JOURNAL OF BACTERIOLOGY, Mar. 1970, p. 755-762 Vol. 101, No. 3Copyright a 1970 American Society for Microbiology Prinited ill U.S.A.

In Vitro Protein Synthesis and Aging inRhizoctonia solani

T. G. OBRIG AND DAVID GOTTLIEBDepartmentt of Plalit Pathology, Untiversity of Illinois, Urbana, Illintois 61801

Received for publication 25 November 1969

A study was made of the ability of cell-free protein synthesis systems from vegeta-tive cells of different age of the fungus Rhizoctonia solani to produce polyphenyl-alanine. Polyuridylic acid-directed phenylalanine incorporation into peptides de-creased linearly with cell age. The 105,000 x g supernatant fluid and ribosomalfractions were equally responsible for the total loss of synthetic activity of the oldercells. Initial rates of phenylalanyl-transfer ribonucleic acid (tRNA) synthetaseactivity decreased with increasing cell age, which accounted for the defect of thesupernatant fraction. An accelerated degradation of soluble phenylalanyl-RNA wasassociated with the ribosomes of the older cells. In vitro systems from cells ofdifferent age transferred phenylalanine from phenylalanyl-tRNA to polyphenyl-alanine at similar rates. Of the 15 specific aminoacyl-tRNA synthetases assayed, 5 in-creased and 5 decreased in specific activity with increased age; 3 others did notchange during aging and 2 were below acceptable detectable levels.

Previous studies have delineated some of thebiochemical changes that occurred in Rhizoctoniasolani and Sclerotium baaticola with increasingage of mycelial cells (8, 10, 20, 28). In bothspecies, a general decline in cellular metabolismaccompanied the aging process. On a dry-weightbasis, deoxyribonucleic acid (DNA), ribonucleicacid (RNA), soluble amino nitrogen, and proteindecreased gradually with age in R. solani. De-creases with age in the content of soluble aminonitrogen and protein per unit (dry weight) arequite common in fungi (2, 5, 9, 16, 26). More-over, the rate of incorporation of '4C-phenylala-nine and '4C-leucine into the R. solani cellularprotein decreased gradually with increasing age(8). This apparent decrease of synthetic activitywas due neither to an amino acid impermeabilityfactor in old cells nor to an isotope dilution froman increase in the amino acid pool size. Reductionin specific activity of protein fractions with agesuggests that the decrease in protein synthesiswas due to the protein synthesizing mechanismitself. Decreases in the protein synthetic capacitywith increasing age has been noted in cells ofhigher plants (17) and in cell-free preparations ofmuscle (3), liver (4), and reticulocytes (23). Anin vitro system was prepared from the myceliumof the fungus R. solani, with all parameters ad-justed for optimal protein synthesis (22). Thepresent investigation was made to determine thelocation of the defect in protein synthesis as cellsof R. solani age.

MATERIALS AND METHODS

Six-day-old surface cultures of R. solani Kuhn weregrown at 26 C in 30-cm (diameter) culture vesselswhich contained 4 liters of liquid media. Compositionof the chemically defined media is described elsewhere(22). The mycelial pad was divided by two concentriccuts into round segments of cells 0 to 2 (young),3 to 4 (mature), and 5 to 6 (old) days old with radialwidths of 4.3, 3.9, and 1.5 cm, respectively.

Preparation of the 105,000 X g supernatant fluid(S-105) and ribosomal pellet (P-105) has been de-scribed (22). Protein content of the S-105 fractionwas estimated by the Biuret (12) or Folin phenolmethod (15) by using bovine serum albumin as astandard. RNA content of the P-105 fraction wasdetermined spectrophotometrically by assuming thatI mg of ribosomal RNA in 1 ml of water equalled25 optical density units at 260 nm in a I-cm lightpath.

