JOURNAL OF BACTERIOLOGY, May 1967, p. 1662-1670Copyright © 1967 American Society for Microbiology

Vol. 93, No. 5Printed in U.S.A.

Macronmolecule Synthesis in Temperature-sensitiveMutants of Yeast

LELAND H. HARTWELL

Department of Molecular and Cell Biology, University of California, Irvine, California 92650

Received for publication 16 January 1967

Approximately 400 temperature-sensitive mutants of Saccharomyces cerevisiaewere isolated. The mutants were unable to form colonies on enriched media at36 C, but grew normally, or nearly so, at 23 C. The mutants were tested for lossof viability, change in morphology, increase in cell number, and the ability to syn-

thesize protein, ribonucleic acid (RNA), and deoxyribonucleic acid (DNA) after a

shift from 23 to 36 C. Mutations were found which resulted in a preferential loss ofability to carry out protein synthesis, RNA synthesis, DNA synthesis, cell division,or cell-wall formation. Diploid cells heterozygous for the temperature-sensitivemutations were constructed and tested for their ability to form colonies at 36 C.Four mutations dominant to their wild-type allele were identified.

Horowitz and Leupold (6) first suggested thatthe isolation of temperature-sensitive mutantsmight provide a technique for obtaining muta-tions in indispensible (i.e., nonauxotrophic)genes. The work of Edgar and co-workers withbacteriophage T4 (2, 4) demonstrated that tem-perature-sensitive mutants can be used effectivelyto gain information about indispensable genefunctions. These studies demonstrated thattemperature-sensitive mutations can occur inmany different genes in an organism. For in-stance, 39 genes were identified in bacteriophageT4 by amber mutations; 26 of these were shownto be able to carry mutations leading to a tem-perature-sensitive phenotype. Neidhardt de-scribed the isolation of temperature-sensitivemutants of Escherichia coli and some of the pat-terns of macromolecule synthesis that resultwhen the mutants are shifted to the nonpermissivetemperature (9). The biochemical lesion in threeof these mutants has been identified as being dueto thermolabile valine-activating enzyme, phen-ylalanine-activating enzyme (3), and fructose1, 6-diphosphate aldolase (1). An extensiveinvestigation of 400 temperature-sensitive mu-tants of E. coli has recently been published byKohiyama et al. (7). Lesions which preferentiallyaffected cell division, cell wall formation, andthe synthesis of protein, ribonucleic acid (RNA),and deoxyribonucleic acid (DNA) were identi-fied through a study of macomolecule synthesisand changes in morphology after the mutantswere shifted to the nonpermissive temperature.

Temperature-sensitive mutants should provide

a useful probe into the poorly understood proc-esses of macromolecule biosynthesis and itsregulation in the eucaryotic cell. The organismof choice for this type of study should fulfilltwo conditions. It should have the smallestamount of genetic material that is compatiblewith the complexity of a eucaryotic cell. Itshould possess a genetic system that allows oneto study complementation and dominance versusrecessiveness of mutations.

Saccharomyces cerevisiae appears to fulfillboth of these criteria. It is one of the simplesteucaryotic organisms, having only 2.2 X 10-8 Agof DNA per haploid nucleus (10). If one assumesthat an average protein contains 500 amino acids,this DNA content corresponds to enough genesto produce 13,000 different proteins. From thesame assumption, an E. coli cell is able to produce3,000 different proteins. Thus, the eucaryoticyeast cell is only about four times as complex asone of the simpler procaryotic cells. Geneticstudies of yeast have reached a high level ofsophistication (for a review, see 8). The yeastgenetic system is ideally suited for study of thecomplementation patterns and determination ofthe dominance or recessiveness of mutations.

This paper reports the isolation and prelim-inary characterization of about 400 temperature-sensitive mutants of yeast. The mutants formcolonies at 23 C but are unable to form coloniesat 36 C on an enriched medium containing amixture of vitamins, yeast extract, peptone,adenine, uracil, glucose, and succinate; the wildtype can grow at either temperature. The mutants

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were grown up at 23 C, shifted to 36 C, and testedfor loss of viability, change in morphology, in-crease in cell number, and the ability to synthe-size protein, RNA, and DNA. Each mutationwas examined to determine whether it was domi-nant or recessive with respect to the wild-typeallele. F. LaCroute (personal communication) hasisolated a large number of temperature-sensitivemutants in yeast and is carrying out studiessimilar to those reported here.

