling, sensitivity of their synthesis to I µg/ml ethidium bromide, and lack of sensitivity to 0.04 µg/ml actinomycin D (which selectively suppresses ribosomal RNA synthesis), their nucleotide composition, and base sequence homology to mito­chondrial DNA appear to be synthesized on a mitochondrial DNA template; (b) 285, 185, and 55 components of nuclear origin, pertaining to ribosomes which are in their great majority, if not exclusively, bound to membranes of the rough endo­plasmic reticulum; (c) 235 and 215 components, of extra-mitochondrial origin, which, tor their base composition and methylatlon level, may be degradation products of 285 ribosomal RNA.

Analysis of tho 165 and 125 mltochondrlal RNAs by polyacrylamido gel elec­trophoresis has shown that whl le the 125 RNA migrates in the gel, with respect to the 285 and 185 rRNA markers, as would be expected from its sedimentation properties in tho absence of conformational differences, tho 16$ RNA moves slightly behind the 185 RNA, that is, slCNJer than would be expected on the basis of Its sedimentation behavior. From their electrophoretlc mobilities In gel relative to 285 and 18S RNA, the molecular weights of the 165 and 12S RNA species have been estimated to be about 7 x 105 and 4 x 105, respectively. The sedimentation behavior of formal­dehyde-denatured 165 and 12S RNAs In sucrose gradients containing formaldehyde has indicated that they are composed of continuous polynucleotide chains. The l6S and 125 RNA appear to correspond to the "215" and "125" electrophoretlc components pre­viously described in HeLa eel Is; hCNlover, in contrast to what has been reported tor tho latter components, the 16S and 125 RNA have been found to be methylated and to have a relatively long half-life. Elsewhere in this report (Ojala and Attardi) evidence indicating tho ribosomal nature of these components ls discussed.

References:

Attard!, B. and Attard!, G. (1969) Nature 224: 1079-1083.

Penman, S., Vesco, C., and Ponman, M. ( 1968) J. Mel. Biol. 34: 49-69 •

• 226. EVIDENCE FOR COM?LETE TRANSCRIPTION OF MITOCHONDRIAL

DNA IN HELA CELLS

Investigators: Yosef Alon I, Giuseppe Attard!

Support: International Agency for Research on Cancer National Institutes of Health, Public Health Service

Tho traction of mitochondrial DNA which Is complementary to mitochondria­associated RNA In exponentially growing HeLa cells has been investigated by RNA-DNA hybridization experiments uti Ii zing mitochondrial-DNA stran_ds separated by alkaline CsCI density gradient centrifugation and RNA from cells uniformly labeled with 3H-5-urldino. It has been found that mitochondrial RNA hybridizes almost exclusively with the heavy strand, In agreement with tho complementarity of their base composi­tions. Less than 2% of the I lght strand purified by two cycles of alkaline CsCI gradient centrifugation was found to form hybrids with mitochondrial RNA.

In hybridization saturation experiments, the RNA sedimenting slower than 22S or faster than 305 gave about 100% saturation level, whereas the components in the intermediate range of sedimentation constants gave about 85% saturation level. An analysis in Cs2S04 gradients of the density of the RNA-DNA hybrids formed at saturation revealed that tho hybrids formed with the RNA <22S and >305 banded at a density of 1.491 g/cm3, i.e., the density expected tor fully base-pal rod hybrids, whereas the hybrids formed with tho components of intermediate sedimentation

227

constants banded at a slightly lower density (1.485 g/cm3), in agreement with the lower saturation level obtained with these components.

An analysis by electron ml.sroscopy of the hybrids separated In a Cs2so4 density gradient showed that essentially al I the material from the I ,491 g/cm3 band consists of nucleic acid duplexes (Biology 1970, No. 227).

In view of tho aval lab le evidence for tho absence of major (ntornal repe­titions in mammalian mitochondrial DNA, tho results obtained by tho throe approaches described above Indicate that the whole or almost whole mitochondrial genome is transcribed in Hola cells.

227. ELECTRON MICRJSCCPIC VISUALIZATION OF MITOCHONDRIAL RNA-DNA HYBRIDS

Investigators: Yosof Aloni, Donald L. Robberson, Giuseppe Attardi

Support: International Agency for Research on Cancer National Institutes of Health, Public Health Service

Hybrids between mitochondrial RNA and heavy strand of mitochondrial DNA banded in a CszS04 gradient wero examined by electron microscopy for their content of duplex structure. For comparison, the heavy strand of mitochondrial DNA and double-stranded !JX RF DNA mounted under tho same conditions were also analyzed. An examination of a population of 480 molecules isolated from tho band p = I .491 g/ml in the Cs2S04 density gradient, and thoroforo presumed to bo RNA-DNA hybrids, re­vealed that the groat majority had a uniform duplex appe~rance. Single-stranded DNA appeared col lapsed in contrast to the uniform extended appearance of duplex DNA (see plate).

228

On the basis of a quantitative analysis of the observed "bushes" of single-stranded DNA regions, and of a comparison of tho length distributions of nonhybridized heavy strand of mitochondrial DNA and of RNA-DNA hybrids, it was estimated that 85% is the lower limit for the fraction of DNA that is base-paired to RNA in the hybrids isolated by buoyant density centrifugation.

228. TITRATION OF MITOCHCNDRIAL GENES FOR 16S, 12S AND 4S RNA IN HELA CELLS

Investigators: Yosof Aloni, Giuseppe Attardi

Support: International Agency for Research on Cancer National Institutes of Health, Public Health Service

In order to obtain information concerning tho fraction of mitochondrial DNA from HeLa eel Is which is homologous to each of tho three mitochondrial DNA­coded discrete RNA components described elsewhere in this report (BiolQQy 1970, No. 225), hybridization saturation experlrronts wore carried out uti I izing l'lc-labelod heavy or I ight strand of mitochondrial DNA and 3ii-laboled highly purified RNA species. Tho 125 and 165 RNA saturated the heavy strand of mitochondrial DNA at levels of about 10% and 15%, respectively. Those levels correspond fairly closely to those expected if there were one clstron for each of these RNA species per mito­chondrial DNA molecule, assuming molecular weights of 4 x 105 for the 125 and 7 x 105 for the 165 RNA, as estimated from their electrophorctic mobilities. The saturation level obtained with combined 125 and 16S RNA indicates that these cis­trons are distinct. The hybridization levels obtained for the 125 and 165 RNA with the light stranQ amounted to about 5% of those obtained with the heavy strand, and wore presumably duo to contamination of the light strand by heavy strand mate­ri a I •

Mitochondria-associated 4S RNA gave saturation levels of about 4% with the heavy strand and 1% with the light strand, corresponding to about 8 genes and 2 genes, respectively, for molecules of 25,000 average molecular weight per mito­chondrial DNA molecule.