Incubation mixtures for the R. solanii peptide-synthesizing system contained, in a final volume of0.5 ml, tris(hydroxymethyl)aminomethane(Tris)-chlo-ride buffer, 50,moles (pH 7.8); magnesium acetate,10 ,umoles; ammonium chloride, 25 ,umoles; 2-mer-captoethanol, 7.5 sAmoles; reduced glutathione, 0.5Mmole; adenosine 5'-triphosphate (ATP), 1.5 /Amoles;guanosine 5'-triphosphate (GTP), 0.1 jAmole; phos-pho(enol) pyruvate (PEP), 2.5 ,moles; pyruvatekinase, 10 ug; yeast-soluble RNA (sRNA), 200,g;polyuridylic acid (poly U), 60 Mg; L-phenylalanine-U-14C, 0.3 Muc, 800 pmoles; 0.005 ,umoles of each of theother 10 unlabeled amino acids; P-105, 300 Ag ofRNA; and S-105, 300 .Ag of protein. When synthesesby different age systems are compared, all mixturescontained the same amount of S-105 protein and

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OBRIG AND GOTTLIEB

P-105 RNA. The mixtures were incubated for 2.5 to45 min at 28 C.

For measurement of the incorporation of "4C-aminoacids into "C-peptides, the reaction was stopped bythe addition of 20,moles of unlabeled amino acidin cold (0 to 4 C) trichloroacetic acid so that the finaltrichloroacetic acid concentration was 8 to 10%.After standing for I hr at 0 to 4 C, the mixtureswere heated for 10 min at 90 C and cooled to 21 C.The mixture was filtered through a type HA Milli-pore filter (0.45 ,um pore diameter), the precipitatewas washed with three 5-mi portions of 5% trichloro-acetic acid, and the filters were dried in scintillationvials at room temperature for 2 hr and at 80 C for 10min. The cold trichloroacetic acid assay differed fromthe hot trichloroacetic acid assay in that all proceduresprior to the filter drying were carried out at 0 to 4 C.A counting efficiency of 70% was attained by usingliquid scintillation counting conditions previouslydescribed (22).

Incorporation of "C-amino acids into 14C-amino-acyl-tRNA ("charging reaction") was followedeither (i) directly with a cold trichloroacetic acidassay in the absence of peptide synthesis, or (ii)indirectly by subtracting the hot trichloroaceticacid-precipitable radioactivity ("4C-peptide) from thecold trichloroacetic acid-precipitable radioactivity(14C-peptide plus "4C-aminoacyl-tRNA). In the directmethod, poly U, GTP, and ribosomes were omittedfrom the reaction mixture. Where incubations of2 to 3 min are reported, the reaction was stopped bysimultaneously placing all reaction tubes into anacetone-dry ice mixture for 5 to 10 sec and then add-ing cold trichloroacetic acid as described above. Ahot and cold trichloroacetic acid assay was also com-bined for measuring the incorporation of "4C-phenyl-alanine from "4C-phenylalanine-tRNA into 14C_polyphenylalanine. During this "transfer" reaction,the reaction mixture was complete except that la-beled and unlabeled free amino acids were omitted.The assay of aminoacyl-tRNA synthetases with

hydroxylamine was carried out generally as de-scribed by Clark (6), with modifications. The 2.0-mlassay mixture contained: ATP, 20 ,umoles; Tris-chloride buffer, 50 J,moles (pH 7.2); hydroxylamine-hydrochloride, 2 mmoles; magnesium acetate, 20jAmoles; potassium acetate, 133 jAmoles; L-aminoacid, 40 ,umoles; and S-105, 3 to 5 mg of protein.Incubation was at 30 C for 30 min. L-Tyrosine hy-droxamate was used as a standard.

For the preparation of "4C-phenylalanine-tRNA,amino acid-activating enzymes from Saccharomycesfragilis (ATCC 10022, NRRL-Y665) were used tocharge stripped yeast sRNA with "4C-phenylalanineby the methods of So and Davie (25) and Downeyet al. (7).

Reagent grade chemicals and sources were: Tris,2-mercaptoethanol, reduced glutathione, pyruvatekinase, ATP, GTP, PEP, sRNA (unstripped), ribo-nuclease, L-tyrosine hydroxamate from Sigma Chem-ical Co., St. Louis, Mo.; poly U from Miles Labora-tories, Inc., Elkhart, Ind., yeast sRNA (stripped) fromGeneral Biochemicals Corp., Chagrin Falls, Ohio;tetracycline from Charles Pfizer & Co., New York,

N.Y.; L-phenylalanine-U-'4C (368 /Ac/jumole), L-"C-phenylalanine-tRNA from E. coli (UL, 0.19 ,uc/mg),and 15 individual L-14C-amino acids (10 mc/mmoleeach) from New England Nuclear Corp., Boston,Mass.; and liquid scintillation reagents from PackardInstrument Co., Downers Grove, Ill.