MATERIALS AND MErHODS

Yeast strains. The yeast strains used in this workwere haploid strains of S. cerevisiae. The parent fromwhich the 400 temperature-sensitive mutants wereisolated was obtained from Rochelle Esposito and isdesignated A364A (a ade-l- ura-lk gal-l- tyr-l-his-7- Iys-2- trp-l-). Strain X1069-2D (a ade-l-ura-l- his4- leu-2- thr4- met-2- trp-S-) was ob-tained from Robert K. Mortimer. The following ab-breviations for genetic markers are used: a and aindicate mating types; ade-, ura-, his-, lys-, trp,leu-, thr-, and met- indicate inability to synthesizeadenine, uracil, histidine, lysine, tryptophan, leucine,threonine, and methionine, respectively; gal- indi-cates inability to ferment galactose; ts- indicatesinability to grow at 36 C.

Media. YM-1 medium contained the followingcomponents (grams per liter): yeast extract, 5; pep-tone, 10; yeast nitrogen base (filter-sterilized), 6.7;adenine, 0.01; uracil, 0.01; succinic acid, 10; sodiumhydroxide, 6; glucose, 10; final pH, 5.8. YM-5 me-dium was the same as YM-1, except that the concen-trations of peptone and yeast extract were reducedfivefold. YEPD-TAU plates contained (grams perliter): yeast extract, 10; peptone, 20; glucose, 20;threonine (filter-sterilized), 0.04; adenine, 0.04; ura-cil, 0.04; agar, 20. Sporulation plates contained(grams per liter): potassium acetate, 3; raffinoseacetate, 0.22; yeast extract, 10; and agar, 20.

Chlemicals. Yeast extract, peptone, yeast nitrogenbase, malt extract, Brain Heart Infusion, and agarwere all obtained from Difco Laboratories (Detroit,Mich.). N-methyl-N'-nitro-N-nitrosoguanidine wasobtained from Aldrich Chemical Co. (Milwaukee,Wis.). All radioactive compounds were the productsof Schwarz Bio Research Inc. (Orangeburg, N.Y.).Glusulase is a commercial preparation of snail diges-tive juice, obtained from Endo Laboratories (GardenCity, N.Y.).

Mutant isolation: A culture of S. cerevisiae, strainA364A, was grown from a small inoculum overnightat 36 C in YM-1. While still growing logarithmically,50 ml of culture was shifted to 23 C, and 0.2 ml of asolution containing 4 mg (per ml) of N-methyl-N'-nitro-N-nitrosoguanidine was added. Samples con-taining 0.5 ml of the culture were immediately dis-tributed to a large number of tubes. The tubes werethen rotated for a period of 5 hr (survival, 0.2 to1.0%), after which time samples were removed,diluted, plated on YEPD-TAU plates, and incubatedat 23 C. When colonies appeared, the pattern was

replicated onto two plates, the first of which was in-cubated at 36 C and the second of which was incu-bated at 23 C. Colonies which grew up on the latterbut not the former (approximately 1% of the totalnumber of colonies) were picked, diluted with water,and streaked onto two YEPD-TAU plates, whichwere again incubated at the two temperatures. Cloneswhich formed approximately 1,000 colonies on the23 C plate and no colonies on the 36 C plate werepicked from the low-temperature plate and desig-nated as ts- mutants. Only one or two mutants wereisolated from a single mutagen-treated culture tube.

Genetic techniques. Haploid cultures were mated bymixing cultures of opposite mating type on YEPD-TAU plates, and the resulting diploid was isolated bythe prototrophic selection method of Pomper andBurkholder (11). The products of meiosis were ana-lyzed by a random-spore technique. Diploids weresporulated on sporulation medium. The ascus wasdigested with glusulase, and the spores were separatedby intense sonic oscillation. Microscopic counts indi-cated that this technique resulted in a preparationconsisting of (80 to 90%) single spores, with the re-mainder being spore aggregates. Unsporulated cellswere destroyed by the digestion and sonic procedure.

