JOURNAL OF BACTERIOLOGY, Sept. 1969, p. 807-814Copyright © 1969 American Society for Microbiology

Vol. 99, No. 3Printed in U.S.A.

Temperature-sensitive Yeast Mutant Defectivein Ribonucleic Acid Production

H. TERRY HUTCHISON,1 LELAND H. HARTWELL,' AND CALVIN S. McLAUGHLIN2

Department of Genetics, University of Washington, Seattle, Washington 98105, and the Department of Molecularand Cell Biology, University of California, Irvine, California 92664

Received for publication 9 June 1969

A single, recessive mutation in a nuclear gene confers a temperature-sensitivegrowth response in a mutant of Saccharomyces cerevisiae, ts- 136. The mutant grows

normally at 23 C, but exhibits a rapid and preferential inhibition of ribonucleic acid(RNA) accumulation after a shift to 36 C, demonstrating a defect in stable RNAproduction. Cultures of the mutant which were shifted from 23 to 36 C display thefollowing phenomena which indicate that messenger RNA (mRNA), as well as stableRNA production, is defective. The entrance of pulse-labeled RNA into cytoplasmicpolyribosomes is even more strongly inhibited than is net RNA accumulation. Therate of protein synthesis, at first unaffected, decreases slowly; this decrease isparalleled by the decay of polyribosomes to monoribosomes with a half-time of 23min. The polyribosomes which remain after a 30-min preincubation of the mutantat 36 C are active in polypeptide synthesis in vivo, whereas the monoribosomeswhich accumulate are not. Furthermore, ribosomes isolated from a culture of themutant preincubated for 1 hr at 36 C are inactive in polypeptide synthesis in vitro,but can be restored to full activity by the addition of polyuridylic acid as mRNA.We conclude that mutant ts- 136 is defective either in the synthesis of all types ofcytoplasmic RNA, or in the transport of newly synthesized RNA from the nucleusto the cytoplasm, and that the mRNA of a eucaryotic organism (yeast) is meta-bolically unstable, having a half-life of approximately 23 min at 36 C.

Conditional lethal mutants provide valuableobjects for the study of essential gene functions.Not only does the conditional nature of thedefect allow one to maintain a mutant whichnormally would not survive, but also an investi-gation of the behavior of the mutant at therestrictive condition allows one to examine thefunction of the defective gene in cellular physiol-ogy. The behavior of a distinct group of tem-perature-sensitive mutants of Saccharomycescerevisiae, thosewhich exhibit a rapid inhibition ofprotein synthesis at the restrictive temperature,was previously examined (3). Sixteen mutantswere divided into four classes based upon certainphysiological properties; three mutants in classIA (ts- 136, ts- 171, ts- 187) displayed propertiesconsistent with the hypothesis that they weredefective in the initiation of protein synthesis, orin ribonucleic acid (RNA) synthesis. A subse-quent report (4) demonstrated that two of thesethree mutants (ts- 171, ts- 187) were defective

1 Present address: Department of Genetics, University ofWashington, Seattle, Wash. 98105.

2 Present address: Department of Molecular and Cell Biology,University of California, Irvine, Calif. 92664.

in the initiation of polypeptide chains. Here wepresent evidence that the third mutant, ts- 136,is defective in RNA production.

MATERIALS AND METHODS

Strains and media. Mutant ts- 136 was obtainedfrom strain A364A (a ad, ad2 ur, ty1 hi7 lY2 gal) bymutagenesis with nitrosoguanidine (2). Strain 2262-2A(a ad, ur, his lyll le2 gal) was obtained from D. C.Hawthorne, University of Washington, Seattle. Allexperiments with whole cells were carried out inYM-5 medium, and those with spheroplasts werecarried out in YM-5 supplemented with 0.4 M MgSO4(2). Spheroplasts were prepared and incubated undergrowing conditions for 3 hr prior to the beginning ofeach experiment (6).

Genetic analysis. Techniques used for the isolation,sporulation, and tetrad dissection of hybrids, and thetechniques used for scoring the segregation of variousgenes, including intergenic complementation and thecomposition of diagnostic media, were taken fromthe methods of Hawthorne and Mortimer (5) andMortimer and Hawthorne (9). Only data from com-plete tetrads are reported.

Macromolecule synthesis, cell fractionation, andpolyribosome analysis. Techniques used to quantitate

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the incorporation of radioactive precursors intomacromolecules were previously reported (2).