RESULTSR. solani did not produce spores under the

described culture conditions. Surface culturesgrew in a typical sigmoidal-growth-curve fashionwhen measured by diameter of the fungal pad.The growth rates (Fig. 1) increased until the 5thday and then decreased sharply. This inherentgrowth characteristic was not due to the depletionof nutrients in the medium or to the synthesis ofan exoenzyme (unpublished data). Viability ofindividual cells was previously shown to be almostidentical for the three different age groups ofR. solani.There was decreased peptide synthesis in cell-

free systems with increasing age (Fig. 2). After 30min, the young system had incorporated twice asmuch "4C-phenylalanine into peptides than didthe old system. An attempt was made to locatethe rate-limiting step of polyphenylalanine syn-thesis in the older cells. The ribosomal (P-105)and supernatant (S-105) fractions from young,mature, and old cells were used in all possiblecombinations, and the relative ability of suchhomologous and heterologous systems to in-corporate phenylalanine into peptides was meas-ured. The results indicate that the activities ofboth the P-105 and S-105 fractions decreased withincreasing age of the cells (Table 1). In addition,50% of the total loss of activity in the homologousmature or old cell systems was due to the P-105fraction; the other half was due to the old S-105fraction.A comparison of the different age preparations

was then made by investigating the individual

EE

a-C

0

0..

a°

32-

28-24-20 -

16

"Ic1 2 3 4 5Incubation (days)

FIG. 1. Growth rate of R. solanti surface culture.

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PROTEIN SYNTHESIS IN R. SOLANI

nr I.I0 5 15 30 45

Incubation (minutes)FiG. 2. Effect of cell age on in vitro polyphenyl-

alanine synthesis by R. solani. The assay was carriedout as described in Materials and Methods. Each assaycontained 360,g ofS-105 protein and 290.ug of P-105RNA; 570 counts/min per pmole of phenylalanine.

TABLE 1. Incorporation ofphenylalanine into poly-phenylalanine by homologous- and heterologous-

age cell-free systems from R. solania

RiobosomesgSupernatant fraction

Young Mature Old

Young.......... 23,600 21,400 18,100Mature ......... 19,800 17,600 15,600Old ........... 17,600 13,500 12,000

a Values represent counts/min per milligram ofribosomal RNA. Each figure is the average of threeseparate experiments (30-min incubation). Con-tents of the reaction mixtures were as stated inMaterials and Methods, with S-105 protein andP-105 RNA ranging from 0.400 to 0.420 mg and310 to 335 ug per assay, respectively; 570 counts/min equals 1 pmole of phenylalanine.

steps involved in the synthesis of polyphenylala-nine. The relative ability of the three homologous-age S-105 and P-105 systems to incorporate14C-phenylalanine into "4C-phenylalanyl-tRNAand "4C-polyphenylalanine was followed over a30-min period. Rates of peptide synthesis andnet phe-tRNA formation decreased with in-creasing age of the preparations (Fig. 3). Between5 and 15 min, phenylalanyl-tRNA formationin the young system was still increasing, but it wasdecreasing in the mature and in the old systems.The data suggest that the ability to carry out anet charging of tRNA might be limiting peptidesynthesis. The data suggest that with increasingage, there is either a decrease in the rate of phenyl-

alanyl-tRNA formation or an increased rate ofdegradation of the phenylalanyl-tRNA, or both.These experiments were conducted in the presenceof saturating quantities of tRNA and '4C-phenyl-alanine.