Analytical methods. Macromolecule synthesis wasstudied in two basic types of experiments. In thepulse-label experiment, protein synthesis was meas-ured by the incorporation of a mixture of '4C-labeledamino acids in one culture flask, and RNA and DNAsynthesis was measured by the incorporation ofadenine-8-14C in another culture flask. In the uniform-label experiment, protein was labeled with 3H-lysinein the same culture flask in which RNA and DNAwere labeled with adenine-8-'4C. The amount ofadenine-8-14C incorporated into RNA was deter-mined by precipitating a portion of culture in an equalvolume of cold 10% trichloroacetic acid, collectingthe precipitate on membrane filters (Millipore, typeHA), and counting the filters in a gas-flow counter.Little or no 14C-adenine was incorporated into pro-tein, as evidenced by the fact that boiling labeledcells in 5% trichloroacetic acid reduced the pre-cipitable counts by greater than 99%. Base hydrolysisof 14C-adenine-labeled cells and chromatography ofthe hydrolysate revealed 95% of the counts as guano-sine-3'-monophosphate and adenosine-3'-monophos-phate. (Only 2% of the counts were in DNA, sinceS. cerevisiae has an RNA-DNA ratio of about 50.)In samples of '4C-adenine-labeled cells from a uni-form-label experiment, the protein was also labeledwith 3H-lysine. Owing to the-poor counting efficiencyof 3H in the gas-flow planchet counter, the contamina-tion of samples being counted for RNA with proteincounts was always less than 0.05%. The sedimentationpattern of 14C-adenine counts from a sodium dodecylsulfate-treated cell lysate in a sucrose density gradientdisplayed the three peaks expected for soluble RNAand the two ribosomal RNA components when longlabeling times were employed.The amount of adenine-8-14C incorporated into

DNA was determined by the incubation of 1.0 ml ofculture in 1 N NaOH at room temperature for 16hr, followed by precipitation with cold trichloroacetic

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acid to a final concentration of about 10%. Thisprocedure solubilizes all the RNA counts. The pre-cipitates were again collected on membrane filtersand counted in a gas-flow counter. Treatment of acell lysate with deoxyribonuclease solubilized about70% of these NaOH-resistant counts. Depurinationof the DNA with 0.02 N HCl at 100 C for 1 hr, fol-lowed by paper chromatography of the hydrolysate,allowed the recovery of 95% of the NaOH-resistantcounts as guanine and adenine. When protein was alsolabeled with 3H-lysine, the contamination of DNAcounts with protein counts was less than 2%.When the protein was labeled with a mixture of

14C-amino acids, a portion of the culture was boiledin 5% trichloroacetic acid for 30 min and the pre-cipitate was collected on a membrane filter. Thefilters were counted in a gas-flow planchet counter.In the uniform-label experiments, when 3H-lysine wasused as a label for protein, a portion of the culturewas boiled in 10% trichloroacetic acid for 30 min tosolubilize the '4C-adenine counts in RNA and DNA.The precipitate was collected on a membrane filter,which was treated in a scintillation vial with 0.4 mlof formic acid. The radioactivity was then counted in atoluene-ethyl alcohol scintillation mixture (5) in ascintillation counter. Although the efficiency of count-ing is low for 8H in this scintillation mixture (about3%), the amount of quenching is influenced onlyslightly by variations in the number of cells on thefilter.

RESULTS

Isolation of ts- mutants. Approximately 400temperature-sensitive (ts-) mutants of S. cere-visiae were isolated by a replica-plating tech-nique. The mutants do not form colonies at 36 Cbut do form colonies at 23 C; the wild type formscolonies at both temperatures. At least 90%of the mutants are known to be of independentorigin, since they were obtained from independentsamples of mutagen-treated cultures. No directselective step was employed in the isolationprocedure. Since the mutant clones have neverbeen exposed to the nonpermissive temperature,mutants which lose viability at high temperaturehave not been selected against. The mutants havebeen assigned numbers from 92 to 489.The ts- phenotype is probably not, in most

cases, due to a lesion in the ability to synthesizesmall molecules such as vitamins, amino acids,purines, or pyrimidines, since the mutants wereisolated on a complex medium containing avitamin supplement, adenine, uracil, yeast ex-tract, peptone, and two carbon sources, glucoseand succinate. Furthermore, 160 of the mutantshave been checked for temperature sensitivityon an enriched medium containing malt extractand Brain Heart Infusion in addition to yeastextract and peptone. All of the mutants exceptfor one was ts- on this medium as well.

