PROTEIN-PROTEIN INTERACTIONS AND PROTEIN PHOSPHATASE A m IN CAENORHABDITIS ELEGANS SEX DETERMINATION

Ian D. Chin-Sang

A thesis submitted in confomity with the requirements for the degree of Ph-D. Graduate Department of Molecular and Medical Genetics,

University of Toronto

@ C o p m t by Ian D. Chin-Sang 1998

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PROTEIN-PROTEIN INTERACTIONS AND PROTEIN PHOSPHATASE ACTI[VITY IN CAENORHABDITIS ELEGANS SE3C DETERMINA'LZON

Doctor of Philosophy 1998

Ian Dexter Chin-Sang

Department of Molecular and Medical Genetics, University of Toronto

ABSTRACT

Male sexual development in rle nematode Caenorhabditis eelegam requires the

activities of the genesfem-l, fem-2 and@-3. Genetic and molecuiar d y s e s of sex

determination suggest that the threefem genes act to link a secreted, masculinizing signal to

the regdation of transcription, but the mechanism by which they act is not yet rmderstood.

1 report the sequence changes in several loss-of-fùnction alleles of-1. Interestingly, 4

temperature-sensitive fem- l alleles carry missense mutations which affect the ANK repeats

in FEM-1. ANK repeats are protein domains that have been implicated in specific protein-

protein interactions in a number of regulatory pathways. My r d t s suggest the ANES

repeats in F E N I are required for its normal role in promoting male development.

In an effort to idente genes that encode products that physically interact with

FEM- I , 1 selected suppressoa oflem--1 (e2003ts). fem-l(e2003ts) carries a missense

mutation within the nrst AM( repeat of FEM-1. 1 identified a novel class offem-3(gB

allele, idDpl. idDpl is 2 duplication and insertional translocation of at least two copies of

jëm-3 onto chromosome V. Both copies have rearrangements in their 3' UTR which

presumably account for the gain-of-fiinction phenotype idDpl coafers.

To isolate cDNAs that encode products that interact with FEM-3,1 used FEM-3 as

bait in a yeast two-hybrid scxeen and isolated cDNA clones that encode products of two C.

e l e g m sexdetennining genes, ka-2 andfem-2. 1 report that FEM-3 physidy associates

with FEM-2, a member of the Type 2C serine/threonine phosphatase f d y , in vitro and 1

demonstrated that FEM-î exhibits ~ c d e p e n d e n t protein phosphatase activity in vibo.

Point mutations that abolish the phosphatase activity of FEM-2 do not interfere with its

ability to bind FEM-3, but they severely impair its ability to promote male development

My resuits present the first evidence of a protein-protein interaction between the FEM

proteins, the first demonstration of the biochernical activity of any of the FEM proteins, and

the nrst proof that the reguiation of protein phosphorylation is important for sex

d e t e e t i o n in C. elegans.

1 thaak my PhD. supervisor and mentor, Andrew M. Spence, for bis guidance, support and

most of d his patience. 1 am gratefid to the members of my supe~sory committee: Joe Culotti,

Brenda Andrews, and John Roder for helpfid suggestions and guidance. 1 would like to express my

thanks to Marc Perry who (with open arms) invited me into his lab to use his cornputer. I thank the

University of Toronto and the Ontario goveniment for providing me with hding during my graduate

career. The Medical Research Council of Canada dso supported this research.

On a more personal note, 1 wish to express my sincere th& to my lab mates and their

partners, and my colleagues in the department for making my stay in Toronto an enjoyable experience.

1 would Like to acknowledge Jeff Boudreau my special fkiend and partner who has always stood by my

side during the good times and the rough times. He has given me the courage and support to tackle the

most difncult of problems and for this 1 am indebted to him.

Lastly, 1 thank my fiimily especially my mother who has always believed in me. 1 apologize

for not visiting her enough.

TABLE OF CONTENTS

.. ABSTRACT ..w.........w.~~~~~~~~..-.rn.rn.**.m.w*w*..m.rn~~.oe~..~**w~mm. * . ~ ~ ~ ~ ~ ~ ~ . e ~ ~ ~ . w ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ . ~ ~ ~ ~ ..................................................................................... ACKNOWLEDGMENTS ......iV

TABLE OF CONTFHTS .m...-. ......w...............*................................................................ ..e.e~

...............................

Primary Se* Deternù'natrin Signai in Ce elegans: The XIA Ratio . ................................................. . 11 Genes fkat Conftol Loth Sex Deteminatm and X Chromosome Dosage Compemation ., ......m....... 17

m&l . . . o . . . . . . . . . . . . ~ . . . . m . . ~ e ~ ~ ~ ~ ~ ~ ~ o ~ . . ~ ~ ~ ~ ~ ~ ~ ~ m . ~ . e . ~ ~ ~ . ~ ~ ~ ~ ~ ~ w ~ ~ * ~ . ~ ~ . ~ ~ ~ * * ~ * m o m ~ ~ ~ . ~ . . . e . . ~ m ~ ~ . ~ . ~ ~ . e ~ ~ . ~ ~ a ~ . ~ . o . * ~ m ~ ~ * . ~ ~ ~ e e . ~ . . e . . e~e . . e .~ .~ . e* .m~. .~~~ .~~~~~~~ .17

The sdc Genes . . ~ ~ , . . e . ~ H . . ~ ~ ~ . ~ o ~ e ~ t e ~ ~ ~ ~ ~ ~ e ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ L ~ ~ ~ ~ m ~ ~ ~ ~ ~ ~ ~ ~ o e ~ ~ ~ a ~ ~ e ~ ~ ~ o a m ~ a ~ ~ ~ e a ~ ~ ~ ~ ~ ~ ~ ~ ~ . e ~ m ~ ~ ~ ~ m ~ ~ e ~ e ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ m ~ ~ ~ ~ ~ ~ ~ ~ ........ .18 ........................................................................... Dosage Compensation dpy (dumpy) Genes ..,..,........ 19

Somafic Sa Detennriation .............................................................. ... .................................................... 22 Genefr'c Funcfion ........e............................................................................................................................. 25

................................................................ The her-1 Gene Product Acts as a Mascuiiaizing Signal ............... 26 ........................................ The @a-2 Gene Encodes a Putative Receptor for the HER-1 Signal ...,.,......... 27

..................... . The b.a-3 Gene Acts as an Accessory to tra-2 Activity ..w............*........~ooe..~e~.~~~a~.~~.wao~m.~e*.....e. 28 ..................................................................... The fem Genes are Required for Male Developmen t. ............... 29

fem-3 ....O . . ~ a . ~ ~ ~ ~ w . e ~ ~ m ~ ~ . ~ ~ m ~ ~ . ~ a w ~ * * * a * . ~ * e * * . * * œ . ~ . ~ ...me~..~.~......~...~~~..~m~..~**~*~aoa~a~.~~~*~~~..~.......~~~~e.~~~.~.~~~~3l

........................................... .. The tra-I Gene is the Terminal Regulator of S o d c Ses Detemination - 32 ................................................................................. MolecUar Model for Somotfc Ser DeternUnation 33

G e d i n e Sex Defermhation ................ .- ............................................................................................... 38 m e r Genes Involved in Sa Detentllinafion ...............a.......................... ... ................... ... ....................... 44

C m 2: IiMuTATIONSAEIFEClZVG TaEANgYRlRrIPEP'TS OFFEM-I LMPLIC4TE PROTE1Rr-PROEl2V~RACTTONS IN TBE Cm eIegans SMDETERMINATION

.............................................. PA-A Y........................H.........e.o...o...o.o........o......o..........o........o.............. 46

Nucleic Acid Isolation . ....~.e....w....~"om~.mw".~e~mem~~ome~~o~~.e.o...omo.t.oo.w.~om~~.~~o..m..~~eew~aee~m.oo~e~.o..o~oe.ee~e~.eoe~~m~.~e" 0.e00S5 Isolittton of Total Genomic DNA ...................................................................................................................................... 55 Isohîion of DNA from Single Wonns ............................................................................................................................... 55 RNA Isolatian ................................. ., ................................................................................................................................ 55 cDNA SyntkLs ............................................................................................................................................................ 56

Detection by Sequencing ..... ...e~.........~..ne....o.....eH...o.oe....o..oe.oo...ee.....e.oe.~oo....o........e..eo..o....e.e.ooo.o..o..eo.~...e.eeo.e ..62 RESULTS ................................................................................................................................................. 63

LIST OF FIGURES

.................................................... Figure 1-1: The Two Sexes of C elegans. ,... .....,...,,.,. 5 Figure 1-2: Gonad and Tail Morphology of the Hermaphrodite and Male . ,... ....................... .a 9 Figure 1-3: Relationship of the XIA Ratio and Senial Development in C eleg ans. ...................... 12 Figure 1-4: The XIA Ratio Anects Three Aspects of Sexual Development in elega m.............. 14

Figure 1-5: The Genetic Control of C elegum Somatic Sex Determination ............................... 23 Figure 1-6: Molecuhr Mode1 of Somatic Ser Determination in C. elegans . ......m......... *.*36 Figure 1-7: Genetic Control of Gerdine Sex Determination . ...................................................... . 42 Figure 2-1: A Schematic Diagram Showhg the Phenotype of fend Null Mutants . ..................... 50

Figure 2-3: Appmximate Locations of Primera Used in this Shidy ................................................ 59 .... F i p r e 2-4: PCRSSCP Deteetion of 4 Temperature-Sensitive Aileles of fenil .........,.,.....,. 64

Figure 2-5: Sequencing Gels Showing the Region of Mutation in fend Alleles . , ...............,........ 66 . Figure 26: Molecular Characterization of Putative NuU Alleles of fem-l ., ............................... 70

Figure 2-7: Schematic Diagram of the FEM-1 Protein Iliustrating Approximate Locations of Mutations Anecting the f e n d Coding Region.... ...................................................... 73

Figure 2-8: Location of Mutations that Aff'ect the ANK Repeats of FEM.1 ................................. 75 Figure 2-9: Sequence Alignment of the Kinesin Light Chain-Like Motif in FEM-1 with ........................... Represeotative Kinesin Light Chain W C ) Family Membem .. .,., 77 Figure 3-1: A Mode1 of Somatic Sex Determination in elegans. ..........................a..........m............ 88