If the amount of phenylalanyl-tRNA formeddetermined the rate of peptide synthesis, thenyoung, mature, or old systems, when suppliedwith "4C-phenylalanyl-tRNA, should transferthe amino acid into peptides at equal rates in the"transfer" reaction. When this reaction wascompared for the three systems with a limitingamount of phenylalanyl-tRNA, after 30 min ofincubation there was less than a 15% differenceamong any of the systems, either in productionof "4C-polyphenylalanine or in decrease of the'4C-phenylalanyl-tRNA substrate (Fig. 4). Inall cases, between 60 and 66% of the radioactivitywhich disappeared as substrate-"IC was recoveredas peptide-14C. Similar results were obtained withcharged tRNA from yeast or bacteria.Amino acid activation with ATP and tRNA

charging by phenylalanyl-tRNA synthetases inthe absence of peptide synthesis was studied inyoung, mature, and old systems. First, whenpeptide synthesis was inhibited 50 to 60% with ahigh concentration of tetracycline by suppressingbinding of phenylalanyl-tRNA to polyribo-somes, "4C-phenylalanine was incorporated into

10 15 20Incubation (minutes)

FiG. 3. Effect of cell age on in vitro incorporation ofphenylalanine into phenylalanyl-tRNA and poly-phenylalanine by R. solani. Assay conditions were asdescribed in Materials and Methods. Each assay con-tained 400 ,g of S-105 protein and 300,g of P-105RNA; 570 countslmin per pmole ofphenylalanine.

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zcr- 25

E0In

° 20.0

E 15

I0- 10x

E

u

0W0 5 10 15 20 25 30

Incubation (minutes)FIG. 4. Effect of'cell age Oli ill vitro inicorpor-ation

ofphentylalaniine Jrom phenylalanyl-tRNA (Escherichiacoli) iitto polyphenylalaitinie by R. solaini. Assay contdi-tionis were as described inl Materials anid Methods exceptthat '4C-pheniylalaninle was omitted aind tRNA was re-

placed with 0.15 mg (6 nimoles) of 14C-phentylalaniniie-tRNA from Escherichia coli; 7,300 coaints/mini per

lmole o/'phenylalanine-tRNA.

'4C-phenylalanyl-tRNA differently by the threedifferent-age systems; the phenylalanyl-tRNAsynthetase activity usually decreased with in-creasing age of the cells (Table 3). After 17 min,the mature and old systems incorporated only 82and 56c/;, respectively, as much amino acid intoaminoacyl-tRNA as did the young system.Second, when peptide synthesis was eliminated byomitting P-105 and poly U from an otherwisecomplete reaction mixture (more than 95r ofsynthetase activity was located in the S-105 frac-tion), '4C-phenylalanyl-tRNA formation bythe young, mature, and old systems was 100, 110,and 74%7,, respectively, after 12 min of incubation(Table 3). Third, an aminoacyl-tRNA synthetaseassay based on the reaction of hydroxylaminewith aminoacyl adenylates indicated that thephenylalanyl-tRNA synthetase activity de-creased in the mature (48%/6) and old (67 %)systems when compared to activity of the youngsystem (Table 4).

Further evidence that phenylalanyl-tRNAsynthetase activity decreases with increasing ageis presented in Table 2 and Fig. 5. Charging oftRNA by a young S-105 enzyme preparationwas from 18 to 22% greater than by an old S-105preparation during the first 10 min of incubation(Table 2). It should be noted that these reactions

TABLE 2. lIicorporacili ot0/ pheli/ / lly/ l(ciiiic, ilitophenylalanyl-tRNA: eJfct of S-105 age oli /Ic

chairgilig reactioir,

Incorporation at minutes of incubationAge -

2.5 i.0 1(.( 15.

Young. 11,500 14,900 15,'950 13,350Old .. 9,450 11,500 13,100 12,400

Values represenlt counts/min per milligram tofS-105 protein. The younlg and old systemiis con-tained 306 and 320 ,g of protein per assay, re-spectively. The incubation mixture was completeas listed in Materials and Methods, except P-1)5and poly U were omitted; 570 counlts/iimm perpmole of phenylalanine.

TABLE 3. Ilicorporatioii of plheny/alanine iltoaminloacyl-iRNA in/ the presence or in tile abscnce

of tetracycline"'

tiounts/min le a- aIncubation Tetracycline n per assy

(min) (400 A.g/ml)Young Mature Old

7 Present', 3,826 4,427 2,61812 Present' 4,483 3,638 2,42917 Presen1t' 3,724 3 053 2,08712 Absenrv 4,325 4,825 3,208

Cold trichloroacetic acid-precipitable radio-activity; 570 counts/min per pmole of' phenylal-t-nine.

b Assay mixtures were complete as listed in Ma-terials and Methods, with 415 jAg of S-l05 proteinand 310,ug of P-105 RNA per assay.

c Assay mixtures were complete exceplt thatribosomes and poly U were omitted.