229. MITOCHONDRIAL MINIRIBOSOMES IN HELA CELLS

Investigators: Deanna K. Ojala, Giuseppe Attard!

Support: National Institutes of Health, Pub I ic Health Service

Tho nature of the submltochondrial structures containing the mitochondrial DNA-coded 16S and 12S RNA species in HeLa eel Is (Biology 1970, No. 225) has been investigated.

Throe types of particles with approximate sedimentation constants of 605, 45S and 35S have boon Identified in Triton X-100 lysates of mitochondria. Those particles are rapidly labeled when tho cells are exposed to 3H-5-uridine, and this labeling is not affected by a low concentration of actlnomycin D which Is sufficient to inhibit completely cytoplasmic ribosomal RNA synthesis. The 60S particles con­tain 165 and 125 RNA, the 455 particles'mainly 165 RNA and the 35S particle mainly 12S RNA. An analysis of tho density of these particles in CsCI density gradient after glutaraldehyde fixation (Baltimore and Huang, 1968> has revealed that they contain protein in varying proportion (from 55 to 70%l depending upon the medium to which they have been exposed.

229

Reconstruction experiments uti lizlng purified l6S or 12S RNA added to unlabeled mitochondrial suspensions immediately before lysis have shONn that the particles described above do not arise from artificial complexing of free RNA with protein during tho extraction procodfrro.

After an in vivo pulse with 3H-loucine in the prosonco of cyctohoximido (to inhibit extra-mitochondrial protein synthesis> tho 60S particles become labeled, and this labeling is suppressed by chloramphonlcol; furthermore, tho label can bo chased by subsequent exposure of the eel Is to puromycin.

The above described results strongly suggest that tho 60S particles repre­sent mitochondria-specific miniribosomes with the 45S and 35S particles being the corresponding subunits.

Reference:

Baltimore, D. and Huang, A. S. (1968) Science 162: 572.

230. THE RELATIONSHIP OF MITOCHONDRIAL RNA SYNTHESIS TO THE CELL CYCLE IN HELA CELLS

I nves ti gators : Livia Pica Mattoccia, Giuseppe Attardi

Support: National Institutes of Health, Public Health Service

•

The study of the mitochondrial RNA synthesis in different phases of tho cell cycle <Biology 1969, No. 28) has been continued.

HeLa cells, synchronized by the mitotic selection technique CTorasima and Tolmach, 1963; Robbins and Marcus, 1964), wore exposed at di fforont phases of the life cycle to 3H-uridino for 20 minutes or for 60 minutes in tho presence of a low concentration of actinomycln D (to inhibit selectively tho labeling of contaminating ribosomal RNAl.

In control experiments, the pulses wore carried out in the presence of tho intercalating dye othidium bromide, in order to specifically inhibit mitochondrial RNA synthesis. Tho rate of labeling of the RNA associated with tho crude mitochon­drial fraction and its sedimentation properties were determined.

The labeling of the extra- and intra-mitochondrial UTP pool was also ana­lyzed by thin layer chromatography in order to correct tho 3H-uridlne incorporation data. Tho results obtained lndlcato that mitochondrial RNA synthesis starts in tho S phase and reaches a maximum In tho G2 phase.

Tho cytochrome oxidaso activity in different phases of tho cot I cycle is at present under investigation.

References:

Torasima, B. and Tolmach, L. J. ( 1963) Exptt. Col I. Res. 30: 344.

Robbins, E, and Marcus, P. L. (1964) Science 144: 1152.

230

231. STUDIES ON THE ISOLATION AND SUBFRACTIONATION OF MITOCHONDRIA OF HELA CELLS

I nvosti gators: Brian Storrio, Giuseppe Attardi

Support: National Science Foundation

Studios begun in the preceding year with tho aim of obtaining a separa­tion of mitochondria from rough endoplasmic reticulum In HoLa eel I mombrano prepa­rations appear to havo yielded an unexpected separation of a transcriptional ly ac­tive mitochondrlon from tho bulk of HoLa col I mitochondria. Tho majority of rap­idly labolod RNA of cytoplasmic membrane preparations, after pulses of 3H-5-uridino of botwaon 5 and 30 minutes duration, is found to bo associ atod with structures which on dextran-sucrose gradients fractionate to the heavy sido of tho mitochondrial enzymo marker prcfi le. This pulse-labeled RNA is in situ resistant to RNaso, its synthesis is sensitive to othidium bromide, and it is similar in sedimentation properties to RNA which has been shown to hybridize to mitochondrial DNA. Tho pulse-labeled RNA Is probably associated with mitochondria which arc either denser or larger than the bulk of mitochondria. Experiments are in progress to test whether tho above separation is basod on size or density and to tost thG possibility that thoso structures arc also active in DNA replication and protGin synthesis.

Tho separation of mitochondria and elements of rough endoplasmic reticu-1 um achieved in those studies should be sufficient to experimentally test tho hy­pothesis of mitochondrial RNA export to elomGnts of tho rough endoplasmic reticu­lum. Experiments on this problem are in progress.

232. STUDIES ON RN~ SYNTHESIS BY RAT BRAIN SYNAPTOSOMES

Investigators: James M. England, Giuseppe Attardi

Support: National Institutes of Health, Public Health Service •

Whan brain tissue is homogenized in an isotonic medium tho prosynaptic terminals resist disruption and form discrete partlclos, synaptosomos, which ro-tal n tho main features of nerve endings, including synaptic mitochondria (Whittaker ct al., 1964). If these mitochondria are active in RNA synthesis, as are mitochon­dria from other difforentlatod cells, e.g., livor coils, tho synaptosomo prepara­tion should provide an advantageous systom for tho study of mitochondrial RNA syn­thesis in tho absence of nuclear RNA synthesis. It is tho aim of this investigation to establish whether RNA is synthosizGd by synaptosomes in vitro and, if so, to characterize it on the basis of size distribution, baso composition and base so­quenco homology with mitochondrial DNA, and to determine its distribution within tho synaptoscmo. Preliminary experiments suggest tho occurrorico of an ethidium bromide­sensltivo incorporation of labeled RNA precursors in synaptosome preparations.