Figure 3-2: Coimmunoprecipitation of FEM-2 with Myc.FEM-3 ................................................. 104 Figure 3-3: Interaction of [35~]-labeled Myc-FEM-3 with GST-FEM-2 . coated beads . ............ 106 Figure 3-4: Casein Phosphatase A M @ of FEM.2. . . . . . . , , . , . . . ~ ~ e o ~ ~ . o ~ e ~ . o ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ . . ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ o ~ ~ ~ ~ ........... 109

Figare 3-5: Sequence Aiigrunent of FEM-2 with Representaüve PP2C F m Members in the Vicinity of Arginine 336 (indicated with an ajterisk). ....................................... 113

. Figure 3-6: FEM-3 Binciing A M t y of FEM-2 Variants Lacking Phosphatase Activi ty. ....... 115 Figure 3-7: Rescue of Male Development by H a t Shock Fapression of Myc-FEM-2 .........,... 120 Figure 3-8: Expression of Wild-Type and Mutant Myc-FEM-2 in Transgenic Nematodes ....... 123 Figure 3-9: SnbceNuiar LoePléation of Heat Shock lnduced Myc.FEM.2 . ..,..,,...................... 126 Figure 4-1: Selection of ferrrI(e2003ts) Srippressors . .,,,..,.,.,,.............m................................... 144 Figure 4-2: Schematic Diagram of PCR Assay to Detect Ectopie fem-3 DN A. ...,.,.,...,........... 154 Figure 4-3 : Sequence of Wild-Type fem3 in gfRegion of 3'UTR, and Sequence Changes ........................... Resulting fkom fem3(gB Mutations .............................................. 161

LIST OF TABLES

Table 44: Summary of Epistasis Tests. ~ ~ ~ ~ ~ e ~ o e o ~ e ~ e e ~ e ~ e e ~ e e e e ~ e o e e o o e ~ ~ e m e m e e e ~ e ~ m ~ e e e m m e ~ e e e e e e e e ~ e ~ o ~ o e o ~ e e e e e e e o o 176

Table 4-7: Linkage of Ectopie fem-3 Sequences to d p p l l and idDp1 e ~ e ~ e ~ m e ~ e e e m e ~ e e e ~ ~ e ~ ~ e o e e e e e e e e e e e ~ - e e o e 181

LIST OF ABBREVIATIONS AND GENE NAMES

ACEDB: A C. elegm Database ANK: ankyrin BLAST: Basic Local Alignment Search Tool bli: blistered BSA: Bovine serum albumin CD: circular dichroism CDC: cell division cycle Co-IF: Coimmunoprecipitation DAE3CO: l,4-diazobicyclo-[2.2.2]-uctane DAPI: 4' ,6'-diamidino-2-pheny lindole DEPC: diethyl pyrocarbonate DIC: differential interference con= mimscopy &y: dumpy DRE: direct repeat element EDTA: ethylenediamine (te-) acetate e g enhanced gain-of-hction EMS : ethylmethanesulfonate fem: feminization FITC: fluorescein isothiocyanate fog feminization of the germline fox: feminizing locus on X & gain-of-hction GFP: green fluorescent protein gld: gemiline defective GLI: glioblastoma GST: giutathione S-ûansferase her: hermaphroditization him: hi& incidence of males HSN: hermaphrodite specinc neuron hsp: heat shock promoter IPTG: Isopropylthio-P-D-gaiactoside KLC: kinesin light chah lq t lethal and fog & loss-of-hction LG: linkage group lon: long a b : monoclonal antibody mog: m a s c ~ t i o n of the germline mot: morphologid abnonnality MYOB: Modined Younger's, only Bactone-Peptone NGM: nematode growth medium NLS : nuclear localization signal

PBS: phosphate-buffered saline PCR: polymerase chah reaction PEG: polyethylene glycol PIPES: 1,4-Pipaazined.iethandonic acid PMSF: PhenylmethyLsulfonyl fluoride PP2C: protein phosphatase type 2C p: revolutions per minute RT: reverse transcriptase sdc: sex and dosage compensation SDS: Sodium dodecyl sulfate SDS-PAGE: SDS polyacrylamide electrophoresis SMC: structural and maintenance of chromosomes smg: suppressor with morphogenetic effects on genitalia SSC: Standard d i n e citrate (0.1 5 M NaCl 1 5 mM sodium citrate) SSCP: single strand conformation polymophism sup: suppressor SWI: defective in rnating type switching TE: Tris EDTA bufffer (1 O rnM Tris-CI, 1 mM EDTA pH=7.4) TCA: Trichloroacetic acid Pa: transformer unc: uncoordinated UTR: untranslated region WT: wild-type X-gal: 5-Bromo4chloro-3-indolyl-P-D-galadoside m l : XO lethal

CHAPTER 1: KNTRODUCTION AND LITERATURE REVIEW

General Introduction

A fundamental question in developmentai biology is: How do ceils acquire

different fates? More specifically, how is the decision to adopt a particular fate made, how

is it executed and how is the fate maintained? Understanding developmental processes

requires an understanding of mechanisrns such as celi to ceii communication and signai

transduction and how the control of gene expression results in changes of ceil growth,

movement and differentiation. Signincant advances in understanding developmental

processes have corne h m dissection of complex genetic pathways in vivo, in conjunction

with in vitro molecular and biochemicai studies. Mode1 organisms such a s the nematode

Caenorhubditis elegans and the fhit fly Drosophila rnelmogaster have been instrumental

in the analysis of genetic pathways and have been extensively used in investigating

developrnental processes.

An extraordinary example of how evolutionarily divergent organisrns use simila.

mechanisms during development is that of the Ras-regulated MAP Kinase pathway

(Ferreli, 1996). The Ras oncogene is a major player in a pathway regulating ce11 growth

and division in mammalian cells. In DrosophiZa Ras is involved in eye development and

many other developmental pathways (Chang et al., 1994), and in C. elegans it directs wlva

developrnent, ceii migrations, and 0th aspects of development (Selfors and Stern, 1994;

Kayne and Sternberg, 1995). The Ras pathway is also a striking exampIe of how many

different organisms use nearly identical pathways to control adoption of different

developmental fates. Other examples of conserved developmental signaling pathways

include the Wnt/wing!ess, and TGF$ pathways (Moon et al., 1997; Hogan et al., 1 994;

Eaton and Cohen, 19%). In view of these discoveries, it is Ucely that a novel signaling

pathway that controls important aspects of development in one species, will have been CO-

opted in evolution to regulate aspects of developrnent in other species.

One of the more productive areas of research into the mechanisms of development

has been the study of sex detennination. SeMiality is a ubiquitous phenomenon, found in

single ceU organisms, plants and animals. Since sex is nearly a universal phenomenon, sex

determination has captured the interest of evolutionary, developmental and molecular

biologists. Sex determination systems are surprisingly diverse, as illustrated by three weIi

studied organisms: the fiuit fly Drosophila melanoguster, the nematode Caenorhabditis

elegans, and the mouse. These three animals dl have extensive sexual dimorphism and sex

chromosome dosage compensation, yet the underlying cellular and molecular mechanisms

that controi sexual fate of these animals are quite unrelated. The shidy of sex determination

in these three animals demonstrates that evolution can produce many solutions to the same

basic problems in development. This thesis focuses on a genetically weli-characterized

signaling pathway that controls sex detennination in the nematode Caenorhabditis elegum.

C. elegons as a Mode1 Organism

In comparison with other animals C. eleguns has many advantages for genetic

anaiysis. It is small(1.5 mm), it has a rapid (3 days) life cycle, it is inexpensive, it is easy

to maintain, and stocks can be fiozen at -70°C indefinitely. These features d o w

researchers to manipulate large numbers of aniIirids, which is a prerequisite for detailed

genetic analysis. Other appealing feahne~ of C. elegans are:

Its s m d genome of about 100 Mb, which is scheduled to be sequenced in its entirety

by the end of 1998.

The complete celi Lineage nom fertilization to adult wonn is characterized (Sulston and

Horvitz, 1 977; Sulston et al., 1983). The development of C. elegmis is airnost invariant

at the single-cell level. Therefore the existence of the cell lineage itseifis extremely

useful because it makes it possible to describe development as a celi lineage in which

the fate of every celi is know~.

C. elegans has a ~e~fert i l izing mode of reproduction which enables strains to survive

while carrying mutations that would be lethal because they prevent rnating.

There are a wide range of sophisticated genetic tools, e.g., duplications (Qs),

deficiencies (m, mosaic analysis and an increasing array of tissue specific promoters and markers of ce11 fate.

Transgenic worms are easily made as DNA can be transfonned into C. elegum by

microinj ection techniques.

These features have encouraged researchers to use C. elegum as a mode1 genetic system to

shidy many aspects of development.

Sexual Dimorphism in C. elegans

C. elegans exists as a self-fertilinng hermaphrodite or a male ( Figure 1-1). The

hermaphrodite can be thought of as a modified female animal; somaticdly it is female, and

its germline is of mixed sex, producing both spem and oocytes. The hermaphrodite first

produces about 300 spem and then switches to oocyte production. The spem are stored

intemaily in a specialued cornpartment, the spematheca, and fertilize the oocytes to yield

about 300 self progeny. The hermaphrodite can also mate with a male to produce cross

Figure 1-1: The Two Sexes of eleganr.

Schematic diagnim showning major anatomid features of the hermaphrodite (top) and

male (bottom) (modifïed with permission h m Hodgkin, 1988).

Hermaphrodite discal gonaci

progeny. Geneticdy, the only diffefence between the hermaphrodite and male

is the nmber of X chromosomes, of which the hermaphrodite has two 0

and the male has one @O). The two sexes mer extensively in most tissues and

organs, including the musculature and nervous system, and ia their behavion.

There are 959 somatic nuclei in the adult hermaphrodite and 103 1 somatic

nuclei in the adult male. About 650 of those ceiis appear to be identical in both

the male and the hermaphroditey such as the main body muscles, pharynx and

excretory system. The remainder of the cells, about 30% in the hermaphrodite

and 40% of male nuclei, are sexually dimorphic. These ciifferences have been

reviewed in greater detail elsewhere (Suiston and HOM% 1977; Kimble and

Hirsh, 1979; Hodgkin, 1988). I WU review only the most distinctive features of

the male and the hermaphrodite.

Somatic Gonad

The overall shape and symmetry of the gonad is quite different in the

male and the hermaphrodite. The hermaphrodite somatic gonad has a

symmetricai bilobed structure, whereas the male has an asymmetrical single-

lobed gonad (Figure 1-2A). For a detailed description of gonadal ce11 lineage,

see Kimble and Hirsh (1979). The hermaphrodite gonad consists of the ovaries,

spermatheca, and a central uterus that conaects to the vulva The male sornatic

gonad consists of reflexed testis, a seminal vesicle and a vas deferens that joins

the cloaca in the tail (Figure 1-2A).