Morphology and viability of ts- mutants.

Microscopic examination of the surface of agarplates indicated that most of the mutants formedless than 32 cells per clone after 3 days at 36 C,and that approximately 10% of the mutantsunderwent morphological alteration during theirexposure to the nonpermissive temperature.Some of the mutants became larger or smallerthan normal, and others appeared to lyse. Tenmutants underwent an extreme alteration inwhich the cells became very long and branched,resulting in a mycelial type of growth (Fig. 8).A simple streak test was used to screen mutants

for loss of viability at the nonpermissive tempera-ture. A suspension of cells was streaked ontotwo plates. One was placed immediately at 23 C;the other was placed at 36 C for 16 hr and thenshifted to 23 C. After 4 days of growth, thecolony density on the two plates was com-pared. Approximately 40% of the mutantsunderwent a greater than 90% reduction incolony-forming ability during their exposure tohigh temperature.Dominance and recessiveness of ts- mutants.

All mutants were tested for dominance or reces-siveness of their ts- mutation. They were matedwith a wild-type haploid of opposite matingtype. The diploid which resulted was isolatedon selective media and streaked on plates athigh and low temperature. The vast majority ofthe mutants displayed a recessive phenotype;that is, the diploid carrying the ts mutation inthe heterozygous state formed approximatelyequal numbers of colonies at high and low tem-perature. Sporulation of these diploids and anexamination of the resulting haploids for tem-perature sensitivity demonstrated that the diploidswhich grew at 36 C were still heterozygous forthe ts- mutation. A total of four dominant ts-mutations were found in the collection of 400mutants (numbers 344, 349, 408, and 453). Thediploids harboring these mutations in the heter-ozygous state formed significantly fewer coloniesat 36 C than they did at 23 C. The ts- phenotypeis either extremely leaky or subject to a high rateof reversion in the heterozygous state, as culturesof these diploids form up to 10% as manycolonies at 36 C as they do at 23 C. Haploidcultures of these four mutants had a low inci-dence of revertent cells, 10-5 to 10 7.

Pulse labeling for macromolecule synthesis.A simple procedure was developed for screeninglarge numbers of mutants for protein, RNA, andDNA synthesis at the nonpermissive tempera-ture. The mutants were grown in liquid cultureat 23 C in YM-5 medium. When they were in alogarithmically growing state, a portion of theculture was diluted threefold into YM-5 medium(at 36 C) containing 14C- amino acids (reconsti-

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tuted 14C-protein hydrolysate); a second portionwas diluted threefold into YM-5 medium (at36 C) containing adenine-8-'4C. After 3 hr at36 C, samples were removed for measurement ofthe amount of radioactive amino acids incor-porated into protein and the amount of radio-active adenine incorporated into RNA and DNA.During this 3-hr period, the growth of the orig-inal culture at 23 C was followed by turbiditymeasurements. About 85% of the mutants grewwith rates between 0.80 and 1.10 of the wild-type growth rate at 23 C. The data obtained forsome of the mutants are listed in Table 1. Figure1 is a plot of the protein value for each of the400 mutants as a function of the RNA value forthe same mutant. Lines have been drawn alongaxes of protein to RNA ratios- (P/R) equal to1, 2, and 4. Almost all of the mutants displayP/R ratios equal to or greater than 1. This ob-

TABLE 1. Growth rate, macromolecule synthesis, andsurvival of colony-forming ability of some

representative ts- mutants as determinedin the pulse-labeling experiment

Mutantno.