Spheroplasts were fractionated into crude nuclearand cytoplasmic fractions by lysing as in polyribosomepreparation. This was followed by zone sedimentationin 11.5 ml of a 10 to 50% (w/w) linear sucrose gradi-ent in lysing buffer [minus poly-L-ornithine (3)] in aSpinco model SW41 ultracentrifuge for 40 min at190,000 X g. The gradient was drawn off the nuclearpellet to obtain the cytoplasmic fraction; the nuclearpellet was dissolved in 0.5% sodium dodecyl sulfate.

This procedure was tested by growing the parentstrain (an adenine auxotroph) from a small inoculumin both "4C-8-adenine to uniformly label the deoxy-ribonucleic acid (DNA) and RNA, and in "4C-recon-stituted protein hydrolysate to label the cellular pro-teins. Spheroplasts were prepared, grown for 3 hr inthe same labeled medium, and fractionated as de-scribed above. The nuclear pellet contained 91% ofthe cellular DNA, 18% of the RNA, and 8% of theprotein, as determined by the distribution of radio-activity; the cytoplasmic fraction contained 9, 82, and92% of the DNA, RNA, and protein, respectively.

Polyribosomes were prepared from spheroplastcultures, fractionated on sucrose gradients, andanalyzed as described previously (3); a slight varia-tion was introduced in one experiment by continuousmonitoring of the OD260 (optical density at 260 nm)profile through a flow cell in a Zeiss PMQ II spec-trophotometer.

In vitro protein synthesis. Cell-free extracts for invitro protein synthesis were obtained from sphero-plasts pregrown at 23 C for 3 hr in YM-5 mediumcontaining 0.4 M MgSO4, and were then grown for1 hr at either 23 or 36 C. A 500-ml amount of aspheroplast culture was collected by centrifugationand was lysed by homogenization in lysing buffer.The particulate matter was removed by centrifugationat 10,000 X g for 10 min; the supernatant fluid thenconstituted the S10 fraction. Ribosome pellets wereprepared from the S10 supernatant fluid by centrifu-gation at 100,000 X g for 60 min. Ribosome pelletsand S1O fractions were stored at -70 C until use.

Protein synthesis was monitored by the incorpora-tion of "4C-amino acids into material precipitable in5% trichloroacetic acid. Each assay tube contained(in a total volume of 0.25 ml): KCI, 0.5 smoles; mag-nesium acetate, 0.14 jsmoles; tris-hydroxymethyl-aminomethane, 1.25 jpmoles (pH 7.6); adenosine tri-phosphate, 2.25 pmoles; guanosine triphosphate,0.0075 ,moles; ,s-mercaptoethanol, 0.3 pmoles; phos-phoenolpyruvate kinase, 2 pg; phosphoenolpyruvate,375 pg. Assays with endogenous mRNA also con-tained "4C-reconstituted protein hydrolysate, 1 lpc.Assays with added polyuridylic acid also contained0.05 pmole of each of the amino acids except phenyl-alanine; 14C-phenylalanine, 0.015 pmoles (50 pc/,4mole); polyuridylic acid, 18 ,g; and spermine, 40jug. S10 extracts (0.80 to 0.88 mg of protein) or ribo-somes (0.18 to 0.24 mg of protein) supplemented withS100 (0.18 mg of protein) previously obtained fromA364A.

RESULTSGenetic characterization. Mutant ts- 136 is one

of many temperature-sensitive yeast mutantswhich have been isolated for their ability to grownormally at 23 C, but not at 36 C, even onhighly enriched medium. As with most of thesemutants, the mutation harbored by mutantts- 136 is recessive (2).