Tho RNA pattern of synaptosomes labeled in vitro wi 11 be compared to that of synaptosomes isolated from animals after in vivo labeling with an RNA precursor in ordor to obtain additional data on tho rolatlvo rolos of nuclear and synapto­somal, presumably mitochondrial, DNA ns tomplatos for axonal RNA synthesis.

At present, methods arc boing Ctevolopod to soparato synaptosomos from free mitochondria and to solectlvoly favor tho in vitro Incorporation of labeled precursor by synaptosomos as opposed to froo mi tochondrl a.

1<'--

,._

Roterenco:

231

Whlttakor, V. P., Michaolson, I. A., and Kirkland, R. J. A. (1964) Bio­chom. J. 90: 293.·,

233. IN VITRJ PROTEIN SYNTHESIS BY HELA CELL MEMBRANE FRACTIONS

I nvestl gators: Muriel Lederman, Giuseppe Attard!

Support: ·National Institutes of Health, Public Health Servico

A protein-synthesizing system has boon developed using a membrane traction from HeLa eel Is and the 229,000 x g supernatant (Biology 1969). Tho characteristics of this system, which arc siml lar to those observed tor tree polysomos, indicate that tho vast majority of polypeptide synthesis, in the presence ct an exogonous ATP-generating system, is occurring on polysomes ct the rough endoplasmic reticu­lum. The synthesis is to a great extent sensitive to RNase and cyclohexlmide, de­pendent upon added supernatant factors and insensitive to chloramphenlcol. The mombrane traction used in the above synthesis is known to contain mitochondr\a. Using incubation conditions described by Kroon et al. (1968), protein synthesis characteristic of mitochondria has boon observed in this traction. This syntjlesis can be entirely supported by oxidatlvo phosphorylation processes, is not dependent upon supernatant factors, is insensitive to RNase and cyclohoxlmlde, sensitive to ch loramphen !col, and part! a 11 y sensitive to din i tropheno I, an inhibitor of resp Ira­tory chain phosphorylation.

Protein synthesis with the characteristics expected for a cytoplasmic sys­tem has been shown to occur in the rough endoplasmic reticulum purified on dextran­sucrose gradients.

The effect of ethidium bromide on the two types of protein synthesis has also been investigated. This drug has been shown to inhibit mltochondrial RNA syn­thesis (Zy lber et al., 1969). It a membrane traction prepared from eel Is treated in vivo with othidium bromide ls used tor protein synthosis, no inhibition of rough endoplasmic reticulum protein synthesis is observed after 4 hours exposure to the drug, whereas mitochondrial protein synthesis is inhibited by 90%.

Tho nature ct the proteins synthesized by these systems is also being in­vestigated with particular attention to the "structural protein." A membrane frac­tion labeled in vivo with 14c-loucine is combined with a membrane fraction labeled with 3H-loucine by in vitro protein synthesis and structural protein isolated and subjected to acrylamido gel electrophoresis after the methods ot Yang and Criddle (1969),

References:

Kroon, A. M., Batman, M. J., and Saccone, C. (1968) In: E. C. Slater, J. M. Tager, S. Papa, and E. Quagl iarlel lo (eds.), Biochemical Aspects of the Blogenesis of Mitochondria. Adriatlca Editrico, Bari.

Zylbor, E., Vesco, C., and Penman, S. (1969) J. Mal. Biol. 44: 195.

Yang, S. and Criddle, R. S. (1969) Biochem. Biophys. Ros. Commun. 35: 429.

•

232

234. STUDIES ON MEMBRANE STRUCTURAL PROTEIN FROM HELA CELLS

Investigators: Sharon T. Vaughan, Giuseppe Attardi

Support: National Institutes of Health, Public Health Service

The suggestion of tho possible involvement of mitochondrial DNA in direct­ing the synthesis of "structural protein" of mitochondria and possibly of other coll membranes has led to the investigation of tho properties of this protein fraction from HeLa coll membranes <Biology 1969, No. 99). Purified structural protein was found to be hotorogonoous In its behavior on 0.1% SDS, 10% acrylamido gels, although three main components with approximate mo/ocular weights of 22,000, 34,000, and 65,000 could be reproducibly recognized. These isolated components migrated as single bands to tho same region when ro-electrophoresed. Uti I ization of the puri­fication scheme and tho disc gel system C0.03% SDS, 8% acrylamide) described by Yang and Criddle ( 1969) for yeast mitochondria "structural protein" has yielded simpler gel patterns. Tho synthesis in vitro by mitochondrial preparations of yeast of the fast-moving component observed in these gels has boon shown to be com-­plotoly sensitive to chloramphenico/ Inhibition, indicating that this component is a product of the specific mitochondrial protein-synthesizing system. The same com­ponent from Ho La structura I proto in, when i sol atod and o loctrophorosed on 12% acry/amlde, 0.1% SDS gels, has yielded 3 components of approximate molecular weights of 7,000, 14,000, and 22,000. In addition to tho fast-moving component, a 35,000 and a 65,000 mo/ocular weight component have boon found in tho disc gel pattern of tho HoLa structural protein. Currently tho fingerprinting patterns of tho various components of tho structural protein fraction arc being investigated.

Reference:

Yang, S. and Criddle, R. s. ( 1969) Biochom. Biophys. Ros. Commun. 35: 529 .

235. STUDIES ON THE STABILITY OF MESSENGER RNA IN HELA CELLS

I nvcsti gators: Wi II i am I. Murphy, Gi uscppe Attardi

Support: National Science Foundation National Institutes of Health, Public Health Service

Because of the cytotoxlclty and side effects of actinomycin D, tho re­sults obtained with this drug in tho study of tho stability of messenger RNA CrrRNA) in po/ysanos arc not unambiguously interpreted. Therefore, we have developed a now technique which wl 11 al low us to investigate tho rate of -degradation of tho rrRNA under conditions closer to tho physlo/ogical state of tho col I.