108159171172187236282289310344349408438453471

Growthrate at23 ca

0.951.051.030.870.991.111.060.990.861.260.730.880.760.920.98

Macromoleculesynthesis at 36 cb

Protein

0.350.880.020.230.021.090.300.780.540.730.070.550.310.660.79

RNA

0.070.720.040.200.080.740.070.810.640.420.020.390.340.730.90

DNA

0.161.230.080.070.060.680.200.230.760.440.030.330.270.460.91

Survival ofcolony-formingability at 36 cc

0.100.301.000.101.000.150.900.100.500.010.000.100.150.050.90

a Growth rate is expressed as k mutant/k wildtype, where k = (ln2 B/Bo)/t.

I Protein synthesis as measured by the amountof 14C from a "4C-protein hydrolysate incorpo-rated into protein for a 3-hr period. RNA andDNA synthesis were measured by the amount ofadenine-8-14C incorporated into RNA and DNA,respectively, over a 3-hr period. All values weremultiplied by (turbidity of A364A/turbidity ofmutant) at the time of the shift to 36 C and thendivided by the value for A364A. Thus, all valuesare normalized to the value of 1.0 for A364A.

r Survival of colony-forming ability on YEPD-TAU plates after exposure to 36 C for 16 hr, fol-lowed by incubation at 23 C for 4 days. A364Awas assigned a value of 1.0.

servation can probably be explained on the basisof control mechanisms which shut down RNAsynthesis as growth slows down for any one of avariety of reasons. The parent strain A364A hasa stringent control over RNA synthesis in thatthe inhibition of protein synthesis by starvationfor a required amino acid results in an immediatecessation of net RNA synthesis. A large numberof mutants have P/R ratios greater than 4. Thesewill be discussed below.

Uniform-label experiment. The data wereanalyzed and the mutants further studied withthe aim of finding mutations which preferentiallyaffect the following processes: protein synthesis,RNA synthesis, DNA synthesis, cell division,and cell wall formation. Certain mutants sus-

pected of falling into one of these five classes, onthe basis of the pulse-labeling experiments, were

chosen and a uniform-label experiment was per-

formed on them. In this experiment, the mutantswere grown at 23 C from a small inoculumin medium containing 14C-adenine and 3H-lysine.It should be recalled that the mutants were allderived from a strain which was auxotrophic forlysine and adenine. When the cultures hadreached a sufficient density and were still growinglogarithmically, they were shifted to 36 C. Atthe time of the shift, the cells protein-is- uniformlylabeled with 3H, and the RNA and DNA isuniformly labeled with 14C. Thus, any increasein radioactivity after the shift to high tempera-ture is directly proportional to the net increasein the particular macromolecule being studied.In contrast to the pulse-labeling experiment,there is no problem with pool equilibration

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after the shift to 36 C. Samples were taken atvarious times after the shift and treated tomeasure the radioactivity in protein, RNA,and DNA. Cell number was determined in aCoulter counter (Coulter Electronics, Inc.,Hialeah, Fla.), and colony-forming ability wasdetermined by dilution and plating. Figure 2presents the results of a uniform-label experi-ment with a culture of the wild type, A364A(ts+). The doubling time of the wild type at 23 Cwas approximately 3 hr. Immediately after theshift to 36 C, the doubling time of all macro-molecules, as well as cell number and colony-forming ability, decreased to about 1.7 hr. Theslowing down of RNA synthesis at 6 hr was dueto an exhaustion of adenine from the medium.In experiments with the temperature-sensitivemutants, the cell density was kept considerablybelow this level.

Mutations preferentially affecting protein syn-thesis. Mutants incorporating less than 10% ofthe radioactivity incorporated by the wild typeinto protein in the pulse-label experiment (Fig. 1)are suspected of being "protein synthesis"mutants. There are 21 mutants in this category.All of these mutants also show low values forRNA synthesis.

Several of these mutants were examined in a

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FIG. 2. Valuesfor protein (P), RNA (R), DNA (D),cell number (N), and cell viability (V) as a function ofthe time after a shift from 23 to 36 C for a culture ofthe wild type, A364A. All parameters were normalizedto a value of I at the time of the shift.

uniform-label experiment. Most of these mutantsshowed little or no net increase in protein aftera shift to 36 C. Figure 3 presents the results of auniform-label experiment with mutant number187. Protein and RNA showed no net increaseafter the shift. DNA increased by about 35%.Cell number remained constant and there waslittle loss of viability. Mutant 171 is similar inphenotype to number 187. Mutants of this typeare currently being examined for their abilityto synthesize protein in vitro.