Tetrad analysis was undertaken to determinewhether this mutant contained more than onetemperature-sensitive lesion and to determinewhether the lesion was in a nuclear or cytoplasmicgene. A hybrid (H28) was constructed by matingmutant ts- 136 with strain 2262-2A (ts+), andisolating the diploid by prototrophic selection.The diploid was sporulated; dissection of theresulting tetrads by micromanipulation was thenundertaken. The dissections were impeded,however, because few aggregates containingfour spores were present after the enzymaticdigestion of the ascus wall, in spite of the factthat many four-spored asci were noted in theoriginal preparation. Observation of asci duringenzymatic digestion revealed that although sporesare customarily very resistant to digestion by theenzyme preparation employed, some of thespores from this hybrid were digested. Sinceother hybrids prepared with strain 2262-2A havenot exhibited this phenomenon, it is apparentthat mutant ts- 136 carries an additional muta-tion which renders spores subject to enzymaticdigestion. Random spore preparations of thishybrid produce approximately equal numbers ofts- and ts+ haploids after enzymatic digestion,indicating that the lesion rendering the sporessubject to digestion is not closely linked to thetemperature-sensitive mutation. By carefullycontrolling the digestion conditions, it waspossible to obtain 22 tetrads for dissection. Sixof these tetrads segregated in a nonmendelianfashion for known nuclear genes; they were notincluded in the analysis. These cases were proba-bly not true tetrads, but resulted from thefortuitous association of spores, a phenomenonwhich has been termed "false tetrads" (7); falsetetrads would be enriched by conditions in whichone or more spores from true tetrads weredigested. The other 16 tetrads segregated 2-:2+spores for known nuclear genes and segregated2ts-: 2ts+ spores. One of the segregating ts-haploids was back-crossed to 2262-2A, and thehybrid was sporulated. The resulting spores werenot digested by enzyme treatment, and 21 com-plete tetrads were examined, all of which segre-gated 2ts-:2ts+ spores. A centromere-linkedmarker, le2, was included in these crosses

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to determine whether the ts- lesion was cen-tromere-linked. The pattern of segregation forthe gene pair ts-:le2 was 4 parental ditype asci: 7nonparental ditype asci: 26 tetratype asci,indicating that the ts- lesion is not closelycentromere-linked. These results indicate thatthe temperature-sensitive lesion is probably dueto a single mutation in a nuclear gene.

Macromolecule synthesis. An examination ofthe incorporation of radioactive precursors intoprotein and RNA by cultures of mutant ts- 136revealed a very rapid cessation of RNA produc-tion at the restrictive temperature (Fig. 1); therate of protein synthesis, however, was notimmediately affected, but gradually decreased. Adetailed investigation of the early kinetics ofprotein and RNA syntheses after the shift to36 C is presented in Fig. 2A and 2B. The rate ofRNA production begins to decrease within 5min after the shift to 36 C, relative to a cultureremaining at 23 C; the rate of protein synthesis isinitially accelerated at 36 C and does not beginto decrease significantly until about 25 min afterthe shift. The kinetics of DNA synthesis at 23and 36 C was examined in a separate experimentand was found to be similar to the kinetics ofprotein synthesis (Fig. 2C). The rate of DNAsynthesis in the culture which was shifted to 36 Cwas at first faster than that in the culture remain-ing at 23 C and then began to decrease afterapproximately 25 min.

Previous results have demonstrated that theincorporation of "IC-glucose into macromolecu-lar material, most of which is polysaccharide, waseven less inhibited in cultures of this mutantincubated at the restrictive temperature thanwas protein synthesis (3).Thus, when a culture of mutant ts- 136 is

shifted to the restrictive temperature, RNAproduction is quickly and preferentially in-hibited in comparison to the synthesis of theother macromolecular components of the cell,protein, DNA, and polysaccharide. Evidently,the function normally performed by the tem-perature-sensitive component in mutant ts- 136is required for RNA production.

Distribution of newly synthesized RNA betweennucleus and cytoplasm. The fractionation ofyeast cells into nuclear and cytoplasmic compo-nents, the isolation of polyribosomes, and theanalysis of RNA metabolism are convenientlycarried out in yeast spheroplasts growing in anosmotically stabilized medium (6). To examinethe distribution of newly synthesized RNAbetween nucleus and cytoplasm, growing cul-tures of parent and mutant spheroplasts were

2000 230 R 36'

1000 /

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R

0 1 2 0 1 2

H O U R S

FIG. 1. RNA and protein synthesis in cultures ofmutant ts- 136. One-half of a culture of mutant ts-136 growing at 23 C was shifted to 36 C and the otherhalf remained at 23 C. 14C8- adenine (0.035 ,c/ml) or'IC-reconstituted protein hydrolysate (0.1 ,uc/ml) wasadded at the time of the shift, and 1-ml samples wereremoved and analyzed for the amount of radioactivityin RNA (R) or protein (P).