TABLE 4. Specific activ,it)y o p,e,nylalanvl1-i RNA,syiltletalses from cdiflierent-age cells of R. soloiti:

hydroxylanmine aissay,

Assay" ~~~Amt of productAssay" forme(l'

Complete, young S-105. 0.86Complete, mature S-105 0. 45Complete, old S-105 0.28Complete, boiled young S-105 <0.05Minus amino acids <0.t)5Minus ATP... <(0.(05Complete, young P-105l <(. 10

1t See Materials and Methods for assay mixturecontents.

h Values are expressed as nanomoles of productformed per 30 min per milligram of proteill.

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Incubation (minutes)

FIG. 5. Effect oJ cell age ont the in7 vitro incorpora-tion1 oj pheniylalaninie inlto phenylalanyl-tRNA and o01the stability ofphenylalanyl-tRNA by R. solani. Assaycontditionis were as described in Materials and Methods,with 310 ug of S-105 proteini antd 300 Ag of P-105 per

assay. Poly U and GTP were omitted; 570 counits/mi,?per pmole oJfphenylalaninie.

were performed in the absence of the P-105fractions. In the same experiment, the time courseof phenylalanyl-tRNA formation was followedby using homologous and heterologous age S-105and P-105 systems. The results (Fig. 5) demon-strate that during the first 2.5 min, phenylalanyl-tRNA synthesis was related to the age of theS-105 fraction rather than to that of the P-105fraction. The data in Fig. 5 and Table 2 wererepeated in other trials in which the age de-pendency of the S-105 fractions in this phenome-non was shown to be consistently reproducible.

In addition, the net synthesis of phe-tRNA wasdependent on the P-105 age (Fig. 5). This wasespecially apparent after the first 5 min of incuba-tion. Whereas charging with a young S-105preparation alone (Table 2) or with homologousyoung S-105 and P-105 (Fig. 5) was almostidentical, a heterologous system of young S-105and old P-105 (Fig. 5) was unable to sustain anet synthesis of product throughout the first 10min. A similar but less pronounced situationwas observed in charging by the three systemsconsisting of old S-105 alone, old S-105 plusyoung P-105, and old S-105 plus old P-105 (Table2, Fig. 5).

Further attempts to compare the relativeability of the different-age systems to produce a

more complete protein were precluded by theinability of R. solani systems to translate TurnipYellow Mosaic viral RNA. The initial chargingof tRNA with 15 different amino acids by youngand old S-105 enzymes was then investigated(Table 5). Young synthetase enzymes were moreactive than were the enzymes from the old S-105preparations for five amino acids (arginine,leucine, lysine, phenylalanine, threonine) and lessactive for five other amino acids (glycine,isoleucine, proline, tyrosine, valine). Of theremaining five amino acids, three (alanine, his-tidine, serine) were transferred almost equallyby the young and old systems, and two (aspartate,glutamate) were not sufficiently incorporatedinto aminoacyl-tRNA for accurate measurements.Optimal requirements for the initial rates of

phenylalanyl-tRNA synthetase activity in youngand old S-105 fractions differed only slightly withregard to ATP (Fig. 6) or Mg++ (Fig. 7) con-centration and to pH (Fig. 8).

DISCUSSIONThe data indicate that polyphenylalanine

synthesis was limited in preparations from old

TABLE 5. Ilncorporationi oJ 15 differelnt aminio acidsin7to aminoacyl-tRNA by aminioacyl-

tRNA sylnthetasesa

Amt of

Amino acid incorporationb Per centdifferencecYoung Old

L-Alanine............. ,360 1,390 +2L-Arginine 7,270 6,220 -15L-Aspartate ........... 23 3L-Glutamate0...........OL-Glycine ..... 200 280 +40L-Histidine 454 482 +6L-Isoleucine 2,410 4,970 +106L-Leucine ...... ..... 5,170 3,370 -35L-Lysine 4,930 3,970 -19L-Phenylalanine 4,590 3,337 -26L-Proline 206 810 +290L-Serine........ 554 520 -6L-Threonine .......... 47 13 -72L-Tyrosine .. ......... 116 136 +17L-Valine.............. 300 2,500 +735

a Each value is the average of two replicates.GTP, P-105, and poly U were omitted from theotherwise complete incubation mixtures; 154counts/min per 10 pmoles of amino acid, 5 nmolesof amino acid, and 300,ug of stripped yeast tRNAwere added per assay.

bValues are expressed as counts/minute permilligram of S-105 protein.