Mutations preferentially affecting RNA syn-thesis. Those mutants displaying protein-RNAratios of greater than 4 in the pulse-labelingexperiment are suspected of being mutants forRNA synthesis. There are 38 mutants in thiscategory. A uniform-label experiment was per-formed on about two-thirds of these mutants.In all cases, the net increase in RNA was in-hibited to a far greater extent than was the in-crease in protein after the shift to 36 C. Figure4 presents the results of a uniform-label experi-ment with mutant 108. Total protein increased100% over the first 3 hr after the shift, whereasRNA increases by only about 5%. DNA in-creased 65 %, cell number increased 35 %, andviability fell off slowly. About 15 mutants werefound with phenotypes very similar to number108. (Number 282 is another example.) Othersshow a slow, but continuous, increase in netRNA. Since ribosomal RNA is the major com-ponent of cellular RNA, it is clear that the for-mation of ribosomal RNA is being preferentiallyshut off in mutant 108. It is unclear at the presenttime whether messenger RNA is being shut off.Experiments are being undertaken to charac-terize the small amount of RNA which is madeafter the shift.

Mutations preferentially affecting DNA syn-thesis. The data from the pulse experiment were

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examined to uncover mutants with a specificinhibition of DNA synthesis. Nineteen mutantshad an RNA-DNA ratio of greater than 2and showed a significant amount of RNA syn-

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ts- mutant 108. All parameters were normalized to avalue of I at the time of the shift.

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thesis (greater than 20% of that of the wild type).These mutants were all examined in a uniform-label experiment. Only four of the mutantsdisplayed a significant preferential inhibition of

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Fio. 6. Valuesfor protein (P), RNA (R), DNA (D),cell number (N), and cell viability (V) as a function ofthe time after a shift from 23 to 36 Cfor a culture ofts- mutant 236. All parameters were normalized to a

value of I at the time of the shift.

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FIG. 7. Valuesfor protein (P), RNA (R), DNA (D),cell number (N), and cell viability (V) as a function ofthe time after a shift from 23 to 36 Cfor a culture ofts- mutant 289. All parameters were normalized to avalue of I at the time of the shift.

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DNA synthesis after the shift to 36 C. Figure 5presents the data from a uniform-label experi-ment with one of these four mutants, number 172.The increase in DNA after the shift was muchless than that of RNA or protein. However,DNA synthesis proceeded at a slow, but sig-nificant, rate after the shift.

Mutations preferentially affecting cell division.Lesions in a number of cellular processes mightbe expected to result in a preferential inhibitionof the increase in cell number. Most entities whichcount as single particles in the Coulter counterconsist of a cell with a bud in various stages of

development. Nuclear division occurs early inbud formation (13), so most of these single par-ticles contain two nuclei. Thus, a lesion in an"early" process such as bud initiation or nucleardivision might result in an increase in cell numberof as much as 100%. A lesion in a "late" process,such as cytokinesis or the separation of the budfrom its parent cell, should result in little or noincrease in cell number. In either case, proteinsynthesis should continue for some time. A num-ber of mutants with protein values of greater than0.6 in the pulse experiment (Fig. 1) were examinedfor increases in turbidity and in cell number after

FiG. 8. Dark-field, phase-contrast photomicrographs of the wild type (A364A) and three mutants after incuba-tion for 12 hr at 36 C in YM-1 medium, all at a magnification of X 770. (a) A364A; (b) mutant 471; (c)mutant 310; (d) mutant 159.