Tai1

To the observer using a dissecting microscope the most obvious

ciiffierence between the dui t hermaphrodite and male is the structure of the tail

(Figure 1-2B). The hermaphrodite tail is a simple whip-like structure, while the

male tail contains several structures that are specialized for mating. The most

striking anatomical feature of the male rail is the copulatory bursa consisting of

an acellular cuticular fan that is supported by nine pairs of sensory rays. Two

copulatory spicules lie agauist the dorsal surface of the cloaca, and during

mathg they aid in locating the vulva and the W e r of sperm (Suiston et al.,

1980).

Germiine

The male germline consists of mitotic precursors, ceiis in various stages

of meiosis and spennatogenesis, and spermatids. Unlike flageilated spem of

m ~ d s , mature nematode sperm fonn pseudopodia and exhibit amoeboid or

crawlhg motility (Roberts and Stewart, 1995). The hermaphrodite germline

consists of mitotic precursors and cells in various stages of gamete

development. The first germ cells to differentiate become sperm, then the

germline switches exclusively to the production of oocytes.

Figure 1-2: Gonad and Taïi Morphology of the Hermaphrodite and Male.

(A) Schematic diagram of the development of the hermaphrodite and male somatic

gonad (modified with permission h m Kimble and Hirsh, 1979). Gonadogenesis in

hermaphrodites (lefi) and males (rïght). The mid-ventrd position of the gonadal

primordium (gp) is the same in both sexes at the L1 stage. Shown is the morphology of the

somatic gonad at successive stages of development (LI to Adult). The adult hermaphrodite

has a symmetrical bi-lobed gonad with a central opening in the uterus that connects to the

vulva on the ventral side; (dtc) distai tip ceii; (ac) anchor celi; (sp) spermatheca ne xiuk

male gonad is an asymmetricai singIe lobed structure; (Ic) Mer ceil. The distal arm of the

somatic gonad lies ventraiiy in the male but occupies a dorsal position in the

hermaphrodite. (B) DIC photo-micrographs showing the hermaphrodite (top) and male

(bottom) taiI morphology. The hermaphrodite has a simple whiplike spike for a tail. In

con- the male tail is complex and is specialized for mating. Distinctive feahires are the

nine pairs of sensory rays (arrows) and a cuticular fan.

Primary Sex Determination Signal in C. elegans: The XfA Ratio

C. elegm embryos that have 2 X chromosomes develop as hermaphrodites,

whereas embryos with only 1 X chromosome develop as males. However, it is not the

absolute number of X chromosomes that determines sex in C. elegm, but the number of X

chromosomes in relation to the number of total sets of autosomes, known as the W A

ratio (Madl and Hennan, 1979). Normdy, a . a l s with an XIA ratio of 0.67 or less are

male. Animais with an X/A ratio greater than 0.75 develop as hermaphrodites (fiom

Nigon, 1 949; Hodgkin et al., 1979; Madl and Herman, 1979; Hodgkin, 198%). The

relationship between sex and XIA ratio is suxnmarized in Figure 1-3.

The X/A ratio affects three aspects of s e 4 development: 1) dosage

compensation, 2) sornatic sex determination, and 3) gerrnline sex detemination. Each of

these three areas of sexud development wiii be discussed separately below. In C. elegm,

the WA ratio sets a weiI defbed cascade of gene interactions to one of two reciprocal States

(outlined in Figure 1-4). The fïrst part of this cascade includes the genes xol-1, sdc-1, sdc-

2, and sdc-3 that regulate both sex determination and dosage compensation. At the level of

the sdc genes the pathway diverges into two separate and for the most part independent

pathways. One branch of the pathway regdates X chromosome dosage compensation. X

chromosome dosage compensation equalizes X-linked gene expression in the two sexes. In

C. e legm at Ieast five dosage compensation dumpy (dpy) genes are involved in the X

chromosome dosage compensation process (Figure 1-4).

Figure 1-3: Relationship of the W A Ratio and Semai Development in C. eleguns.

Sex of the animai is shown as a &don of the XIA ratio, The number of X chromosomes

(X) is shown on the vertical axis, the number of the total sets of autosomes (A) is shown on

the horizontal axis. Ratios are enclosed in brackets. Ln general, animas with an X/A ratio

x0.67 develop as male, and animals with an WA ratio >0.75 develop as hermaphrodite.

Adapted h m Meneely (1 994).

d. maie e"; hm,,,

highmortality ND; not determined

Dead ND (2-0) (1.33) (1.0)

Q Q Q (1*9 (!-O) 0 . . C!*?5l . . Q i s

(1.0) (0.67) Cr

8 . I I . . . .

(0.9

d 8 Dead (0.5) (0.33) (0.25)

2 3 4

Figure 1-4: The X/A Ratio Anects Three Aspects of Senial Deveiopment in C elegans.

The nrst part of the pathway includes 4 gens (xol-1, sdc-1. sdc-2,and sdc-3) that

coordinately control X chromosome dosage compensation and sex determination. The

pathway branches at the level of the sdc genes. One branch (top) includes genes (dpy-21,

dm-26-28, and dw-30) that regulate X chromosome dosage compensatiof~ The other

branch includes the genes that control sex detemination. Seven genes control both somatic

and germline sex determination; their order of genetic interaction in the germiine is

different fiom the order of interactions in somatic tissues, shown below. In somatic sex

determination tra-l is the tenninal regulator that determines male or femaie somatic fates.

The germline sex determination pathway also has a number of genes that are hvolved

excluively in the germline (fog-2,fog-l, fog-3, mog-l and gld-1). In contrast to the

somatic sex determination pathway, the fem genes and two gedine-specific genes, fog-1

and fog-3 are the terminal regulators that promote spermatogenesis in the g e r m e s of both

XX and XO animals. Other minor interactions atfect semai phenotype; however, they

have been omitted for the sake of simpiicity. Barred lines indicate negative influences

while arrows indicate positive influences.

X Chromosome Dosage Compensation

Xy 1 sdc-2 m o

S~C-3 \ - --J'Y fem-1 v Femaie Fate Primsry Signai Coordinate Conml her-1 -( îra-2 -( m-2 4 m-1

Male of Sex Detrminetion &a-3 fem-3 Fate

Intemicdiato mgulators Terminal coordination by cciicalî 8omtic 1 interaction ragulator

Somatic Sex Determination 1

The other bmch of the pathway exclusively regulates sex detennination. At least

seven genes (her-l,tra-2, ira-3, fem-l, fem-2, fem-3 and tra-1) contml somatic sex

determination. These seven genes also have roles in gerrniîne sex determination. In

addition, other known genes (rog-2, mog-I - mog-6, fog-1, fog-3 and gld-2) act specificaliy

to control germliue sex detemination (Figure 1-4).

Very Little is known about the molecuiar nature of the XIA ratio in C. elegans.

However, it is obvious that a comting mechanism must exist. The counting mechanism

may invohe expression of X-Wed numerator and autosoma1 denorninator elements. A

numerator must fWiU three criteria: first, it must be located on the X chromosome, second,

a change in its copy number shouid disnipt dosage compensation, sex determination, or

both, and third, these elements should be the most upstream regulators in the sex

determination / dosage compensation pathway. Hodgkin et al. (1 994) and Akerib and

Meyer (1 994) identified a region on the lefi end of the X chromosome that contributes

strongly to the XIA signal. Duplications fiom this region cause nearly dl males to die h m

dosage compensation defects . One numerator element fiom this region is called fox-l (for

feminizing locus on X ) (Hodgkin et al., 1994). Multiple copies of this locus i nc~ase the

perceived XIA ratio and are lethal or feminizing to XO animals but have no effect on XX

animals. XO animals with duplications of fox4 die because they inappropriately

implement the hermaphrodite mode of dosage compensation. The fox-1 gene encodes a

putative RNA binding protein (Hodgkin et al., 1994; Nicoll et al., 1 997). fox-2 is not an

essential gene, as XO animais carrying null alleles are M y viable (Nicoll et al., 1997).

The molecuiar function of fox4 is presently unknown; however, if it acts as an RNA

binding protein its effects on other gene(s) are presumably post-transcriptional.

No gene characterized so far in C. elegmrs is a candidate for a denominator

element, the "A" part of the H A ratio. One would predict that these genes when in

multiple copies should Iower the perceived X/A ratio and have XX-specifïc dosage

compensation and sex determination effects. Deficiencies in these genes should increase

the perceived N A ratio and have XO-specific effects.

Genes that Control both Sex Determination and X Chromosome Dosage Compensation

Four genes, xol-1, sdc- 1, sdc-2, md sdc-3. coordinately control both sex

detennination and dosage compensation in C. e l e g m Tablel-1 provides a summary of

their genetic and moiecular properties.

ml-I

Genetic epistasis experirnents place ml-l (XO leîhd) as the earliest acting gene in

the genetic pathway that govems sex detennination and dosage compensation in C.

elegam. Therefore, xol-1 may be a direct target of the XIA signal (Miller et al., 1988)

(Figure 1-4). Nul1 mutations in xol-l cause the death of XO aaimals, and the rare suMvors

exhibit feminization. XX animals appear unaffected. The XO-specific lethality results

fiom reduced X-linked transcript levels caused by inappropriate activation of the

hemaphrodite mode of dosage compensation. These data suggest that the wild-type

fiinction of x d l is to promote male development and to ensure that downstream genes

controlling hermaphrodite development and dosage compensation are inactive in XO

anirnals (Miller et al., 1 988). Rhind et al. (1 995) showed that overexpression of ml-l fiom

a heat shock promoter during gastrulation triggers male development in XX animals and

causes death by disrupthg dosage compensation. Overexpression of xol-I at other times

during development did not have this eff- These results demoastrate that xol-l fimctiom

as an early developmentai switch to set the choice of sexuai fate. Assesment of the X/A

ratio must occur early in embryogenesis to determine sex. The fox-I gene does not seem to

have an effect on the transcription of xol-I and thus the regdation of xol-I by fox4 may be

pst-transcriptional (Nicoll et al., 1997). xol-I transcript Ievels are, however, reguiated in

a temporal and sex specifk manner: xol-l transcripts are 10 fold higher in early XO

ernbryos (c 6 hours of development) as c ~ q d tc = y XX embryos (Rhind et al.,

1 995). Therefore xol- I must be transcriptionally regulated by something other than fox-1.