, Incorporation by old system relative to in-orporation by the young system.

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C

E2150.CL

E 12

0.4

E s o Young

(0

0 Old0

0 2 4 6 8 10 12[ATP] (mM)

FIG. 6. Effect of cell age on in vitro phenylalanyl-tRNA synthetase activity by R. solani; optimal adeno-sine triphosphate concentration. Reaction time was 2.5min. Assay conditions were as described in Materialsand Methods, with 300 lig of S-105 protein. Poly U,GTP, and P-105 were omitted from the reaction mix-ture; 570 counts/min per pmole ofpheniylalanine.

14f

.C o-o YoungOld

£%-12-O \

10

0.~~~~~~~~

Es 0

88 0x

EC-)

FIG. 7. Effect of cell age on in vitro phenylalanyl-tRNA synthetase activity by R. solani; optimal mag-nesium concentration. Reaction time was 2.5 min. Assayconditions were as described in Fig. 6.

pHFIG. 8. Effect oJ cell age on in vitro plheiylalanyl-

tRNA synthetase activity by R. solaiii; optimal pH.Reaction time was 2.5 mini. Assay coiiditions were (isdescribed in Fig. 6.

vegetative R. solani cells both by the initial ratesof phenylalanyl-tRNA synthetase activity and byribosome-associated degradation of phenylala-nyl-tRNA. Whether these limitations are actuallyinvolved in the age-dependent decrease in proteinsynthesis of the intact cell remains to be es-tablished. Moreover, the present investigationdeals with the synthesis of a peptide containingonly phenylalanine as directed by a poly U mes-sage and does not reveal the activity of young andold cells in the act of producing a protein com-prised of many different amino acids. However,the results obtained for phenylalanine with wholeand cell-free systems may also apply to some ofthe other amino acids in the cells. The observedreduction in phenylalanyl-tRNA synthetaseactivity with age suggests an alteration in enzymicactivity rather than a change in the tRNA species,because charging was performed with the sametRNA preparation. Transfer RNA has, however,been implicated in the regulation of protein syn-thesis during differentiation in higher plants (29),animals (13, 30, 32), and bacteria (33). In con-trast, Wong et al. (31) reported that if a leucineor tryptophan auxotroph of E. coli was limited bythe supply of these amino acids, there was littlespecific repression or derepression of the tRNAfor the limiting amino acid. Furthermore, thelevel of charged leucyl- or tryptophanyl-tRNAwas found to increase or decrease with the growthrate when amino acids were supplied to the grow-ing cells. This suggested that the specific amino-

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PROTEIN SYNTHESIS IN R. SOLANI

acyl-tRNA synthetase enzymes were controllingthe amount of charged tRNA.Aminoacyl-tRNA synthetase activity has been

shown to change with cell development. VanEtten and Brambl (27) noted that the activity ofsynthetases for 12 different amino acids increasedrapidly as spores of Botryodiplodia theobromaewere germinating. Hartwell and McLaughlin (11)concluded that a temperature-sensitive mutanthaploid yeast would not grow at high tempera-tures because the only isoleucyl-tRNA synthetasewas thermolabile. Evidence is also accumulatingthat dissimilar synthetase enzymes differ in theirrequirements for maximal activity (21, 24, 27).Shearn and Horowitz (24) measured the specificactivity of synthetases for all 20 amino acids inNeurospora crassa mycelium and found thatATP, tRNA, Mg++, pH, reducing agents, and an-ions influenced such activities. In addition, com-petitive inhibition of substrate has recently beenreported for arginyl-tRNA synthetase of N.crassa (19).