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a shift to 36 C. Many mutants showed a parallelincrease in cell number and turbidity for net in-creases of up to 10-fold after the shift. However,some mutants displayed the anticipated pheno-type, with increases in cell number of less thantwofold and much larger increases in turbidity.These mutants were further tested in a uni-

form-label experiment. Figure 6 presents the re-sults of this experiment with mutant 236. The in-crease in cell number is consistent with our ex-pectations for a lesion in an "early" process ofcell division, showing a net increase of 70%. By10 hr after the shift, the cells are much larger thanthey were at the moment of the shift and displayno small buds. The content of protein, RNA, andDNA per cell has increased by factors of 6.0, 2.3,and 2.0, respectively. By 5 hr, there is a 10-foldloss of viability.

Figure 7 presents the results of a uniform-labelexperiment with mutant 289. This mutant dis-plays a pattern which is consistent with a lesion ina "late" process of cell division. The increase incell number stopped by 3 hr, but macromoleculesynthesis still continued at 6 hr. Cell number in-creased by only 20% after the shift, whereas allmacromolecules increased by large amounts. By6 hr after the shift, the culture contained 3.8 timesas much protein, 2.6 times as much RNA, and 2.3times as much DNA per cell as it did at the time ofthe shift. After an initial lag, there was a slow lossof viability.

Mutations affecting cell wall formation. Mu-tations in genes which function in cell wall for-mation would be expected to result in the lysis ofthe cell or in pronounced morphological changesafter extended periods at the nonpermissive tem-perature. Macromolecule synthesis should be rela-tively unaffected initially. Consistent with theseexpectations is the finding that few, if any, mu-tants with low values for protein in the pulse ex-periment showed observable morphological al-teration after long exposure to 36 C. However, anumber of mutants with large values for protein(greater than 0.5) in the pulse experiment showedextensive alterations of shape and appearance.Many of these were detected by microscopic ex-amination of agar plates on which the mutantshad been streaked at high temperature during theinitial isolationprocedure. Figures 8b, c, and d aredark-field, phase-contrast photomicrographs ofmutants 471, 310, and 159, respectively, after 12hr in liquid culture at 36 C. Figure 8a is of thewild-type strain A364A (ts+) growing normally at36 C. Before the shift to 36 C, the mutants weremorphologically similar to the wild type. Mutant159 underwent extensive lysis, and much extra-cellular debris was present in the culture fluid; abroken cell is evident in the Fig. 8d. Mutant 310

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cell number (N), and cell viability (V) as a function of'the time after a shiftfrom 23 to 36 Cfor a calture of ts-mutant 438. All parameters were normalized to a valueof I at the time of the shift.

displays elongated cells and a lack of separationbetween daughter cells; many of the cells in thisculture are lysing. Mutant 471 has undergone ex-tensive elongation and branching; few, if any, ofthe cells have undergone lysis.

Nonspecific lesions. Only about 10 to 20% ofthe mutants display patterns in a uniform-labelexperiment which are consistent with a lesionthat preferentially inhibits protein synthesis, RNAsynthesis, DNA synthesis, cell division, or cellwall formation. In the rest, a preferential effect isnot evident for any of the parameters measured.Mutant 438 is one such mutant (Fig. 9). Protein,RNA, DNA, and cell number all increased severalfold after the shift. However, the rate of increasein these parameters was more linear than ex-ponential.

DISCUSSIONIt was estimated, in the introduction, that a

haploid yeast cell contains enough DNA to pro-duce 13,000 different proteins. Watson estimatedthat the number of proteins needed for small-molecule metabolism in E. coli is 600 to 800 (12).It seems unlikely that the number of small mole-cules or their routes of metabolism would bevastly different in yeast and in E. coli, and we maytherefore assume a similar number in yeast. Ofcourse, a variety of proteins are involved inmacromolecule metabolism and in the structuralproteins of cells such as amino acid-activatingenzymes, ribosomal proteins, polymerases, andmethylating enzymes. However, the number ofidentified proteins in this category would total nomore than a few hundred. If we assume that all ofthe DNA in a yeast cell is active, in the sense thatit produces functional proteins, then it is evident

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HARTWELL

that the functions of the vast majority (i.e., about90%) of the gene products in one of the simplesteucaryotic cells are unknown. The work of Edgarand Lielausis (2) indicates that a large percent-age of the proteins of an organism can mutate toa temperature-sensitive form. It therefore seemsreasonable to conclude that most of the 400 mu-tants which were selected only on the basis of atemperature-sensitive phenotype have lesions inproteins whose function in cell physiology is en-tirely unknown. We hope that the continuedstudy of these mutants will lead to the identifica-tion of the roles played by the products of someof these genes in cellular processes.