This technique consists of pulse labeling a culture of HcLa cells at 37°C with 311-uridino fol lowed by exposure of the culture to low temperature in tho pres­ence of high concentrations of uridlno and cytidino, after which tho coils arc grown at tho original temperature. Our studios have shown that during this cold treat­ment macromolecular synthesis and degradation arc markedly slowed down or arrested, while tho specific activities of tho Ul'R and CTP pools arc reduced to 10% of their values at tho end of tho pulse. This provides a 3-5-fold more effective chase in terms of residual incorporation of label into RNA than simply tho addition of tho urldine and cytidino alone. After tho col ls have undergone this treatment, they grow and divide normally at 37°C and, in particular, their rate of protein synthe­sis continues normally for at least 24 hours.

233

Wo aro presently employing this technique to investigate tho stability of the polysome-associated mRNA In HoLa eel Is.

-, 236. FRACTIONATION OF HELA CELL METAPHASE Q-IROtJOSOMES ON THE

BASIS OF THEIR ELECTRICAL CHARGE

Investigators: * Yosof Alon!, Gluseepe Attard I, Aurora Lande I , Michael A. Raftery

Support: International Agency for Research on Cancer National Institutes of Health, Public Health Service

Previous work from this laboratory (Huberman and Attard!, 1967) has shown that It is possible to fractionate Isolated Hela cell metaphase chromosomes on the basis of sedimentation velocity in sucrose-glycerol gradients. Using this technique we have found that the DNA complementary to 4S and 5S RNA Is not located solely in the smal lor HeLa col I chromosomes, which contain the DNA complementary to the high molecular weight riboscmal RNA, but rather distributed over al I tho chromosomes <Biology 1969, No. 22).

It Is, however, clear that future studies on the chromosomal location of gonos or tho charactorization of gone-specific chromoscmal protein wl II demand much higher resolution than provided by sedimentation veloclty fractionation. Attempts to develop a different approach to chromosome fractionation (Biology 1969) have boen continued. We have separated HeLa eel I metaphase chromosomes on tho basis of their electrical charge. Hybridization with ribosomal RNA is being used as an assay to monitor tho effectiveness of fractionation.

Roferonce:

Huberman, J. A. and Attard i, G. ( 1967) J. Mol. Biol. 29: 487.

PUBLICATIONS

Attard!, B. and Attardi, G. ( 1969) Sedimentation properties of RNA species homolo­gous to mitochondria DNA in Hela col Is. Nature 224: 1079-1083.

Attard!, B., Cravioto, B., and Attard!, G. (1969) ~mbrane-bound ribosomes In Hela col ls. I. Their proportion to total coll ribosomes and their association with messenger RNA. J, Mol. Biol. 44: 47-70.

Attard!, G., Aloni, Y., Attardi, B., Laderman, M., Ojala, D., Plca-Mattoccia, L., and Storrie, B. ( 1970) Properties of mitochondria! RNA in Hela cells. lnternat. Symp. on "Autonomy and Blogenosis of Mitochondria and Chloro­plasts." Academic Press <In press).

Attard!, G., Aloni, Y., Attard!, B., Ojala, D., Plca-Mattoccla, L., Robberson, D. L., and Storrie, B. (1970) Transcription of mitochondria! DNA in Hola cells. Cold Spring Harbor Symp. Quant. Biol., Vol. 35 (in press).

Attardi, G. and Ojala, D. ( 1970) Mitochondria! minlrlbosomos in Hola cells. Nature <in prossl.

* Division of Chemistry and Chemical Engineering, Cal ifomia Institute of Technology.

234

Attardi, G., Parnas, H., and Attard!, B. (!970) Pattern of RNA synthesis In duck erythrocytes In relationship to tho stage of col I dlfforent!atlon. Exptl. Coll Res. 62 <In press).

Hatlen, L. E., Ama!d!, F., and Attard!, G. (1969) Oligonuc!ootlde pattern after pancreatic r!bonuc!ease digestion and the 3' and 5 1 termini of 5S ribo­nucleic acid from Hola eel Is. Biochemistry 8: 4989-5005.

Joantour, Ph. and Attard!, G. ( 1969) Relatlonship between HoLa col I ribosomal RNA and Its precursors studied by high rosolutlon RNA-DNA hybridization. J. Mol. Biol. 45: 305-324.

235

THE FOLLOWING REPORT IS BY A GRADUATE STUDENT IN THE DIVISION OF BIOLOGY WHO ELECTED TO DO HER THESIS WORK IN THE DIVISION OF CHEMISTRY AND CHEMICAL ENGI­NEERING.

237. NUCLEAR MAGNETIC RESONANCE STUDIES OF PROTEIN CONFORMATION AND ALLOSTERIC MECHANISMS

I nvosti gators:

Sup port:

* Wray H. Huestis, Michael A. Raftery

National Science Foundation

Because of Its sensitivity for detection of differences in chemical envi­ronments, nuclear magnetic resonance spectroscopy has considerable potential for dynamic studios of macromclocules, such as proteins. The multlpl !city of different environments for the many hydrogen nuclei in proteins, however, yield very compll­catod spectra and render difficult the extraction of information relating to envi­ronmental changes for specific nuclei. An alternative approach has been initiated. This involves specific labeling of proteins at known sites using reagents contain­ing 19F nuclel. Ribonuclease S has been labeled In this manner through modification of two lysine residues (numbers I and 7 in the sequence), yleldlng a slmple \9F spectrum. Chemical shift changes induced by means of inhibitor binding have boon studied and tho effects observed in tho known labeled regions of the molecul~ there­by related to specific molecular events.

Extension of this method to hemoglobin has been achieved by specific modi­fication of a single sulfhydryl group In the S-chaln (residue 93). Observation of tho 19F spectrum of the modified protein has been carried out ln the absence and presence of ligands, such as oxygen, carbon monoxide, and cyanide Ion. Tho results obtained, combined with further studies in progress, wi II be applied toward under­standing tho mechanisms of subunit Interactions which result in cooperative binding of ligands by hemoglobin.

* Division of Chemistry and Chemical Engineering, California Institute of Technology.