In R. solani, the change with age in the specificactivities of 10 of the 15 different aminoacyl-tRNA synthetases studied most likely was notdue to a general change in protein concentrationof the S-105 fraction. From the present study,it is not possible to ascertain whether control ofsynthetase activity was at the substrate or enzymesynthesis level. The similar and sharp optimalconcentrations of Mg++, ATP, and hydrogenions for phenylalanyl-tRNA synthetases of theyoung and old cells might indicate that one andthe same species of that enzyme is present incells of different ages, unlike Neurospora whichhas two individual cytoplasmic phenylalanyl-tRNA synthetase enzymes, each specific for oneof two different species of phenylalanyl-tRNAfrom that organism (1).The ribosome-activated phenylalanyl-tRNA

degradation process in old cell P-105 fractions isbeing studied further, because endonucleaseshave been characterized from N. crassa (14),ribonuclease T1 is commercially prepared fromthe fungus Aspergillus oryzae, and Nagasaki(18) has demonstrated that such endonucleaseactivities increase in the older vegetative cells ofA. niger.

It is well established that ribonucleases, as wellas other degradative enzymes, become abundantin bacterial and fungal cultures after the log phaseof growth. A similar phenomenon is reportedhere. Further, the factor responsible for phenyl-alanyl-tRNA degradation progressively increaseswith increasing age of the cells within a singleculture.

In summary, the decreased synthesis of proteinfrom "4C-phenylalanine that occurred in older

cells can now be related to the protein-synthesiz-ing mechanism, since such a decrease also takesplace in a cell-free system. The decrease can beexplained, in part, by a defect in the charging oftRNA with some amino acids and, in part, bythe presence of nucleases in ribosomal prepara-tions of older cells.

ACKNOWLEDGMENTS

This investigation was supported by Public Health Servicetraining grant GM1380 from the National Institute of GeneralMedical Sciences.We thank Shirley Bohm for excellent technical assistance and

P. D. Shaw for his helpful suggestions.

LITERATURE CITED

1. Barnett, W. E., and J. L. Epler. 1966. Fractionation andspecificities ot two aspartyl-ribonucleic acids and twophenylalanyl-ribonucleic synthetases. Proc. Nat. Acad. Sci.U.S.A. 55:184-189.

2. Bent, K. J., and A. G. Morton. 1964. Amino acid compositionoffungi during development in submerged culture. Biochem.J. 92:260-269.

3. Breuer, C. B., and J. F. Florini. 1965. Amino acid incorpora-tion by cell-free systems from rat skeletal muscle. IV.Effects of animal age, androgens, and anabolic agents on

activity of muscle ribosomes. Biochemistry 4:1544-1549.4. Cammarano, P., G. Giudice. and Bessie Lukes. 1965. Poly-

ribosomes in regenerating liver. Biochem. Biophys. Res.Commun. 19:487-493.

5. Chayen, R., J. Chayen, and E. R. Roberts. 1959. Turnover ofnitrogen in Torulopsis utilis. Biochim. Biophys. Acta 31:186-194.

6. Clark Jr., J. M. 1964. Acyl activation reactions, p. 171-176.In J. M. Clark Jr. (ed.), Experimental biochemistry. W. H.Freeman & Co., San Francisco.

7. Downey, J. M., A. G. So, and E. W. Davie. 1965. Charac-terization of the polynucleotide-dependent transfer reactionin protein biosynthesis employing a cell-free system from theyeast Saccharomyces fragilis. Biochemistry 4:1702-1709.

8. Gottlieb, D., H. P. Molitoris, and J. L. Van Etten. 1968.Changes in fungi with age. III. Incorporation ofamino acidsinto cells of Rhizoctonia solani and Sclerotium bataticola.Arch. Mikrobiol. 61:394-398.

9. Gottlieb, D., and J. L. Van Etten. 1964. Biochemical changesduring the growth of fungi. 1. Nitrogen compounds andcarbohydrate changes in Penicillium atrovenelum. J.Bacteriol. 88:114-121.

10. Gottlieb, D., and J. L. Van Etten. 1966. Changes in fungiwith age. I. Chemical composition of Rhizoctonia solani andSclerotium bataticola. J. Bacteriol. 91:161-168.

11. Hartwell, L. H., and C. S. McLaughlin. 1968. Mutants ofyeast with temperature-sensitive isoleucyl-tRNA synthe-tases. Proc. Nat. Acad. Sci. U.S.A. 59:422-428.

12. Layne, E. 1957. Spectrophotometric and turbidimetric meth-ods for measuring proteins, p. 447-454. In S. P. Colowickand N. 0. Kaplan (ed.), Methods in enzymology, vol. 3.Academic Press Inc., New York.