Alternative splicing leads to the production of 3 xol-l mRNAs that are predicted to encode

three novel proteins (Rhind et al., 1 995).

The sdc Genes

The three sdc genes act at an early step in the regdatory hierarchy downstream of

xol-I. Their loss-of-function phenotypes are similar but not identical. Loss of fimction

mutations in any one of the sdc genes result in a shift of both the sex determination and X

chromosome dosage compensation processes to an XO mode of expression. Such

mutations have no apparent effect in XO animals, but cause two distinct phenotypes in XX

animals: 1) mascuiinization, reflecting a defect in sex determination and 2) IethaIity or

dumpiness, reflecting a disruption in dosage compensation (Hodgkin, 1983; Meyer and

Casson, 1986). Geneticdy, aIl three sdc genes behave as targets for negative regdation by

xol-1. However, sdc-2 may be a direct target of ml-I as multiple copies of the sdc-2 gene

can suppress the XX I e W t y caused by over expression of XOL- 1 (Rhind et al., 1995).

SDC-1 contains seven zinc fingers and is predicted to fiindon as an embryonic

transcription factor regulating downstream genes involved specificalIy in the sex

deterrnination and dosage compensation pathways (Nonet and Meyer, 199 1). The sdc-3

gene is unique among sdc genes in that its sex determination and dosage compensation

hctions are genetidy separable. Molecular arialysis reveals that separate domains of the

SDC-3 protein control these two developmentai processes. Dosage compensation sdc-3

mutations specifically eliminate a pair of zinc finger motifs at the carboxyl temiinus of

SDC-3, while alleles defective in the sex detemiination hc t ion of sdc-3 have mutations

affecthg a region with limited homology to the ATP-bindiag domain of myosin (DeLong

et al., 1 993; Klein and Meyer, 1993).

Dosage Compensation dpy (dumpy) Genes

In nematodes, flies, and mamrnais, dosage compensation equaiizes X-chromosome gene

expression between the sexes through chromosome-wide regdatory mechanisms that

f'wiction in one sex to adjust the levels of X-linked transcripts. In C. elegam, dosage

compensation is achieved by decreasing the level of X Linked transcripts fiom both

hermaphrodite X chromosomes. Dosage compensation in C. elegum is d e d out by at

least five autosomal genes (see Figure 1-4) c d e d the dosage compensation dumpy genes:

dpy-2 1, dpy-26, Ùpy-2 7, dpy-28, and dpy-30. The activity states of these genes respond

indirectly to the primary sex determination signal, the XIA ratio (Hodgkin, 1983). Ail the

dosage compensation dumpy genes except 4zy-21 cause XX-specific lethality. The genes

were named for the fact that rare XX survivors have a distinctive dumpy phenotype (short

and fat). Mutations in the dosage compensation dumpy genes except dpy-ll increase X

Iùiked gene transcription in XX animals but not in XO animais. In dpy-22 mutants X-

Linked transcription is increased in both XX and XO animals (Meyer and Casson, 1986;

Hsu and Meyer, 1994). Dosage compensation is achieved by a protein complex that

associates with the X chromosome in a sex-specinc fashion to modulate gene expression.

This complex includes at least four proteins, including SDC-2, SDC-3, DPY-26, and DPY-

27 (Chuang et ai., 1996; Lieb et aL, 1996). SDC-3 requires its zinc £hg= motifs for its

association with the X chromosome. Interestingly, DPY-27 is a member of the SMC

(structural maintenance of chromosomes) family of proteins (Chuang et al., 1994). The

SMC proteins are involved in assembly and structural maintenance of Xenopus

chromosomes in vitro, and in the segregation of yeast chromosomes in vivo (Hirano et al.,

1995). These fkdings suggest that C. eIegm may have adapted an evo lu t io~ ly

conserved mechanism of chromosome condensation to achieve dosage compensation.

Table 1-1: Properties of Genes that Coordinatety Control Sex Determination and Dosage Compensation.

Gene Phenotype Gene Product Regulation and Function References

XO: lethal, ferninized XX: wild-type

XO: wild-type XX: Mascuiinization and high X- linked gene expression

Like sdc-l but more severe, some alleles lethal in XX

XO: wild-type XX: Sex determination (Tm) and dosage compensation (Let or Dpy) defects genetically separable. Nul1 alleles are lethal and have no overt sex determination nhenotwe.

Novel proteins 355,417, and 425 a.a.. The first 322 a,a. common to all three proteins. The 4 1 7 a.a. protein sufficient for ml-l activity.

1203 a.a. protein with 7 Zinc fingers

Novel

2 150 a.a. protein with 2 Zn fingers (required for dosage compensation)and an ATP binding motif (required for sex determination)

Early switch gene in XO (Miller et al., 1988; 8Nmals. Direct target of XIA Rhind et al., 1995) ratio? Negative regulator of sdc genes, speci fically sdc-2?.

Matemal, sex-specific. Non ce11 autonomous. Negative regulator of her-l . Positive regulator of dosage compensation dpy genes.

A switch gene. Negative regulator of her-1 . Positive regulator of dosage compensation dpy genes.

Maternal, sex-specific. Negative regulator of her-1. Positive regulator of dosage compensation dpy genes.

(Villeneuve and Meyer, 1987; Villeneuve and Meyer, 1990b; Trent et al., 1991)

(Nusbaum and Meyer, 1989; Rhind et al., 1995; Lieb et al., 1996)

(Klein and Meyer, 1993; Davis and Meyer, 1997)

Somatic Sex Determination

In this section 1 summarize genetic and molecular data about the seven genes that

regdate sex determination in ail tissues. I WU first rariew the genes' roles in somatic sex

determination; germline sex determination will be discussed separately below. Hodgkin

deduced nom epistatic interactions arnong the dominant and recessive mutations in these

genes that the global sexdetermining genes act in a negative regdatory pathway (Figure 1 -

4 and Figure 1-5) (Hodgkin, 1980; Hodgkin, 1986). The last gene in the pathway that

controis somatic sex is tra-1. If hpa-I activity is high, it promotes femde somatic

development, and if ho-I activity is low this le& to male development. In XO animals,

three genes, fem-1, fem-2 and fem-3 down-regdate tra-l activity to allow male somatic

development. Thefem genes are targets of two upstream negative regdators, -2 and tra-

3. The fia-2 and tro-3 genes are under the negative influence of her-l activity. The state

of the pathway is set by the X/A signal, which is relayed to her-l by the sdc genes. The

genetic and molecular properties of the global sex determination genes are summarized in

Table 1-2 and Table 1-3 respectively.

Figure 1-5: The Genetic Control of C elegauis Somatic Sex Determination.

The two states of the pathway that lead to femaie or male somatic development are

illustrateci. Gene activities are indicated as either high or low. Barred lines indicate

negative influences, whiie arrows indicate positive innuences. The broken line h m the

X/A signal indicates that the genes upstream of hep-l (Figure 1-4) have been omitted for

sake of simplicity. Other rninor interactions have been left out for simplicity.

I - h l -( -2 ratio f for-3 tra-3

Low H%h

mm Low

The Ber4 Gene Product Acts as a Mascalinmng Signal

her-l is the fïrst gene in a hierarchy of g e n s that exclusively regulates sex

detexmination (Figure 1 4 and 1-5). N d mutations of her- l cause complete ûmdormation

of XO males into self-fertilizing hermaphrodites, but they have no effect on XX animals

(Hodgkin, 1980). Therefore the her-l gene is required for male sexual development. Gain-

of-hction alleles of her-l behave as if they have escaped the negative influence of the sdc

genes and cause inappropriate rnasciilini7iition of XX animas. Since the gainsf-fhction

mutations cause an oppsite phenotype to that of loss-of-fhction her-l mutations, hep-l

acts as a switch gene.

A rare 1.2kb mRNA and an abundant 0.8kb mRNA are transcribed fiom two

promoters Pl and P2 respectively. Both are sex-specific as they are more abundant in XO

animds than in XX animals (Trent et al., 199 1 ; Perry et al., 1 993). The two gainef-

function alleles (n69.5 and yl0l) carry identical single base mutations that map to the Pl

promoter of her-l (Perry et al., 1994). The gain-of-hction promoter mutation may

identify a contact site for a repressor(s) of her-l transcription because the gfalleles are

inappropriately expressed in XX anirnals, resulting in masculini7iition of XX

hermaphrodites (Trent et al., 1988; Trent et al., 199 1 ). Good candidates for transcriptional

repressors of her-l are the upstream negative regdators of the her-l gene: sdc-1. sdc-2 and

sdc-3. XX animals that carry (fl mutations for either sdc-l sdc-2 or sdc-3 inappropriately

express both her-l transcripts (Trent et al., 1 991 ; DeLong et al., 1993). This implies that

the sdc genes act to control her-l at the transcriptional level.

Hunter and Wood (1992) showed that her-l acts non ceU-aidonomously. The

Iarger her-1 transmipt is predicted to encode a small 175 aa protein that is cysteine-rich

and has a signal sequence at the N-terminus. The signai sequence is essential for HER-1

activity. Furthermore, expression of HER-1 fiom the muscle-specific rmc-54 myosin

promoter causes inappropriate m a s c ~ t i o n of many tissues in XX animals (Perry et al.,

1993). These results suggest that the larger product of the her-l gene may act as a secreted

product to signai a male development program. The genetic target for her-2 activity is tra-

2. Since tm-2 encodes a putative receptor protein, HER-1 is a candidate for a ligand that

binds the TRA-2A receptor.

The -2 Gene Encodes a Putative Receptor for the HER-1 Signal

Null mutations of ha-2 transfomi animals into incomplete maies and have no

effect on XO male animals (Hodgkin and Brenner, 1977). Therefore, PU-2 is required for

female sexual development in XX animais The tra-2 gene is weakly haplohmfficient.

Some tra-2 / + heteroygous XX animals exhibit slight m a s c ~ t i o n (Doniach, 1986).

These animals have abnormal or missing hermaphrodite-specific neurons (HSNs).

Programmed celi death of the HSNs in the male is the nrst visible sign of sexuai

dïmorphism in C. elegrnrî, and is usuaily a sensitive indicator of weak mascuIini7ation in

XX animals because it results in an egg laying defective (Egl) phenotype (Trent et al.,

1983).

The tra-2 gene encodes two proteins, TRA-2A and TRA-2B. The larger protein,

TRA-2A, is predicted to have 9 membrane-spanning domains, with an amino-termiml

extracellular domain and a carboxy-terminal cytoplasmic domain (Kuwabara et al., 1992).