236

VISITING LECTURERS

David Apirlon, Washington University, St. Louis: "Messenger RNase Associated with E. col I Ribosomes."

Michael W. Berns, Pasadena Foundation for Medical Research: "Laser Micro-I rradi­ation of Chromosomes, Nucleoli, and Mitochondria of Tissue Culture Ce I ls."

Dean Bek, University of California at Los Angeles Medical School: "Visual Pigment Renewal in the Frog Retina."

Suzanne Bourgeois, Salk Institute: "Repressor--Operator I nteractlon In the Lac Dperon."

Joseph V. Brady, Walter Reed Army Institute of Research: "Emotion: Some Conceptual Problems and Psychophyslological Experiments."

Roy Britten, Carnegie Institution of Washington: "DNA Evolution and Regulation."

Leon J. Bruner, University of California, Riverside: "Blocking Phenomena and Charge Transfer Through LI pi d B 11 ayer Membranes."

Harris Busch, Baylor University College of Medicine: "Nuclear RNA of Cancer Ce I ls."

Mel Cohn, Salk lnstltote: "How Does a Mammal Distinguish Tolerance from Induction of Antibody Synthesis?"

John Cronin, Washington University, St. Louis: "Mutants of E. coli Defective In Mentirane Lipid Synthesis."

R. L. DeHaan, Carnegie Institution of Washington: "Development of Pacemaker Ac­t! vi ty in the Embryon I c Heart."

George Eisenman, University of California at Los Angeles Medical Schoel: "Lipid Composition and Ion Permeation Through Bl layers."

Dona1d M. Engelman, University of London King's Coi lege: "X-ray Diffraction Evi­dence for the Structure of a Biomembrane."

Harold P. Erickson, University Postgraduate Medical School, Cambridge, England: "Compensation of Defocussing and Aberrations by Fourier Processing of EfecTron Micrographs."

Stuart F. Goldstein, University of California, Berkeley: "Laser Mlcrobeam I rra­diation of Unidirectional and Bidirectional Flagella."

Marcus Goodal I, A.M.A. Institute for Biomedical Research, Chicago: "Action ot Two Cyclic Peptides on Lipid Bi layers."

Terrel I HI 11, University of Cal I fornla, Santa Cruz: "On the Theory of Steady State Transport Across Membranes."

f J 1 i

i l '

Maurice R. Hi I leman, Merck Institute for Therapeutic Research, West Point, Pa.: "Vaccines, Host Resistance and Chemotherapy In the Control of Viral Di seas es . "

J. Al Ian Hobson, Harvard Medical School: "Single Unit Activity of Cortex, Cere­bellum, and Brain Stem During Natural Sleep in Cats."

237

D. E. Hurst, University of California, Berkeley: "Gene Conversion and Recombina­tion in Yeast."

Theodore L. Jahn, University of Cal I fornla at Los Angeles: "Locomotion of Fl a­ge I I ates and c i 11 ates."

N. K. Jerne, Institute for Immunology, Basel, Switzerland: "The Generation of Se If-Tolerance and of Anti body Divers I ty."

Laurence Kedes, Laboratorlo di Embrlologla Molecolare, Naples, Italy: "Synthesis of Chromosomal Protel ns and The Ir Tempi ates During Early Embryonl c Deve I opment."

Regis Kelly, Harvard Medical School: "Acetyl Choline Esterase In the Neuremuscular Junction."

• Edward Kravitz, Harvard Medical School: "An Informal Discussion of His Current

Research In Neurochemistry."

Peter U!uger, University of Constance, Germany: "Electrochemical and Optical Studies of Lipid Bl layer Membranes."

Ted Mar, University of I I llnols, Urbana: "Primary Events in Photosynthesis."

Donald A. Marvin, Yale University: "Replication of Filamentous Bacteriophages."

Roger S. Payne, Rockefeller University: "Communication Among Wi Id Whales."

Karl Pribram, Stanford University School of Medicine: "The Problem of Distributed Memory Storage."

Jean Paul Revel, Harvard Medical School: "Fine Structure of Tight and Not-So-Tight Junctions . 11

Alexander Rich, Massachusetts Institute of Technology: "Structure and Function in the E. coll Ribosome and Its t-RNA."

Richard L. Russell, University of Cambridge, England: "Neurogenetics of a Nema­tode. 11

H. Saedler, Cologne University, Germany: "A Stop Signal for the RNA Polymerase Created by a Mutation With In the Ga lactose Operon of E. col I."

Stanley W. Sajdera, Rockefel !er University: "The Macromolecular Organization of the Matr Ix of Cart I I age."

Heinz Schal !er, Max-Planck Institute fUr Vi rusforschung, TUbingen, Germany: "A New System for DNA Synthesis In Vitro."

238

WI 11 iam Ste I I, National Institutes of Health: "Photoreceptor Response Mechanisrrs In Limulus: Ultrastructural and Electrophysiologlcal Studies."

Curt Stern, University of California, Berkeley: "Experiments In Developmental Genetics. 11

WI I I lam C. Summers, Yale University School of Medicine: "Regulation of Phage T7 RNA Metabol Ism In Vivo and In Vitro."

J. Thomas Ungerleider, University of California at Los Angeles, Neuropsychiatric Research Institute: "Drug Use In the Seventies."

Elton T. Young, University of Washington, Seattle, Washington: "T4 mRNA Synthe­sis.11

239

GRADUATES

Thirty-three students In &lology were awarded the B.S., M.S. or Ph.D. degrees In June, 1970. The names, degrees conferred, the titles of doctoral theses, and the plans of the recipients for future work are as folle><s:

Ml chael J. Abrams, B.S.

Robert W. Brackenbury, B.S. with honor. Graduate work In biology at Brandeis Uni varsity.

Victor L. Bresson, B.S.

Steven D. Flanagan, B.S. Graduate work In biology at U.C., San Diego.

Yee Wing-Fong, B.S. with honor. University of Southern Cal lfornla Medical School.

James L. Gould, B.S. with honor. Winner of George W. Green Memorial Award. Graduate work In biology at Rockefeller University.

Michael A. Henerey, B.S.

Allen G. Hirsh, B.S. Graduate work In biology at Columbia University.

WI I I lam B. Inwood, B.S. with honor.

David I. Lewin, B.S. with honor. Graduate work In blol~gy at Yale University.