u

I

0 10 20 0 10 20 0 10 20M N U T E S

FIG. 2. Early kinetics of protein, RNA, and DNAsynthesis in mutant ts- 136, after a shift from 23 to36 C. One-half of a culture of mutantt ts- 136 growingat 23 C was shifted to 36 C and the other half re-mained at 23 C. Reconstituted "C-protein hydrolysate,0.1 Mc/ml, (A) or "C-8-adenine, 0.1 ,uc/ml, (B) wasadded at the time of the shift, and the radioactivityincorporated in 1-ml samples was determined. DNAsynthesis (C) was examined by centrifuging a cultureof ts- 136 grown at 23 C and suspending at 107 cells/mlin YM-5 medium containing 0.5 ,uc of "C-adenine perml without carrier adenine. After a 15-min incorpora-tion period at 23 C to allow equilibration of nucleotidepools, one-half of the culture was shifted to 36 C andthe other half was left at 23 C. Samples of a 1-mlamount were removed at various times and analyzedfor radioactivity in DNA; the data are plotted as theincrease in radioactivity after the time of the shift(time zero).

shifted to 36 C, and "C-adenine was added.Samples were collected by centrifugation atvarious times, and were lysed to prepare crudenuclear and cytoplasmic fractions. In the parentculture, the majority of the labeled RNA wasfound in the nucleus at early times (less than 7min), whereas later the majority was found in the

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cytoplasm (Fig. 3A). Since most of the RNA issynthesized in the nucleus of eucaryotic cells andthen transported to the cytoplasm, this was theexpected result. Nuclear RNA labeling followedroughly the same time course in the mutant, andthe amount of radioactivity was only 2.5-foldless than in parent nuclei (Fig. 3B). In the mu-

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FIG. 3. Distribution of newly synthesized RNA be-tween nucleus and cytoplasm of A364A and mutantts- 136 at 36 C. Spheroplast cultures of A364A (A)and mutant ts- 136 (B) growing at 23 C were shiftedto 36 C and 14C-8-adenine (0.2 pc/ml) was added.Samples (5 ml) of each culture were collected, lysed,and centrifuged to prepare crude nuclear (N) andcytoplasmic fractions (C). Counts/min in 0.5 ml ofsample are recorded.

I0

N

a

0

tant, however, a much smaller proportion of thenewly synthesized RNA appears in the cyto-plasm; even after 30 min of incorporation, theamount of radioactivity in the mutant cyto-plasm does not equal that in the nucleus.

Entrance of newly synthesized RNA intopolyribosomes. The entrance of newly syn-thesized RNA into the cytoplasmic componentswas further examined by sucrose density-gradientcentrifugation. Parent and mutant spheroplastswere labeled with 14C-8-adenine for 15 min,after a shift to 36 C; as an additional control,mutant spheroplasts were labeled for 15 min at23 C. Cytoplasmic extracts were prepared anddisplayed on sucrose density gradients (Fig. 4).Newly synthesized RNA appears in the polyribo-somal region (fractions 11 to 24), in the mono-ribosomal region (8 to 10), and in three peakswhich sediment more slowly than the monori-bosome; by analogy with studies in mammaliancells and in bacteria, it is likely that the latterthree peaks represent transfer RNA and the tworibosomal subunits. The radioactivity of thecytoplasmic components prepared from themutant culture at 36 C is much less than that ofthe parent culture at 36 C, or the mutant cultureat 23 C (Table 1). Whereas the spheroplastculture of the mutant incorporated 36% as muchradioactivity into newly synthesized RNA duringthe 15-min period at 36 C as did the parentculture, the mutant polyribosomes incorporatedonly 12% as much radioactivity as did the poly-

a

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F R A C T I 0 N

FIG. 4. Entry of newly synthesized RNA into the polyribosomes of strain A364A and mutant ts- 136. Sphero-plast cultures of A364A and ts- 136 growing at 23 C were centrifuged and suspended at 108 cells/ml in mediumcontaining 2 pg of adenine per ml. The spheroplasts were preincubated in this medium for 5 min at 23 C and shiftedto 36 C (or left at 23 C); "4C-8-adenine (I juc/ml) was added at the time of the shift. Samples of 0.1-mi wereremoved and analyzed for total radioactivity incorporated into RNA. After 15 min of incubation, polyribosomeswere prepared from 4 ml of each culture and centrifuged for 3 hr in the Spinco model S W25 ultracentrifuge at24,000 rev/min. Fractions were collected and analyzed for absorbancy and radioactivity; the top of each gradientis at fraction 1.