Full-length TRA-2A can rescue XX Pa-2(-) animais and is &cient to feminize WT XO

animals. Furthermore, overexpression of the carboxy-termiral region of TRA-2A cm

partiaiiy feminize XX 1ra-2QJ mutant and XO tra-2(+) males (Kuwabara and Kimble,

1995). Therefore the intraceliular domain of TRA-2A praumably promotes female

development by negatively regulating one or more of the FEM prote&. Mehra et al. (in

preparation) showed that the C-temiinal portion of TRA-2A interacts with FEM-3, and this

interaction serves as the primary means of negative regdation of FEM activity.

Hodgkin and Albertson (1 995) screened for enhancers of tra-i(e2046gB and

isolated a unique class of na-2 gain-of-hction alleles c d e d the "eg" (enhanced gain-of-

function). "eg" aileles of ha-2 appear to be insensitive to the negative influence of her-l

activity. AU ten @a-2(e@ mutations encode the identical missense change, R177K, in the

predicted extracellular domab of TRA-2A (Kuwabara, 1996). The site of the eg mutation

may define a major negative regdatory site within TRA-2A. Since HER-1 is predicted to

be a soluble ligand that interacts with TRA-2A, the arginine residue at position 177 may

identifjr a specific amino acid in TRA-2A that is required to contact the HER-I protein.

The ha-3 Gene Acts as an Accessory to tra-2 Activity

Nul1 aüeles of PU-3 are Mly recessive and masculinize XX animais to an extent

simîlar to that of weak loss-of-function alleles of tra-2. Very linle of the fra-3 product is

required for femaie somatic development, because fia-3 ndi mutants exhibit cornpiete

maternai rescue, and amber aileles are completely suppressed by weak tRNA amber

suppressors (Hodgkin, 1986). Genetic evidence suggests that tra-3 acts as a positive

reguiator of ira-2, and may act as an accessory to ira-2 fbnction. F i a tra-3 is dispensable

in the presence of certain tru-2(gB alleles (Doniach, 1986). Second, genetic epistasis

experiments place @a-3 upstream of -2. Goodwin et al. (1 997) showed that a mutation in

the Id-l (lethal and fog) gene is epistatic to ha-3 but not to tru-2, thus fonnaly separating

ira-3 from tra-2 (figure 1-5). The laf-I gene behaves as a putative translationai repressor of

ha-2, however it also has at least one other essential d e in development (Goodwin et ai.,

1997).

The bu-3 gene product is similar in sequence to the large subunit of a family of

calcium-regdated proteases, the calpains (Barnes and Hodgkin, 1996). Calpains are

processing proteases that cleave a substrate at only a d number of sites and are capable

of causing either protein activation or inactivation. The TRA-3 protein lacks the regdatory

calcium binding domain found in most calpains and to date there is no biochemical

evidence that TRA-3 can act as a calpain.

The fem Genes are Required for Male Development

The threejZm genes are required for ai i aspects of male development (Doniach and

Hodgkin, 1984; KimbIe et al., 1984; Hodgkin, 1986). In the soma the three fem genes

promote male development by down-regulating the activity of tra-1. Nul1 mutations in any

one of thefern genes cause XX and XO animais to develop as fertile females. AU threejëm

genes exhibit maternal effects; wild-type matemal products can p d y rescue homozygous

fem progeny of heterozygous hermaphrodites.

Complete feminization by fem-l n d alleles is only seen when the mother is

homozygous for fem-l(-). These animals are referred to as fem-l(m-z-), following the

convention of Hodgkin (1986) to indicate that there is no maternai (m) or ygotic (2) fem-I

activity. About 20% of the homozygous fem-I(m+ z-) progeny of afem-l / + heterozygous

hermaphrodite can develop as hermaphrodites giving rise to broods that are 100% femaie

(Doniach and Hodgkin, 1984). The matemal contribution of the fem-l gene product is

capable of causing somatic m a s c ~ t i o n in XX h a l s but is normdy prevented h m

doing so by its negative regdators tra-2 and fia-3. Therefore tra-2 and or ira-3 must be

capable of exercising post-transcriptional control over at least one of thefem gene products

(Doniach and Hodgkin, 1984; Hodgkio, 1986)

The fem-l gene encodes a single protein of 656 aa and is predicted to be a soluble

intcacellular protein (Spence et al., 1990). The most striking feahire of the gene product is

the presence of 6 copies of the cdc lO/SWI6 motif(ANK v a t ) near its N-temiinus. The

ANK repeats in many other proteins forrn a structural domain that mediates protein-protein

interactions @ennet& 1992; Michaely and Bennett, 1992). Gaudet et al. (1996) reported

that the expression offem-l mRNA and protein is constant throughout development in both

sexes. The fact that XX hermaphrodites express bothfem-1 mRNA and protein in their

soma, but nonetheless stiU adopt female fates, argues that the activity of*-1 is regulated

pst-transcnptionally and most likely pst-translationaliy. h e d with the laiowledge that

FEM-1 contains 6 AM< repeats, and that its regulation is most LikeIy post-trauslational, it is

reasonable to suggest that FEM-1 engages in protein-protein interactions, specifically

through its AM< repeats to regulate its own activity or to regulate other proteins.

fem-2

Genetic data suggest that very littlefim-2 activity is required for male development

and under certain conditions pamal male development can occur independent offem-2

activity. First, the fem-2 gene shows 100% maternal rescue (Hodgkin, 1986). This implies

that the matemal contribution of WTfem-2 product (mRNA or protein) to the oocyte is

sufficient for spermatogenesis in the XX animals, albeit less spermatogenesis than wild-

type XX animais. The materna1 contribution can also allow extensive male somatic

development in XO animals that are homoygous forfm-2(-1 at 2S°C. Second, null deles

of fem-2 are temperature-sensitive; that is, XO homozygous&m-2(-1 animals are

compietely feminizeâ only at 25OC (Hodgkin, 1986; Pilgrim et al., 1 995).

The fem-2 gene is expresseci as a single t r ans~p t that is detectable at alI stages but

most abundant in adult XX animals d h g oogeaesis. The fem-2 gene encodes a 449 aa

protein related in sequence to the type 2C serine/threonine protein phosphatases (Pilgrim et

al., 1995). The role offim-2 and its product is discussed in Chapter 3 of this thesis.

fem-3

Although all three fem genes are required for male development in C. elegans,

genetic evidence mggests that fem-3 is limiting among the fem genes, and that its activity is

dose sensitive. First, uniikefem-I andfem-2, the fem-3 gene exhibits a maternai absence

effect: among fem-3 / + heterozygous progeny fiomfem-3(-) fernales, about 15% of the

XX animals are female, and about 30% of the XO anirnals are partiaily feminlled in both

their soma and germline. Therefore, the maternai contribution of WT fem-3 is required for

male development and zygotic fem J(+) is sometimes not enough for nomial male

development. In addition to its matemal effects,fem-3 exhibits haploinsufficiency for

hermaphrodite spermatogenesis; about 5- 10% offem-3 / + XX animals are femaie even

when descended from mothers carrying a WT copy of fem-3 (Hodgkin, 1986; Barion et al.

1987; Rosenquist, 1989). Second, overexpression offem-3 fiom a heat shock promoter

causes a dominant gain-of-function phenotype resuiting in inappropriate masculinization of

XX animais in the soma (Andrew M. Spence p o n d communication). Third, several

gain-of-fhction alleles of fem-3 exist that behave as if they caüsefem-3 activity to be

constitutive in the germline parton et al., 1987). XX animals that are heteroygous or

homozygous forfem-3(@ deles only produce spem and the switch to oogenesis does not

occur (Barton et a%, 1987). This phenotype is referred to as the Mog phenotype (for

r n a s c u l ~ t i o n of the germhe). The gain-of-fûnction phenotypes offim-3 suggest that

fem-3 acts as a switch gene in both the soma and the germiine.

The predicted product of thefem-3 gene is a protein of 388 amino acids (Ahringer

et al., 1992) that exhibits no significant simiiarity to any other known protein.

The tra-I Gene is the Terminal Regulator of Somatic Sex Determination

The tra-1 gene is the terminal reguiator of somatic sexual development because its

activity or lack of activity specines female or male somatic development irrespective of the

0 t h gens in the pathway. NuIl alleles of RU-l cause XX animals to develop into fertile

males (Hodgkin, 1 987a; Schedl et al, 1 989). Therefore the d e of wild-type ira4 activity

in the soma of XX animais is to promote female development. A number of gainsf-

h c t i o n deles of ka-] dorninantIy feminize both XX and XO animds (de Bono et d,

1995). Geneticaily, the gain-of fiuiction RU-l alleles behave as if they have escaped the

negative influence of the upstream regdators, the fem genes, allowing ha4 activity to be

constitutive.

The tra-l gene encodes two zinc finger proteins, TRA- 1 A and TRA-1 B, that are

translated from two aiternatively spliced transcripts (Zarkower and Hodgkin, 1992). TRA-

1A has 5 Zn fhgers, while TRA-1B is equivalent to the N-temiinal portion of TRA-1A and

contains ody the first 2 N-temillial Zn fingers. The Zn fïnger domain of TRA-IA is most

similar to those encoded by the vertebrate Gli genes, (KiBzter et al, 1988; Hui et al., 1994;

Vortkamp et al., 1993, the Drosophila segment polanty gene cubitus i n f e r r u - (cf) and

the pair d e gene odd-paired (opa) (ûrenic et al., 1990; Benedyk et d , 1994; Cimbora and

Sakonju, 1995)- TRA-IA protein binds to a specinc DNA sequence in vitru, but, the

shorter 2 Zn finger protein, TRA-IB, does not bind DNA in vitro (Zarkower and Hodgkin,

1993). The data available on TRA-1 suggest that TRA-IA acts as a transcription factor, to

activate femaie-specifk genes or to inactivate de-specific genes or both.

The expression of each na-l transcript is similar in both sexes, suggesting that the

regdation of ku-1 is post-transcriptionai (Zadcower and Hodgkin, 1992). de Bono et al.

(1 995) reported that several ira-1 gain-of-fiinction deles cany mutations that affect a shoa

sequence of amino acids (1 6 aa) close to the amino terminus in both TRA-1 A and TRA-

1B. This site has been named the GF region (for gain-of-hction), and it may define a site

of interaction for negative regdators of TRA-1, such as one or more of the FEM proteins.