13. Lee, J. C., and V. M. Ingram. 1967. Erythrocyte t-RNA:change during chick development. Science 158:1330-1331.

14. Linn, S., and I. R. Lehman. 1965. An endonuclease fromNeurospora crassa specific for polynucleotide lacking anordered structure. 11. Studies on enzyme specificity. J. Biol.Chem. 240:1294-1304.

15. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folin phenolreagent. J. Biol. Chem. 193:265-275.

16. Mariat, F. 1959. Nucleic acid contents of the yeast-like andmycelial forms of Sporotrichum schenckii. C. R. SeancesAcad. Sci. 248:3468-3469.

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OBRIG AND GOTTLIEB

17. Medvedev, Z. A. 1967. Molecular aspects of ageing, p. 1-28.In H. W. Woolhouse (ed.), Aspects of the biology of ageing.Academic Press Inc., New York.

18. Nagasaki, S. 1968. Physiological aspects of various enzyme

activities in relation to the culture of Aspergillus niigermycelia. J. Gen. Appl. Microbiol. 14:147-161.

19. Nazario, M. 1967. The accumulation of arginosuccinate inNeurospora crassa. Ir. Inhibition of arginyl-tRNA synthe-tases by arginino-succinate. Biochim. Biophys. Acta 145:146-152.

20. Nicolas, G., and D. Gottlieb. 1968. Changes in fungi with age.

IV. Role of coenzymes in the respiratory decreases inRhizoctonia solani and Sclerotium bataticola. J. Gerontol.23:544-550.

21. Novelli, G. D. 1967. Amino acid activation for proteinsynthesis. Annu. Rev. Biochem. 36:449-484.

22. Obrig, T. G., J. Cerna, and D. Gottlieb. 1969. Characteristicsof in vitro protein synthesis systems from Rhizocioniia solaniand Sclerotium bataticola. Phytopathology 59:187-192.

23. Rowley, P. T. 1965. Protein synthesis in reticulocytes maturingin vivo. Nature 208:244-246.

24. Shearn, A., and N. H. Horowitz. 1969. A study of the transferribonucleic acid in Neurospora. I. The attachment of aminoacids and amino acid analogs. Biochemistry 8:295-303.

25. So, A. G., and E. W. Davie. 1963. The incorporation otfaminoacids into protein in a cell-free system from yeast. Bio-chemistry 2:132-136.

26. Suskind, S. R., and D. M. Bonner. 1960. Effect of mutation on

RNA, protein, and ribonuclease formationi in Neuirospora

crassa. Biochim. Biophys. Acta 43:173-182.27. Van Etten, J. L., and R. M. Brambl. 1968. Protein synthesis

during fungal spore germination. IX. Aminoacyl-solubleribonucleic acid synthetase activities during germination of'Botryodiplodia theobrom7ae spores. J. Bacteriol. 96:1043-1048.

28. Van Etten, H., P. Molitor-is, and D. Gottlieb. 1966. Changes in

fungi with age. 11. Respiration and respiratory enzymes ot'Rhizoctoniia solaiti and Sclerotiuns bataticola. J. Bacteriol.91:169-175.

29. Vold, B. S., and P. S. Sypherd. 1968. Modification in transfei-RNA during the differentiation of wheat seedlings. Proc.Nat. Acad. Sci. U.S.A. 59:453-458.

30. Weisblum, B., F. Gonano, G. von Ehrenstein, aind S. Benzer.1965. A demonstration of coding degeneracy for lcucine inthe synthesis of protein. Proc. Nat. Acad. Sci. U.S.A. 53:328-334.

31. Wong, J. T., M. Mustard, aind E. Herbert. 1969. Regulation of'amino acid specific transfer RNA's in Fseherichi(a coli.Biochim. Biophys. Actai 174:513-524.

32. Yang, S. S., and D. G. Comb. 1968. Distribution of imtultipleforms of lysyl transfer RNA during early emiibryogeniesis ofsea urchin, Lytechiitus roriegatus. J. Mol. Biol. 31:139-142.

33. Yegian, C. D., and G. S. Stent. 1969. Diflerential amlino-acylation of three species of isoleucine transfer RNA fronEscherichia coli. J. Mol. Biol. 39:59-71.

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