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TABLE 1. Radioactivity in RNA of sphereoplasts,cytoplasm, and polyribosomes after IS min of

"4C-8-adenine incorporationa

Radioactivityb

Strain and temp Total in Poly-Total in gradient ribosomalculture (cyto- region

plasm)

A364A, 36 C 168,000 77,200 50,300ts- 136, 23 C 141,000 53,200 29,500ts- 136, 36 C 60,600 18,500 5,840

a Data have been normalized for small differ-ences in total ribosomal OD260 recovered.

b Expressed as counts/min in 4 ml of culture.

ribosomes from the parent strain. Similarly, thespheroplast culture of the mutant incorporated43% as much radioactivity into newly synthe-sized RNA at 36 as at 23 C, but only 20% as

much into polyribosomes at 36 as at 23 C.Decay of polyribosomes. The rapid inhibition

of RNA production in cultures of mutant ts- 136and the preferential effect upon the entrance ofnewly synthesized RNA into polyribosomessuggest an explanation for the slow decay in therate of protein synthesis which occurs in culturesof this mutant at 36 C (see Fig. 1). If the mRNAof yeast is metabolically unstable as it is inbacteria, then an inhibition of mRNA synthesis,or its transport into the cytoplasm, would resultin a decay of cytoplasmic polyribosomes with atime constant which is characteristic of theinstability of mRNA (11). Therefore, polyribo-some preparations from spheroplasts of mutantts- 136 were examined at various times after a

shift to 36 C for any change which might becorrelated with the decreasing rate of proteinsynthesis. The percentage of ribosomes in poly-ribosome structures decreases progressively as a

function of time after the shift to 36 C (Fig. 5).The results obtained in a detailed examination ofthe kinetics of polyribosome decay are presentedin Fig. 6. Ribosomes present in polyribosomestructures decrease from 90% at time zero toabout 10% at 2 hr. The decay is approximatelyexponential, exhibiting a half-time of 23 min; thekinetics correlate well with the rate of decay ofprotein synthetic capacity (half-life about 26min; Fig. 1).

Protein synthesis in vivo. If the decay of poly-ribosomes is due to a deficiency in mRNA pro-duction, then the decreasing rate of proteinsynthesis reflects a decreasing number of ribo-somes attached to mRNA, rather than a declinein the synthetic activity per ribosome. The hy-pothesis of a deficiency in mRNA predicts that

the accumulating monoribosomes will be inactivein polypeptide synthesis, but that the remainingpolyribosomes will be as active as those at thepermissive temperature. Spheroplast cultures ofmutant ts- 136 were preincubated for 30 min at23 and 36 C, and then were pulse-labeled for 2min with reconstituted 'IC-protein hydrolysate to

a

N

0

F RACTI O N

FIG. 5. Decay of polyribosomes in mutant ts- 136at 36 C. Polyribosomes were prepared from 40 ml ofaspheroplast culture of ts- 136 and then centrifuged for 3hr in the Spinco model SW25 ultracentrifuge at 24,000rev/min. The spheroplasts were harvested at 0, 30, and60 min after a shift from 23 C to 36 C.

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0 30 60 90 120 0 30 60 90

M N U T E S

FIG. 6. Kinetics of polyribosome decay in mutantts- 136 at 36 C. Polyribosomes were prepared andanalyzed in several experiments. The percentage ofribosomes contained in polyribosomes from eachgradient is plotted as a function of the time after theshift from 23 C to 36 C on a linear scale (A) and a

logarithmic scale (B). To obtain the data for part B,the plateau value of 10% was subtracted from eachpoint in part A, and the data were then normalized to100% at zero time.

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label nascent polypeptide chains. Most of theribosomes were present as polyribosomes in theculture incubated at 23 C, whereas approxi-mately 50% had decayed to monoribosomes inthe culture incubated at 36 C (Fig. 7); the spe-cific activity of the nascent polypeptide chainslocated on polyribosomes was approximately thesame in the two cultures, being 224 count/minper OD260 unit on the 23 C polyribosomes, and238 counts/min per OD260 unit on the 36 C poly-ribosomes. The specific activity of the monoribo-somes which had accumulated in the 36 C cul-ture was only 37 counts/min per OD260 unit;this amount might be due to newly synthesizedribosomal proteins which appear on ribosomesvery quickly in yeast (4). This result indicateseither that most of the monoribosomes do notcarry nascent polypeptide chains, or that thenascent chains on the monoribosomes are muchshorter on the average than those on the poly-ribosomes. It seems unreasonable to think thatthe temperature shift would induce a progressivechange in the size distribution of the polypeptidechains being synthesized; we conclude, there-fore, that polypeptide synthesis occurs to a sig-nificant extent only on polyribosomes. The cor-relation between the rate of polyribosome decayand the rate of decay of protein synthesis is,therefore, explained by the finding that the ac-cumulating monoribosomes are inactive in pro-tein synthesis.