Molecular Mode1 for Somatic Sex Determination

The molecular data, summarized in Table 1-3, combined with genetic mosaic

analyses of her-l and PU-1 strongly suggest that the C. e l egm somatic sex determination

pathway is a cell-ceii communication pathway (see reviews by Hodgkin, 1992; Kuwabara

and Kimble, 1992). In this model, a secreted masculiniPng signal, HER-1, acts at the cell

membrane to regulate a transcription factor in the nucleus, TRA-1A. Figure 1 6 illustrates

the model of somatic sex determination in C. elegm. According to this model, in XX

anirnals the high X/A ratio causes the sdc genes to repress transcription of her-1. In the

absence of HER-1, TRA-2A, a putative receptor protein, is active. TRA-2A exerts its

negative influence on the FEMs by binding FEM-3 (Mehra et al., in preparation.). The

binding of FEM-3 by TRA-2A is sufncient to inhibit the masculinizhg activity of the

FEMs. TRA-lA, a putative transcription factor, is fke h m the negative influence of the

FEMs and therefore can activate fernale-specinc or repress male-specific genes or both.

In XO anhnals the XIA ratio is iow, causing inactivation of the sdc genes, which

Ieads to the transcription of her-l. Kuwabara (1996) proposed that HER-1 is an inhibitory

ligand for TRA-2A. The binding of HER-1 has the eflect of inactivating TRA-2A, causing

the release of FEM-3. FEM-3 dong with FEM-1 and FEM-2 then act upon TRA-1 A.

How the FEM proteins inactivate TRA-1 is unknown. However, direct protein-protein

interactions with TRA-1 and one or more of the FEMs may be involved (de Bono et al.,

1 995, David L u - personal comxnulzication).

Table 13: Molecular Properties of Global Sex-Determining Genes.

Gene her-1

tra-2

&a-3

fem-1

fem-2

fim-3

tra-1

Transcripts Gene Product Regulation and Function References. - Sex-specific: rare 0.8kb in HER-1 A: 175 a.a., secreted Transcnptionally regulated. (Trent et al., 199 1 ; X X , rare 1.2kb and abundant 0.8kb in XO, XX: 4.7kb and 1.8kb. The 1.8kb transcript is germline- specific. DRE in 3' UTR. XO: 15X lower expression than XX, also a male- specific 1.9kb tmnscript 2.2kb expressed at equal levels in XX and XO.

2.4kb expressed at equal levels in XX and XO during al1 stages in development. 1.8kb expressed at equal levels in XX and XO.

1.7kb and 1 S5kb expressed at qua1 levels in XX and XO. Gain -of-fiuiction site in 3' UTR. 5.0 kb expressed at equal levels in XX and XO. 1.5 kb peaks in L2 but found throughout developrnent.

HER- 1 B: C-teminal(64 a.a.) of HER- 1 A TM-2A: 1475 a.a., Putative receptor, TRA-2B: Predicted intracellular domain of TRA- 2A

TRA-3 : 648 a.a. Calpain-like protease.

FEM-1: 656 a.a., Six copies of the ANK repeat.

FEM-2: 449 a.a., Protein serinelthreonine Phosphatase Type 2C. FEMJ: 388 a.a., Novel

TRA-IA: 11 10 a.a., 5 zinc finger protein, related to Gli family of transcription factors. TRA-2B: N-terrnnal288 a.a., 2 zinc finger protein.

HER- 1 : putative ~ k a n d for TRA-2A receptor. Post-transcri ptional and translational reguiation. TRA-2A: Putative Receptor for HER-1 ligand. Intracellular region needed for down regulation of FEMs. TRA-2B: Not detemined Positive regulator of tra-2 activity. Negative regulator of lufil.

Post-transcriptional regulation. Signal transducer; negative regulator of tra-1.

Post-transcriptional regulation. Signal transducer; physically interacts with FEMJ; negative regulator of rra-1. Post-transcriptional regulation. Signal transducer; physical interaction with TRA-ZAY and FEM-2; negative regulator of tra-1. TRA- 1 A: Binds DNA in vitro. May act as a transcription factor. TRA-1B: Does not bind DNA in vitro. Includes the GF site,

Perry et al., 1993)

(Okkema and Kimble, 199 1 ; Kuwabara et a/. , 1992; Goodwin et al., 1997)

(Barnes and Hodgkin, 1996; Godwin et al., 1997) (Spence et al., t 990; Gaudet et al., 1996)

(Pilgrim et al, 1995; Chin-Sang and Spence, 1996)

(Mehra et al. in prep. Rosenquist and Kimble, 1988; Ahringer et al., t 992)

(2arkower and Hodgkin, 1992; Zarkower and Hodgkin, 1993)

Figure 1-6: Molecular Mode1 of Somatic Sex Determination in C elegam.

Molecular model for somatic developmnt, XX female (lefi), XO male (right). On top of

each mode1 is the genetic pathway indicating the predicted activity (high or low) of each of

the seven genes. (Left) A high X/A ratio prevents HER-I synthesis, aiiowing TRA-2A to

inhibit FEM-3 (Mehra et al., in prep.), and TRA-IA, a sequence-specific DNA-binding

protein, directs female somatic development by activating fede-specific genes andor

inadvathg male-specific genes. (Right) A low XIA ratio results in the synthesis of HER-

1, a srnall secreted protein that inactivaies the membrane protein TRA-ZA, thereby

releasing FEM-3 fiom negative regulation. The three FEM proteins then inhibit the

activky of TRA-1A by an unknown mechanism to cause male development There is no

evidence for the FEM proteins forming a complex as depicted, but d three are required for

male development. TRA-3 does not appear to play a direct role in the signal transduction

pathway and is left out of the model. Diagram generously provided by Jeb Gaudet.

NOTE TO USERS

Page(s) not included in the original manuscript are unavailable from the author or university. The manuscript

was microfilmed as received.

UMI

oogenesis and blocks spermatogenesis, as in the case of adult XX animals (Figure 1-7c).

This idea is supported by the properties of gain-f-fimction deles of tra-1. fra-l(gB alleles

behave as ifthey have escaped regulation by the- genes and cause both XX and XO

animals to produce only oocytes in their germlines.

In addition to the seven genes involveci both in somatic and gemiline sex

determination, at least ten genes (fog-2, mogl - 6, fog-1. fog-3 and gld-1) specifically affect

germiine sex determination. The fog (Ceminkition of the germline) genes are required for

spermatogenesis (Schedl and Kimble, 1988; Barton and Kimble, 1990; Ellis and Kimble,

1999, and the mog (mascuiinization of the g e h e ) genes (Graham and KLnble, 1993;

Graham et al., 1 993) and gld-2 (germline defective) (Francis et al., 1995a), me required for

oogenesis (Figure 1-7).

The hermaphrodite poses a very interesting problem - it has to switch its sex in the germline during development. Thus the hermaphrodite requires additional levels of

regdation that are not necessary in males. Two genes that play major roles in this germiine

switch are tro-2 and fem-3. Dominant gain-of-hction alleles of both genes cause sexual

transformation of the germline. (Doniach, 1 98 6; Barton et al., 1 987).

During the L3 to L4 stage of hermaphrodite development, ka-2 activity is repressed

transientiy to allow spermatogenesis (Doniach, 1986). A key player in hermaphrodite

spermatogenesis is thefog-2 gene. Null mutations infog-2 have no affect on males but

aboli& spermatogenesis in the hermaphrodite. Genetic epistasis experiments place fog-2

upstream of ha-2 and suggest that fog-2 is either an activator of the fems or a negative

reguiator of ha-2 (Schedl and Kimble, 1988) (Figure 1-7b,c). Once tra-2 activity is

repressed, thefem genes together withfig-l andfog-3 (Barton and Kimble, 1990; Ellis and

Kimble, 1995) can activate spermatogenesis.

Gain-of-fùnction aiieles offent3 completely masculini7e the XX germhe (the

Mog phenotype) suggestuig that the switch to oogenesis is controlled by down-regulating

fim-3 activity. AUfem-3(g;B alleles have mutations affecting a 5bp region in the 3'UTR of

the fem-3 gene temed the gain-of-fhction region. Ahringer and Kirnble (1 991) showed

that the level offem-3 mRNA is not incfeased infem-3(gB mutants. Mead the extent of

polyadenylation of fem-3 mRNA increases. The authors suggested that this increase in

polyadenylation leads to increased translation offem.3 mRNA in the germline. The

sequence of these aiieles and the resulting phenotype suggest that the gfregion in the

3'UTR offem3 serves a s a target for negative regulation. This mode of regulation may be

specific to the germline, or-3 activity may be sllnilarly regulated in the soma of XX

animals. However, regulation by fia-2 is suficient to prevent somatic masculini7sition by

gfmutations affecthg the fem-3 3'UTR. Putative negative regulators of thefem-3 3'UTR

are the products of the rnog genes (Graham and Kimble, 1993; Graham et al., 1993). Loss

of hc t ion mutations in any one of the six mog genes cause a Mog phenotype similar to the

fem-3(gB phenotype. Epistasis aualysis places the mog genes at the same Ievel in the

genetic hierarchy as ira-2 (Figure 1-7). AU six mog genes exhibit materna1 effects on

embryogenesis as well as their zygotic eEécts on the sperm-oocyte switch, suggesting that

these gens regulate other processes.

Nuil mutations in the terminal regdators of gemiline sex determination result in

oogenesis: Therefore oogenesis appears to be the defauit pathway. One known gene, gld-

I, is essential for oocyte development. A gld-I null mutation abolishes hermaphrodite

oogenesis and confers a tumorous gemiüne phenotype in which presumptive female gemn

c e k exit the meiotic pathway and r e m to the mitotic ceii cycle. The product of gld-I

gene, GLD- 1, is a putative RNA binding protein (Jones and Schedl, 1995) and acts

dowmûeam of sexual fate specification to regulate oocyte Merentiation (Francis et al.,

199%; Francis et al., 1995b; Jones et al., 1996) (Figure 1-7c).

Figure 1-7: Genetic Control of Germline Ses Determination.

The activities of the genes involved in germline sex determination are dependent on the

XIA signal and stage of development. Bold type indicates the genes are active, smaller

type indicates the genes are inactive. h o w s indicate positive interactions, barred lines

represent negative interactions (A) In XO animais spemuitogenesis commences at L3 and

is constitutive throughout the rest of development The terminai regdators @ml-3, fogl,

fog3 and to some extent ira-1) promote abundant spermetogenesis and block oogenesis.