James H. Richards, B.S. To Nepal with the Peace Corps.

David P. Rossum, B.S. with honor. Graduate work In biology at U.C., Santa Cruz.

Michael Ruth, B.S. with honor. To medical school.

David P. Schor, B.S. Case Western Reserve Medical School.

Jeffrey M. Schredder, B.S.

H. Peter Zassenhaus, B.S.

Elizabeth Denise Campbell, M.S. Will continue graduate study at C.l.T.

Tommy Charles Douglas, M.S. WI II continue graduate study at C.I .T.

Moises Eisenberg-Grunberg, M.S. WI 11 continue graduate study at C.l .T.

John B. Jamieson, M.S.

Denis R. Rydjeskl, M.S.

David A. Clayton, Ph.D. Thesis: "Occurrence and structure of complex mitochon­drial DNA in human leukemic leukocytes and normal mammalian tissues. I I. Use of alkal I metal salts of trichloroacetic acid as buoyant denaturing solvents for DNA." Postdoctoral research at the City of Hope Med I ca I Center.

240

Sydney P. Craig, Ph.D. Thesis: "Mitochondria and the development of sea urchin embryos." Postdoctoral research at the National Institutes of Health.

Ernest Wi I I lam Goode I I, Ph.D. Thesis: "Processes affecting growth of Phycomyces sporanglophores." Postdoctoral research at Swedish Forest Products Research Laboratory, Stockholm.

Jack D. Griffith, Ph.D. Thesis: of chromosomal fibers."

"High resolution electron microscope studies Postdoctora I research at Corne 11 Un I vars I ty.

Loren E. Hatlen, Ph.D. Thesis: "I. Structural studies on HeLa 5S RNA. 11. Hybrid­ization studies with HeLa 4S and SS RNA." Postdoctoral research at the Salk Institute.

Richard D. K. Jessi In, Ph.D. Thesis: "The lysls mechanism of phage T4." Post­doctoral research at UCLA.

Joyce B. Maxwell, Ph.D. Thesis: "I. Synthesis of L-amlno acid oxldase by a serine­or glycine-requiring strain of Neurospora. 11. Studies concerning multiple electrophoretlc forms of tyroslnase In Neurospora." Now an Assistant Professor In the Biology Department at San Fernando Valley State College.

Jerre Levy Nagylakl, Ph.D. Thesis: "Information processing and higher psycho­logical functions In the disconnected hemispheres of human commlssuroto­my patients." Postdoctoral research at the VA Hospital, Denver.

John E. Newbold, Ph.D. 'Thesis: "I. The abortive Infection of bacteriophage (1X174 at low temperatures. I I. The early stages In the process of Infection by bacteriophage (1Xl74." Postdoctoral research at the Salk Institute,

John S. Parkinson, Ph.D. Thesis: "Organization and function of the phage lambda • chromosome." Department of Microbiology, Oregon State University.

' John E. Smart, Ph.D. Thesis: "Studies on the role of hlstones in the structure and function of chromatin." Postdoctoral research at C.I .T.

Lois s. Toevs, Ph.D. Thesis: "Identification and characterization of the egg­laylng hormone from the neurosecretory bag eel Is of Aplysla."

! !

l

j

241

HONORS AND AWARDS

Giuseppe Ati"ardi and Charles Brokaw h·ave received Guggenheim Fel IO#shlps to spend the 1970-71 academic year abroad. Dr. Attardl wl 11 do research at Gif-sur-Yvette and Dr. Brokaw wi II be at Cambridge University.

Max DelbrUck was awarded the Nobel Prize In Physiology and Medicine, and the Louisa Gross Horwitz Prize of Columbia University.

Sterling Emerson was elected to membership in the National Academy of Sciences.

Arie J. Haagen-Smlt received the B. Y. Morrison Memorial Award given by the U.S. Department of Agriculture, and the Hodgkins Award from the Smithsonian Institution for research In atmospheric science.

Norman H. Horowitz was elected to membership In the American Academy of Arts and Sciences.

Robert L. Sinsheimer was awarded the Beijerinck Virology Medal of the Royal Netherlands Academy of Sciences and Letters. He was elected Presl~ent of the Biophysical Society for 1970-71 and a member of the Counci I of the National Academy of Sciences. •

Jerome Vinograd was the recipient of the American Chemical Society Award In Col lold or Surface Chemistry sponsored by the Kendal I Company.

Irving Brown, Research Fe II ow w I th Dr. VI nograd, has been- des I gnated a King George V Jubl lee Fellow during the tenure of his current tel lowship from the National Cancer Institute of Canada.

A P P 0 I N T M E N T S

Felix Strull"lilasser was appointed to the Fellowship Review Committee of the National Institutes of Health and to the Editorial Board of Physiological Reviews.

242

F I N A N C I A L SUPPORT

The financial support available tor the work of the Division of Biology comes from many sources: from general Institute endowment and from special endow­ment funds tor broad areas of work; from grants or contracts with individuals, companies, foundations and U.S. governmental agencies tor specific projects; from unrestricted annual gifts; from fellowships tor the support of individual scholars; and from contributions to general funds provided by Industrial Associates and Institute Associates, as follows:

Fund - Research Support

American Heart Association

John E. Barber Fund for Biological Research

Beckman Instruments, Inc.

James G. Boswell Foundation

Louise Jane Church Fund

Norman W. Church

Commonwealth Fund

Corser Fund tor Biology

Josephine V. Dumke •

Herman Frasch Foundation

The Goodyear TI re and Rubber Co.