In vitro protein synthesis. The foregoing resultsargue strongly that the monoribosomes whichaccumulate in cultures of mutant ts- 136 afteran incubation at the restrictive temperature aredeficient in mRNA. This hypothesis was con-

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firmed by an experiment which tested the abilityof ribosomes prepared from mutant and parentcultures to stimulate protein synthesis in vitro.Extracts were prepared from spheroplasts ofthe mutant preincubated at 23 and at 36 C for 1hr, after which 90 and 15%, respectively, of theribosomes would be present as polyribosomes.Extracts were also prepared from spheroplasts ofthe parent strain incubated under the same con-ditions, after which 90% and 70%, respectively,of the ribosomes would be present as polyribo-somes based on previous results. Extracts of S10were prepared by homogenizing the spheroplastsin lysing buffer and then centrifuging out theparticulate debris at 10,000 x g for 10 min;ribosome pellets and S100 supernatant fluids werein turn prepared from S10 extracts by centrifuga-tion at 100,000 X g for 120 min. The assay mix-ture supplied all components necessary for poly-peptide synthesis except ribosomes, supernatantfactors, and mRNA. Extracts of S10 or ribo-somes prepared from parent spheroplasts pre-incubated at 36 C were approximately the samein activity (when using either endogenous mRNAor polyuridylic acid) as the comparable extract,or ribosomes prepared from parent spheroplastspreincubated at 23 C (Table 2). On the contrary,the SlO extract prepared from mutant sphero-plasts preincubated at 36 C was only 10% asactive when using endogenous mRNA as theS10 extract prepared from mutant spheroplastspreincubated at 23 C. The ribosomes isolatedfrom mutant spheroplasts were only one-third asactive when using endogeneous mRNA as thosefrom mutant spheroplasts preincubated at 23 C.The fact that ribosomes did not show as great a

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10 20 30 10 20 30

F R A C T 0 N

FIG. 7. Pulse-labeling ofnascent polypeptide chains on polyribosomes of mutant ts- 136. A spheroplast cultureof mutant ts- 136 was divided in two parts, one of which was incubated at 23 C for 30 min (A); the other wasincubated at 36 C for 30 min (B). The cultures were concentrated 10-fold by centrifugation, preincubated for 5min at 23 and 36 C, respectively, and labeledfor 2 min with reconstituted '4C-protein hydrolysate (S Jc/ml), after.which time the cultures were collectedfor polyribosome analysis.

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difference as did the S10 estract may be becausethe S100 supernatant liquid added to the mutantribosomes perhaps contains some mRNA, sinceit was prepared from parent spheroplasts growingat 23 C. The addition of polyuridylic acid as anmRNA restored the activity of both the S10extract and the ribosomes prepared from mutantspheroplasts preincubated at 36 C to levels equalto, or greater than, either the S1O extract orribosomes prepared from spheroplasts preincu-bated at 23 C, demonstrating a deficiency inmRNA in the former perparations.

DISCUSSIONThe rapid and preferential inhibition of net

RNA accumulation exhibited by cultures of ts-136 after a shift to 36 C indicates that the syn-thesis of stable RNA species is dependent uponthe gene product that is defective in this mutant.The kinetics of net RNA accumulation, how-ever, provide no information about the metab-olism of unstable RNA species, since the synthesisand degradation of these forms may be accom-plished by turnover of the intracellular nucleotidepools without the uptake of radioactive pre-Cursors from the medium (10). Thus, the datapresented here do not distinguish between acessation of nuclear RNA synthesis on the onehand and a continuation of nuclear RNA syn-thesis balanced by RNA degradation on theother. It is clear that a much smaller proportionof the RNA synthesized at 36 C reaches thecytoplasm of the mutant than is the case in theparent strain. One plausible model to explainthese results is that the primary lesion in mutantts- 136 is in the transport of RNA to the cyto-plasm and, as a result of this defect, nuclearRNA synthesis ceases or is balanced by degrada-tion. On the other hand, a model can be postu-lated in which a primary defect in nuclear RNAsynthesis results in the cessation of transport.In fact, some experimental evidence for such amodel is provided by the observation that actino-mycin, a well characterized inhibitor of RNAsynthesis, inhibits both synthesis of new RNAand transport of previously synthesized RNA inmammalian cells (1).