(B) XX animals during L31L4 produces spem tmnsiently. Thefog-2 gene plays a role in

down-regdation of ha-2 or activating thefem, fog-land fog-3 directly to promote

spennatogenesis. (C) In the addt XX animal oogenesis is constitutive. This is achieved

by repressing fem-1-3, fog-l andj5g-3. Whenfem activity is absent tra-l promotes

abundant oogenesis. The gld-1 gene is essential for oogenesis and acts downstream of

sexual fate determination (Jones et al., 1996). Figure adapted h m C h e and Meyer

(1 996).

A &a-1 Spematogenesis 6

WA - ,y+ her-14 *a-2 f e m - 1 3 ratio trtr-3 -1 f e m - l ' y ûogenesis

m 0 ~ 4 - 6 fem-3 gld4 3

WA ratio

XX Larva (lm

Spennatogenesis

C fog-2 1 \ A spennatoge~1e~is

fm-l fl

A - a . 1 -,th€-2 ratio tra-3 /d k oogenesis fem-3 mog-1-6 gld-1 8

Other Genes Involved in Sex Determination

Over the past 20 years a number of screens for mutations that cause sexual

transformation have been carried out. It is iikely that al1 the genes involved exclusively in

sex detemination have been isolated. However, there are presumably more genes that are

involved in sex determination as weli as other processes. Many previous genetic screens

for sex-àeterrnination mutations were designed to detect sexual transformation in

homozygotes. Genes that are redundant or have essential roles in addition to sex

determination would have been missed in these screens. One way to isolate these genes is

to carry out genetic modifier screens. In certain genetic backgrounds where the activity of

one gene is reduced to a threshold level, the function of other components of the pathway

become dosage dependent. Dominant enhancers or mppressoa of phenotypes resulting

from threshold Ievels of sexdetermining genes can idente components in the sex

determination pathway. Biochemicd approaches, such as coimmunoprecipitations and

affinity chrornatography (Phizicky and Fields, 1995) may i d e n e proteins which

physically interact with sexdetemllning proteins or approaches such as the yeast two-

hybrid system (Fields and Song, 1989) may identify genes whose products directly interact

with sexdetermining prote&.

The primary focus of this thesis is on how the three fem genes and their products act

to bring about male development in C. elegans. In Chapter two of this thesis 1 descnbe the

characterization of 7 Loss-of-function mutations and two putative nuil mutations of the fem-

I gene. Four temperature-sensitive deles of fem-1 carry missense mutations within the

ANK repeats. 1 provide the fkst direct evidence that the ANK repeats are f h c t i o ~ y

required for the activity of FEM-1 in promoting male developmental fates. In Chapter

uiree 1 use molecular approaches to show that two of thefem gene products, FEM-3 and

FEM-2, can physically associate in vitru. 1 go on to characterize the FEM-2 protein,

demonstrating that it is a member of the Type 2C serine/threonine protein phosphatase

f d y and that its phosphatase activity is required in vnto for normal male development. In

Chapter four 1 descnbe the use of a genetic approach in an attempt to idente genes which

encode proteins that physicdy interact with FEM-1. I selected for suppressors of a

temperature-sensitive allele of fem-l that encodes a missense mutation in a highly

conserveci residue in the first Adcyrin repeat of FEM-1. Although 1 did not identiQ any

new genes that encode products that physicaliy interact with FEM-1, I did isolate a mique

class of-3 gain-of-fiuiction dele. Chapter five provides the reader with some

concluding remarks and focuses on fuhire projects.

CHAPTER 2: MUTATIONS AFFECTING THE ANKYRIN REPEATS OF FEM-1 IMPLICATE PROTEIN-PROTEIN INTERACTIONS IN THE C. elegans SEX DETERMINATION PA-AY

1 did al the experiments reported in this chapter with the following exceptions:

David LM identified the mutation in two aiieles,fem-l (el1 77) and fem-l (elW8).

ABSTRACT

The fem-1 gene of Caenorhabditis eelegons is one of thnx gens required for all

aspects of male sexual development. Thefem-1 gene encodes a protein with 6 copies of an

evolutionarily conserved sequence motifcalled the ANK repeat ANK repeats are

important mediators of specific protein-protein interactions in s e v d protein familes In

this chapter 1 report the sequence of 9 loss-of-fiuiction (fl mutations offem-1. Two

putative null alleles contain a nonsense mutation and a splice acceptor mutation

respectively. The nonsense mutation is predicted to tnuicate the FEM-1 protein to about

U3 of its length, while the splice acceptor mutation causes aberrant splicing offem-1

mRNA. Interestingly, four temperature-sensitive aiieles offem-l are missense mutations

that affect 3 of the 6 ANK repeats in FEM-1. These mutant deles establish the fùnctionai

importance of the ANK repeats in FEM-1. In view of the roles of ANK repeats in other

proteins, 1 suggest that FEM-1 engages in protein-protein interactions for its normal role in

development.

INTRODUCTION

Genetic and molecular data support a model for the elegans sex detennination

pathway as a ceil-celi communication pathway (see Figure la). The current model

portrays the FEM proteins as transducers of a mascuiinizing signal, HER-I , at the ceil

membrane to TRA-1 in the nucleus. The activity of the tra-l gene is necessary and

sufncient for fernale somatic development independent of the activity of any of the other

global sex-determining genes (Hodgkin, 1987a; Hunter and Wood, 1990; Zarkower and

Hodgkin, 1992). Therefore, tra-l acts as the terminal regulator of somatic sex

determination in the C. elegcns sex detennination pathway. Thefem genes encode the

most direct known regulators of #a4 activity in the soma. In XO animals the three fem

genes negatively regulate ha4 activity to d o w male somatic development to occur. How

the activities of the FEM proteins exert their negative influence on TRA-1 is not

understood.

Figure 2-1 provides a schemaîic diagram of the sexual transformation redting

fiom nuil mutations in fem-l. Putative nul1 mutations infem-l cause both XX and XO

animais to develop as fernales. The loss-of-fùnction phenotype suggests that the activity of

the fem-l gene is required in the hermaphrodite germiine for spemiatogenesis and in both

the soma and gemiline for male se& development.

The fem-l gene encodes a protein with 6 copies of a 33 amino acid motif caiied the

cdcl O/S WI6 motif or ANK repeat (Spence et al., 1990)(Figure 2-2). Breeden and

Nasmyth (1 987) fkst described the cdc 1 O/SWI6 motif in a yeast transcription factor and

since then the motifs have been found in a wide variety of proteins of diverse hction fiom

many different organisms. The human erythrocyte protein, has 24 copies of the

cdc 1 O/SWI6 motif and the motif was ~named the at&ah or ANK repeat (Lux et al.,

1990). For the remainder of this thesis 1 will refer to the cdc l O/SWI6 motifi of FEM- 1 as

ANK repeats.

The ANK repeat is usually found in four or more contiguous copies (Bo& 1993).

The ANK repeat has been show in some proteuis to mediate specific protein-protein

interactions. These proteins include the p 1 OSIp 100 precutsors of the p50Ip52 s u b ~ ~ & ~ of

NFKB (Hatada et al., 1 992; Baeuerle and Baltimore, 1 996) Id3 (Inoue et al., 1992; Beg

and Baldwin, 1993; Gilmore and Morin, 1993), cactus (Kidd, 1992) members of the Notch

family including lin-12 and glp-I fkom C. elegans (Wharton et al, 1985; Austin and

Kimble, 1 989; Yochem and GreenWald, 1989; Matsuno et al., 1999, and the structurai

proteins of the ankyrin family (Davis and Bennett, 1990; Lux et al., 1990; Bennett, 1992).

In view of the role of the ANK repeats in other proteins, it is quite probable that FEM-1

participates in protein-protein interactions through its ANK repeats.

In this chapter 1 report the sequence alterations in 9 loss-of-hction (ZJ alleles of

the fem-l gene. Of these 9 alieles, 7 are temperature-sensitive, and the other two are

putative n d alleles. hterestingly all the temperature-sensitive mutations are missense

mutations that affect, or map close to, the ANK repeats or a newiy identifieci kinesin light

chain-like motif of FEM-1. These ~ d t s provide the first direct evidence that the ANIS

repeats and the kinesin light chah motif in FEM-1 play a fiuictional role.

Figure 2-1: A Schematic Diagram Showing the Phenotype of fem-l N d Mutants.

(A) Wdd type XX anirnals develop as hermaphrodites. These animais have a female soma

with a niixed gemiiine producing both sperm and oocytes. (B) Wdd type XO anirnals

develop as males. (C) Both XX and XO animais that are homozygous for a ndi mutation

infem-2 develop as femdes. These are tnie fernales as they can mate with a male to

produce 50% XX hermaphrodite and XO male cross progeny. The female is identical to

the hermaphrodite with the exception that she does not produce sperm in the germline.

Fernales have a characteristic appearance resulting fiom the accumulation of stacked

oocytes. Drawings rnodified with permission fiom Hodgkin (1 988).

Hermaphrodite d i i gonad

Maie

Femaie

Figure 2-2: The fem-l Gene and its Product.

(A) Thefem-l gene is located on linkage group N (LGIV). A 5.5 kb Nsfi-Hpd genomic

fiagrnent 60m CB#1099 comtîtutes thefein-1 gene and is sufncient to rescue male

development in a fem-1 (mII) animal in the germline and soma (Spence et al-, 1990).

Exons are represented as boxes. Shaded boxes represent untranslated regions. (El) The

fem-l gene has 1 1 exons that are spliced to give a mRNA of about 2.4 kb, including the 5'

and 3' UTR (C) The product of the fem-1 gene is a 656 aa protein. Near its N-terminal

end are six copies of the ANK repeat (shaded). Encoded almost entirely by exon 9 is the

kinesin light chah-like (KLC) motif.

ANK motifs (1-6) KLCmotif FEM-1 656 aa

MATERIALS AND lMETHODS

Worm Strains and Culture Methods

C elegmrr var. Bristol strain N2 was the wild type progenitor of aU strains used in this

study (Brenner, 1974). Genetic nomenclature follows Horvitz et uL (1 979), with the

following additional conventions: gfrefers to gain-of-fûnction (Barton et al., 1 98î), ts

refers to temperature-sensitive and am refers to amber-suppressible. The mutant alleles

used in this study are as foilows:

LGIV: dm-1 j(e184) (Brenner, 1974); und(e53) (Brenner, 1974);@n~-Ific17, e 1918,

e196.5, e1969, e1988, e199lm e2003) (Doniach and Hodgkui, 1984); fem-l (e2177,

e2179) (Hodgkin ,unpub lished); mor-Z(e1125); mc-24(e138);fem-3(q20ts, q95 gfts)

(Barton et al., 1987); d?20(e1282).