Lois Gosney Castle Fund

Frank P. Hixon Fund

Irma Hoef ly Fund

Lasker Award Fund

Clara Lockwood Fund

Medlsche Facultelt Rotterdam

Merck Company Foundation

National Aeronautics and Space Administration

Purpose

Genera I support

Research, particularly as related to the brain

Funds tor equipment

VI rus research

Research In biology

Research in chemical biology

General support

Genera I support

Cancer research

Plant growth research

Bl ocheml stry research

Research in behavioral biology

Neurobiology, physiological psychology, and re I ated research

Cancer research

Chemical genetics

General support

Neu rob i ol ogy

Faculty development

Hrl>ernatlon studies

l I j

j

j l I

J

National Science Foundation

The President's Fund

Rocke fe I I er F oundat I on

Gordon S. Ross Foundation

Edwin H. Schneider Fund

Harlan Shoemaker Fund for Para I yt lcs

Sloan Fund for Basic Research

Stone Foundation

Sundry donors

Albert Tyler Memorial Fund

Union Carbide Corporation

U.S. Atomic Energy Commission

U .s. Pub Ii c Hea I th Service

Joseph G. Venable Fund for Arthritis Research

Martin Webster Fund

Jean Welgle Memerial Fund

Wright Foundation

243

Studies in animal physiology, biochemistry, biophysics, developmental biology, genetics, neurobiology, plant and cell biology; under-

' graduate research parti cl pat I on program

Research and development at JPL

Chemical biology research

Medical research

Study and research in go net I cs

Research on muscle relnnervatlon

Genera I support

Psychobiology research

Chemical biology research

Annual lectureship In biological resa,arch

Genera I support

Research in radiation genetics, protein synthesis

Studies In basic experimental biology, animal physiology, biochemistry, biological systems analysis, biophysics, developmental biology, genetics, neurobiology, plant and col I blology, psychobiology, and vlrology; graduate research tral n i ng, b I omedlca I sciences support grant

A rth r It Is research

Immunology and virology

Genera I support

Medical science

244

Fund - Fel lowshlp Support

Amer I can Cancer Soc I ety

Earle C. Anthony

Mary K. Beschorman Fund

California Foundation for Biochemical Research

Jane Coffin Chi Ids Memorial Fund for Medical Research

Lucy Mason Clark Fund

Dartmouth Co I iege

Deutscher Akademischer Austauschdlenst

Deutsche Forsch ungsgeme I nschaft

Ford Foundation

Free University Amsterdam

Gloria Gartz Fund

E. S. Gosney Fund

lnternatlonal Atomic Energy Agency

International Research and Exchanges Board

Los Angeles County Heart Association

Helen G. and Arthur McCal lum Fund

National Aeronautics and Space Adm In l strati on

National Defense Education Act

National Science Foundation

Netherlands Organization for the Advancement of Pure Research

North Atlantic Treaty Orga~lzation

Nutrition Foundation

Damon Runyon Memorial Fund

Smith, Kline and French Laboratories

South African Councl I for Scientific and Industrial Research

University of Indiana

U.S. Public Health Service

Helen Hay Whitney Foundation

World Health Organization

245

AUTHOR INDEX (By page n umbo r l

Al oni, y.' 226-228' 233 Fong, Y.-w., 210 Arch, s .• 184

·-.., Foster, K. W., 44-4)

Arora, H. L., 204-205 Fro Ii ngcr, J • A.' 40 Attard i, B. F.' 225 Froehner, s. c.' 28 Attard i, G., 225-233 Fukuda, A., 59, 6 I Beckendorf, s. K.' 104 Ge I tosky, J. E.' 123 Benbow, R. M.' 55-56. 58 Go I ub, E. E., 109 Benedict, B. R. ' 95 Goode I I, E. w •• 45, 51 Bonawitz, L. I . ' 206 Goodwin, A. w •• 157 Benzer, s .. 129-131, 133-136 GOpc I, G., 113 Borgmun, K., 46, 49 Gordon, H. W., 20 1-202 Bi rdvic I I , c. R., 146 Gould, J . L. , 141

l Bissel I, s. A., 48 Gray, H. B. ' Jr., 219-220 Blankcnhorn, E. p •• 79 Gray, W. R., 148-152 Blaschke, w .• 113 Haagon-Smi t, A. J •• 117 Bonner, J . ' 21-23 Hal Iberg, R. L., 218 Brackenbury, R. W., 32 Hanson, T. E.. 132-133 Brokaw, c. J •• 94-95 Harris, s.' 34-35 Brown, I. H., 222-223 Heath, R. T. ' 60 Brown, w. M.' 217-218 Henoroy, M. A.' 141 Burke, P. y.' 52 Hiatt, D. E., 90 Charlang, G.' 108 Higgins, R. C., 33 Chong, M.-T., 29 Hodgctts, R. B.' 138, 140 Ch ristcnscn, P. T., 129. 135 Holmos, D. s.' 24 Clayton, D. A., 215, 217 Horn, G., 107 Cm on, N. s.' I 18 Horowitz, 1L H.' 107-108 Cdlon, r' . J . ' 48 Hatta, y .• 130. 136' 138 Cross, J. w .• Jr•, 111 Huestis, W. H.' 235 Dafny, N., 85, 167 Hurn, J . ' 23 Darby, G.' 61 I ngcrsol I, s. M •' 190 De I b ruck, M.' 47, 53 I to, M., 82 dcl Val le, u. E., 150 Jack lot, J. w.' 186 Denni son, D. s .• 47 Jcsaitis, A. J • ' 45 De YI i ager, T. A., 176 Johnston, c .• 39 Ooug I as, T. C., 42 Janos, R.' 86 Dreyer, w. J .• 143-145, 147-148 Jordan, J • M., 216 Dumas, L. B.' 61 Joss I in, R. D. K.' 106 Dusonbo ry, D. B.' 41 Joyce, N. c .• 21 Eason, R., 223-224 Kauffman, J • w.' 53 E i chonbc rgcr, E.' 129 Kavanagh, R. N.' 157 Eisenberg, M. G. I 56 Kci gh Icy, G.' 118 Elgin, s. c. R.' 27 Kol ls, S., 105 Emerson, s .. 98 Konopka, R. J .• 136-137, 139 England, J. M., 230 Ko rnb Ii th , C. L.' 89 Eskin, A.' 180 Kaw, L .-M., 168 Espejo, E. c .• 68 Kreuter, c. D.' 194 Espejo, R. T., 68 Kumar, s .. 199-201 Fnbri cant, J. D.' 55-56. 59 Laico, M. T.' 143-144 Fagan, L.' 203-204 Lande I, A.' 233 Fe i gonson, G.' 40 Langstroth, P. T.' 114 Fonder, [) • H. , 157 Latta, J. E.' 103 Fitkova, E.' 161-164 Lederman, M., 231 Fi rte I , R. A.' 31-33 Lee-Teng, E., 207-208 FI ory, P. J .• Jr., 219 Levine, M.' 40