Regardless of whether the primary lesion is inthe synthesis or in the transport of RNA, theresponse of the protein synthetic system to thecessation of cytoplasmic RNA productionstrongly indicates that the mutational defectextends to the production of unstable cytoplasmicmRNA. This response includes a slow decay inthe rate of protein synthesis, which is paralleledby the decay of cytoplasmic polyribosomes to

TABLE 2. Protein synthesis in vitro with S1O andribosome fractions prepared from parent and

mutant spheroplasts after preincubationfor 1 hr at 23 or 36 C

Protein synthesisa

Strain Kind of extract Growthtemp Endo- Plus poly-geneous uridylicmRNA acid

A364A S10 23 896 356S10 36 355 412Ribosome 23 6,609 12,793Ribosome 36 6,133 13,653

ts 136 S10 23 487 321510 36 53 511Ribosome 23 5,528 16,985Ribosome 36 1,986 21,171

a Measured as counts/min incorporated intopolypeptide per milligram of S10 or ribosome pro-tein present in the assay tube.

monoribosomes. The polyribosomes which re-main after 30 min at 36 C are fully active in poly-peptide synthesis in vivo, but the monoribosomeswhich accumulate are inactive. The monoribo-somes can be restored to full activity in vitro bythe addition of polyuridylic acid, an artificialmRNA. These results are completely analogousto those obtained after the addition of actino-mycin, a well-characterized inhibitor of RNAsynthesis, to cultures of Bacillus subtilis (8, 11),and indicate that the mRNA of a eucaryoticorganism (yeast) is metabolically unstable, hav-ing a half-life of about 23 min at 36 C.

In experiments not reported here, we havebeen unable to demonstrate any striking defectin an aggregate RNA polymerase preparationfrom this mutant, or in its ability to carry outnucleotide metabolism at 36 C. A negative resultin this case is inconclusive, however, since invitro conditions undoubtedly do not accuratelymimic in vivo conditions. Although we are con-tinuing our efforts to locate the lesion in mutantts- 136, it should be pointed out that the prop-erties of this mutant offer some novel approachesfor the investigation of RNA metabolism, re-gardless of the site of defect. For example, therapid inhibition of RNA transport imposed bythis mutation allows one to follow the movementor the maturation of prelabeled cytoplasmicRNA components uncomplicated by the entranceof newly synthesized RNA into the cytoplasm.

ACKNOWLEDGMENTS

We thank W. L. Fangman, Jonathan A. Gallant, and J.Warner for their criticism of the manuscript.

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This investigation was supported by grant GB8028 from theNational Science Foundation and by Public Health Service grantCA10678 from the National Cancer Institute. H. T. Hutchisonwas the recipient of a Public Health Service predoctoral fel-lowship.

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2. Hartwell, L. H. 1967. Macromolecule synthesis in tempera-ture-sensitive mutants of yeast. J. Bacteriol. 93:1662-1670.

3. Hartwell, L. H., and C. S. McLaughlin. 1968. Temperature-sensitive mutants of yeast exhibiting a rapid inhibition ofprotein synthesis. J. Bacteriol. 96:1664-1671.

4. Hartwell, L. H., and C. S. McLaughlin. 1969. A mutant ofyeast apparently defective in the initiation of protein syn-thesis. Proc. Nat. Acead. Sci. U.S.A. 62:468-475.

5. Hawthorne, D. C., and R. K. Mortimer. 1960. Chromosomemapping in Saccharomyces: centromere-linked genes.Genetics 45:1085-1110.

6. Hutchison, H. T., and L. H. Hartwell. 1967. Macromoleculesynthesis in yeast spheroplasts. J. Bacteriol. 94:1697-1705.

7. Johnston, J. R., and R. K. Mortimer. 1959. Use of snail di-gestive juice in isolation of yeast spore tetrads. J. Bacteriol.78:272-280.

8. Levinthal, C., A. Keynan, and A. Higa. 1962. MessengerRNA turnover and protein synthesis in B. subtdlus inhibitedby actinomycin D. Proc. Nat. Acead. Sci. U.S.A. 48:1631-1638.

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