Meles are described in Hodgkin et al. (1 988) and Hodgkin (1 997) unless otherwise noted.

Nematodes were maintained on NGM plates streaked with Escherichia coli strain

OP50 using standard techniques (Brenner, 1974). Homoygotes carrying temperature-

sensitive deles were maintained at 1 SOC. Strains that couid not be maintained at 1 SOC as

homozygotes were maintained as bdanced heteroygotes at 20°C. Homozygotes carrying

thefim-l(el918) ailele are self-fertile at 15OC ifthey dso carry afem-3(& mutation. 1

rnaintained e l 918 as a homozygous strain AS57: unc-S(e53) lem-1 (e 19 18) fem3(q20) dpy-

20(e1282) (Andrew M. Spence unpublished observations).

Nucleic Acid Isolation

Isolation of Total Genomie DNA

Nematode genornic DNA was isolated as descnbed by Suiston and Hodgkin

(1 988). For the seven temperature-sensitive alleles (hc17, e 1918, e 1969, el988 e2003,

e2177, and e2179), genornic DNA was isolated f?om homozygous stocks grown at 1 SOC.

For alleles that could not be maintained in homozygous stocks (el965 and e1991),

homozygous female animals were hand-picked, and total genomic DNA was prepared as

described below.

Isolcrtion of DNA from Single Worms

DNA fiom single worms was prepared using methods described by Barstead et ai.

(1991). Single animals were picked to 5 pl of wom lysis buffer containhg 60 p g l d

Proteinase K, 50 mM KCI, 10 mM Tris-HCI, pH 8.2,2.5 m M MgCl2,0.45% NP-40,0.45%

Tween 20,O. 1% gelatin, in a 0.5 ml PCR tube. A drop of minerai oil was added and the

tubes were put at -70°C for 10 min, then incubated at 60°C for 1 hour followed by heat

inactivation of the Proteinase K at 95OC for 15 min.

RNA Isolation Samples of 50 to 1,000 worins were rinsed in M9 buffer and pelleted at 5000 rpm

for 30 seconds in a microcentnfuge tube. The pellet was fiozen at -70°C. One volume of

acid-washed, baked glass beads and three volumes each of GITC solution (5 M guanidine

and T.E. saturated pheno1:chloroform (1 : 1) were added and vortexed immediately for

several minutes. Samples were spun in a microcentrifuge for 5 minutes to separate the

phases. Phenol:chloroform extraction of the aqueous phase was repeated twice and to the

fioal aqueous extract, two volumes of ethano1 were added to precipitate the RNA. The

RNA was pelleted by centrifbgation at 12K rpm in a microcentrifuge for 5 minutes, rinsed

with 70% ethanol, and resuspended in DEPC- treated water. An equal volume of 5 M LiCl

was added to the RNA solution, mLved and kept on ice for one hour. The RNA was

pelleted by centrifugation for 10 minutes at 4OC, rinsed with 2 M LiCl, 10 rnM EDTA, and

collected by centrifùgation as before. RNA was dissolved in DEPC-treated water and

precipitated with 0.5 M ammonium acetate and 2.5 volumes of EtOH. The RNA wsis

peileted by centrifbgation as before, and rinsed with 70% EtOH, dried and resuspended in

DEPC-treated water. The RNA was subjected to a DNase treatment [2 units RQ1 RNase-

Free DNase (Promega) in 50 pl containing 40 mM Tris-HCI (pH 7.9) 10 m M NaCl, 6 mM

MgCh and 10 mM CaClz] for 2 hours at 3TC, foilowed by a phenol:chlorofom extraction

and ethanol precipitation as described above.

Total RNA (up to 5 pg) was used as template for cDNA synthesis, using random

hexamer primem. Reactions were canied out in 50 pl volumes with the foilowing £inal

concentrations: 1 X RT bUner (50 mM Tris-HCI, pH=8.3,75 mM KCI, 3 mM MgCW, 10

mM DTT, 40 units of RNasin (Promega), 400 m M dNTPs, 3 pM random hexamer and 400

mi ts of Moloney Murine Leukemia Virus Reverse Transcriptase (Gibco BRL). Reactions

were incubated at 23OC for 10 min, foiIowed by 42OC for 1 hour. The cDNAs were

pheno1:chIoroform extracted foiîowed by EtOH precipitation and resuspeaded in T.E..

Cloning of fem-1 Mutant Genes

Five overlapping DNA fkgments containing nearly the entire fem-l genomic

region (with the exception of part of intron 8)' or three overlapping cDNA firagments

containing the entire coding region of fem- l (see Figure 2-3 A and B), were generated by

polyrnerase chab reaction (PCR) (Saiki et al., 1988) using Taq DNA polymerase

(Promega) and primes indicated in Figure 2-3.

Figure 2-3 illustrates the approximate locations of the fem-l oligonucleotide

prime= that 1 used. Their sequences follow:

(+) = sequence corresponds to the sense strand of the fem-l gene.

(-) = sequence corresponds to the anti-sense strand of the fem-lgene.

Numbers at the 3' end correspond to the position (or complementary position) of the 3'

nucleotide with respect to thefem-l sequence as numbered in Spence et al. (1 990)

[GenBank accession # JO3 1721.

oAS 1 O3 5'-TGG ATC ClT TTC TTC TCG AAT TTC TGC43g-3 '(+)

oAS 104 5'-TGG ATC CGT ACT TCT TGT G'TT AGT TGC lo3r3'(-)

oAS 1 05 5 '-TGG ATC CGC TGC TGG ACA CAT TG939-3 '(+)

oAS 1 O6 5'-TGG ATC CCA ACA GCT TCA ACG CAT C 153r3'(-)

oAS 1 1 1 5'-GGC CGC; ATC CAT TGA TGG GTG GAC C1471T3'(+)

oAS 1 12 5'-GGC CCT CGA GTA CAT TCA CAA GAT GC3786-3'(-)

0AS113 5'-GGT TGG AT C CAG GAT ACG GAA TGC TGT GG37243'(+)

oAS 114 S-CCG GCT CGA GCG ACG ATA ATG AAA GAA G-5~3'(-)

oAS I 15 5'-ATC GAC AAT GGA TAA TTG65f 3'(+)

oAS 1 16 5'-GAT GAT TGC ATC CTA CAGIP6-3'(+)

0AS118 5'-TGT ACG AAT ATC AAC GTG175r3'(+)

oAS 1 19 5'-TAC TAT GGT ACA ATA ACC3536-3'(+)

oAS 120 5'-GAC TI% CCA AAG CTG CAG398f3'(+)

oAS 1 2 1 5 '-T'TG ATG ATC TAC CAC TAG421r3 '(+)

0AS126 5'-GGC CGG TAC CTT CGC TAC ATC AAA GAT GGn71-3'(-)

0AS127 5'-AT?' CAT GTG AAC GGA ATT' GTC G2l89-3'(-)

0AS128 5'-GGT TGG ATC CAG CTA TTC AAG GAC ACT cTG3m3'(+)

0AS129 5'-GGC CGG AGC CI'G TGA GAA TAO GAT TAG AGC4n4-3'(+)

PCR products were cloned into Bluemipt vectors (Stratagene) using convenient

restriction enzyme sites at the 5' end of the primers (indicated in bold-type). Standard

molecular biology procedures for cloning were used as describeci by Sarnbrook et al.

(1 989). PCR products acquired fiom primers oAS 103 to oAS 1 O6 were cloned into

Bluescript (Stratagene) vectors using the "A/T" cloning approach descnbed by Marchuk et

al. (1991).

Figure 2-3: Approximate Locations of Primers Used in th% Stady.

(A) Schematic diagram of thefem-I locus and the approximate position of primers relative

to the gene. Boxed regions represent the cDNA with shaded boxes correspondhg to the 5'

and 3' untranslated regions; lines between the boxes represent introns. Pnmers are not

drawn to scde. (B) The five overlapping PCR fkgments amplined h m genomic DNA.

(C) The three overlapping firagments amplified h m cDNA. Numbers in parenthesis

indicate the size of the amplinedfern-l sequence.

Standard PCR Protoc01

The conditions used for amplification were as foilows: 0.1 4 . 5 pg of genomic

DNA, cDNA, or total genomic DNA exûacted h m one worm was amplilied for 25 cycles

of 95OC for 1 min, 60°C for 1 min, and 72OC for 1 min. Each reaction containexi 20 pmoles

of each of the appropriate primers, 1X Taq buffer [50 mM KCl, 10 rnM Tris-HC1, pH=8.8,

O. 1% Triton X-1001 (Promega), 0.25 rnM dNTPs, 2.5 mM MgCh, and 1.25-2.5 units of

Taq DNA polymerase (Promega). Reactions were carried out in 50-100 pl volumes.

Identification of fend(&) Mutations

PCR products fiom al1 deles except fem- l (e l96.S) and fem-1(19 18), were subject

to PCR-SSCP (Polymerase Chain Reaction-Single Strand Conformation Polymorphism)

anaiysis using the procedures described by Orita et aL(1989). The part offem-I encoding

the AM( repeats was amplified f?om genomic DNA using primers fiom the oAS 103 to

oAS 106 region (Figure 2-3) and the cDNA spanniag the remainder of the gene was

amplified using primer pairs oAS 1 1 l/l12 and oAS 1 l3/ll4 (Figure 2-3). About 0.1 pg of

worm DNA was used as template in a 10 pl PCR assay. Each 10 p1 reaction containeci 3

pmoles of each appropnate primer, 1 X Taq b a e r (1 0 mM Tris-HCl, pH=8.0,0.1% Triton

X- 1 O0 (Promega), 0.08 m M clNTPs, 1.5 mM MgCh, 1 pl of [ a - 3 2 ~ ] d ~ ~ ~ (3000 Cilmol,

10 mCVm1, Amersham) and 0.25 units of Taq DNA polymerase (Promega). Reaction

conditions were 30 cycles of 9S°C for 1 min, 57C for 1.5 min, and 72OC for 1.5 min. PCR

products were subjectea to restriction enzyme digestion (Ah1 in all cases except the

oAS 1 1 1/112 amplification product which was digested with RraI) in 25 pl. The reaction

was incubateci for 2 hours at 37"C, and then stopped bl adding 75 pi of 0.1% SDS, 10 mM

EDTA. Then 2 pl of this solution was mixed with 2 pl of loading bufTer (95% deionized

fornamide, 20 rnM EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol). Prior to

loading, the samples were heated to 80°C f