246

Levy, J., 195, 197-199 Low is, E • B. , 21 0 Lowis, R. K., 94 Li, H.-J., 25-26 Li ob I i ch , I . , 88 Lindsay, D. E., 79 Linscman, M. A., 89-90 Lowy, P. H., I 18 Lu, C., 157 Macleod, M. C., 141 Magnus, J. G., 208 Mattoccia, L. P., 229 Mayfield, J. E., 34 Mayol, R. F., 55, 58 McConnol J, D. J., 29-30 Md~ahon, D., 113-114 Moad, C. A., 53 Meech, R. w., 185 Moltzor, P. S., 121 Melvin, S. L., 38-40 Mc rr i am, J . R. , I 29 , I 35, I 38 Moyer, R. L., 206 Miller, M. J., 112 Miller, R. L., 95 Mitchel I, H. K., 120-121 Mitcholl, M. B., 98-99 Murphy, W. I., 232 Murthy, L. D., 23 Naka, K.-1., 159-160 Nobcs, R. D., 188-189 Newman, R. S., 146 Nye, P. W., 160 O'Har~. K. R., 129 Ojala, D. K., 228 Olds, 'M. E., 79 Olds, J., 78-79, 82, 84, 88, 90 Owen, R. D., 38 Papcrmastor, D. s., 143, 145 Peretz, B., 185-186 Pctzuch, M., 47 Phi I I i ps, A. G., 87 Phi 11 l ;is, M. I . , 83-86, 90 Piorco, J. S., 73 Pik6, L., 217 Pischol, K. D., 33 Poddar, R. K., 62 Raftery, M. A., 233, 235 R"'11, J-. L., 183 Razin, A., 63, 66 Roau, P. M., 49 R6vot, B. M. J • , 221 Roach, J • L. , I 77 Robberson, D. L., 224, 227 Rohwer, R. G., 66, 68 Roth man, B. S • , 18 I Ruosiahti, E. I., 143-144 Rush, M. G., 223-224

Sandor, G., 27 Sarmiento, L. A., 120-121 Scheidt, G. C., 212 Schmir, M., 221 Schrcddcr, J. M., i46 Searcy, D. G., 29 Searcy, K. B., 129 Sadat, J. w., 64, 66 Seybold, W. D., i26 Simmons, D. T., 72 Sinshoimcr, R. L., 61 Sivolap, Y. M., 22 Smart, J • E. , 146- 14 7 Smith, C. A., 216 Smith, L. H., 64 Sperry, R. W., 195, 197-199, 204-205 Stewart, B., 129 Storrio, B., 230 Strauss, E. G., 69 Strauss, J • H. , Jr., 72- 74 Strumwassor, F., 186 Topi itz, M. R., 215 Thomasson, W. A., 124-125 Ting, K.-P., 27 THkds, Z. A., 153 Tracy , U. w. , I 20 - i 21 Trovarthon, C. B., 189-192, 194-195,

197-199 Trubatch, J., 165-i66 Tuchman, J., 115 Upholt, W. B., 219-221 van dcr Eb, A., 216 Van Harrovoid, A., 161-163, 165, 167 Vaughan, S. T., 232 Vi I let, R. H., 50-51 Vinograd, J., 216-223 Wa I I aco, J . M. , 2 15 Ward, S., 101-102 Webb, S. M., 39, 41 Wo i go I , A. H. , 146 Wes torgaard, M., 126 Wiersma, C. A.G., 172-175, 177 W i I son, D. L., 182 Wi Ison, J. H., 105 Wini cur, S., 122 Wood, W. B., 100 Wu, J .-R., 23 Yanagisawa, K., 171-173, 175 Yoon, M., 205-206 Yuan, L. C • , I 31 , I 33 Yuwei lor, A., 79

' l

FINANCIAL SUPPORT INDEX (By page number)

American Cancer Society, 143, 145 ·, American Heart Association, 182, 185 Ear lo C. Anthony Fol lowshlp, 55-56, 58,

105 Mary K. Boschorman Fund, 101-102 Centro de Recherches sur lo Cancer, 221 Jane Coffin Chi Ids Fund for Medlcal

Research, 138, 140 Lucy Mason Clark Fund, 29-30 Council for Scientific and Industrial

Research, South Africa, 50-51 Deutsche Forschungsgemei nscha.ft, 27 Ford Foundation, 141 Free University Amsterdam, 176-177 Gosney Fund, 50-51, 126 Frank P. Hixon Fund, 195, 197-199,

204-205 International Agency for Research on

Cancer, 226-228, 233 International Atom! c Energy Authority,

23 International Research and Exchanges

Board, 22 Los Angelos County Heart Association,

185 McCal lum Fund, 24, 29, 44, 126, 168 Med I scho F acu I to it Rotto rdam, 174-175 National Aeronautics and Space

Administration, 108, 180, 182, 184-185

National Cancer Institute of Canada, 222-223

Natlonal Defense Education Act, 42, 45, 64, 103-104, 123, 206

247

National Institutes of Health, Public Health Service, 21-35, 38-41, 45-49, 52-53, 55-56, 58-64, 66-69. 72- 74. 78-79. 82-90. 94-95. 98, 101-103, 106, 109, 111-115, 118, 122, 124-125, 129, 135, 141, 143-148, 150-153, 157, 159, 160-161, 163, 165-166, 172-173, 177, 180-186, 188-192, 194-195, 197-208, 215-233

National Research Counci I of Canada, 27, 45

National Science Foundation, 27, 31-33, 40, 45, 47, 49, 51, 72-74, 104-105, 107, 113, 120-121, 129-140, 146-147' 162-163, 164, 167, 171-177, 183, 210, 212, 216, 221, 224, 230, 232, 235

Netherlands Organization for tho Advancement of Pure Roseartjl, 216

North Atlantic Treaty Organization, 113, 221

President's Fund, 149-151, 152 Rockefol ler Foundation, 56 Gordon S. Ross Foundation, 120-121,

126 Damon Runyon Mcmori al Fund for Cancer

Research, 23, 73, 223-224 Sloan Fund for Basic Research, 44 Stiftung Volkswagonwork, 47 U.S. Atomic Energy Commission, 38-42,

210 He I on Hay \vh i tnoy Foundation, 182