In this context, it is interesting that about 80%of the mutants do not show a preferential inhibi-tion of any of the parameters measured after theshift to 36 C. Only the determination of furthervariables, such as oxygen uptake, active transport,and lipid metabolism, will provide a clue as to thesite of the lesion in these mutants.

In fact, of the 20% which do appear to bepreferentially affected in protein synthesis, RNAsynthesis, DNA synthesis, cell division, or cellwall formation, it is entirely possible that theirdefect is only indirectly related to the processwhich is inhibited. Further characterization ofthese mutants is continuing.

ACKNOWLEDGMENTSI thank Herschel Roman and Robert Mortimer for

allowing me to visit their laboratories and learn alittle yeast methodology. The competent and en-thusiastic assistance of Pamela Michaels and MaureenDalzell was much appreciated. A computer programfor data-processing was kindly written by TerryHutchison, and the computer time was provided bythe Computer Facility at the University of California,Irvine. Equipment for the photographic work wasgraciously made available by Donald Kaplan.

This investigation was supported by Public HealthService research grant GM 13304 from the NationalInstitute of General Medical Sciences.

LITERATURE CITED1. BOCK, A., AND F. C. NEIDHARDT. 1966. Isolation

of a mutant of Escherichia coli with a tempera-ture-sensitive fructose-1 ,6-diphosphate aldo-lase activity. J. Bacteriol. 92:464-469.

2. EDGAR, R. S., AND I. LIELAUSIS. 1964. Tempera-ture-sensitive mutants of bacteriophage T4D:their isolation and genetic characterization.Genetics 49:649-662.

3. EIDLIC, L., AND F. C. NEIDHARDT. 1965. Proteinand nucleic acid synthesis in two mutants ofEscherichia coli with temperature-sensitiveaminoacyl ribonucleic acid synthetases. J.Bacteriol. 89:706-711.

4. EpsTEIN, R. H., A. BOLLE, C. M. STEINBERG, E.KELLENBERGER, E. Boy DELA TOUR, R. CHEV-ALLEY, R. S. EDGAR, M. SUSMAN, G. H.DENHARDT, AND A. LIELAUSIS. 1963. Physio-logical studies of conditional lethal mutants ofbacteriophage T4D. Cold Spring HarborSymp. Quant. Biol. 28:375-394.

5. HALL, T. C., AND E. C. COCKING. 1965. Highefficiency liquid-scintillation counting of 14C.labelled material in aqueous solution and de-termination of specific activity of labelled pro-teins. Biochem. J. %:626-633.

6. HOROWITZ, N. H., AND U. LEUPOLD. 1951. Somerecent studies bearing on the one gene-oneenzyme hypothesis. Cold Spring Harbor Symp.Quant. Biol. 16:65-72.

7. KOHIYAMA, M., D. COUSIN, A. RYTER, AND F.JACOB. 1966. Mutants thermosensibles d'Esch-erichia coli K12. I. Isolement et characterisa-tion rapide. Ann. Inst. Pasteur 110:465-486.

8. MORTIMER, R. K., AND D. C. HAWTHORNE. 1966.Yeast genetics. Ann. Rev. Microbiol. 20:151-168.

9. NEIDHARDT, F. C. 1964. The regulation of RNAsynthesis in bacteria, p. 145-181. In J. N.Davidson and Waldo E. Cohn [ed.], Progressin nucleic acid research and molecular biology,vol. 3. Academic Press, Inc., New York.

10. OGUR, M., S. MINCKLER, G. LINDEGREN, ANDC. C. LINDEGREN. 1952. The nucleic acids in apolyploid series of Saccharomyces. Arch. Bio-chem. Biophys. 40:175-184.

11. POMPER, S., AND P. R. BURKHOLDER. 1949.Studies on the biochemical genetics of yeast.Proc. Natl. Acad. Sci. U.S. 35:456-464.

12. WATSON, J. D. 1965. Molecular biology of thegene. W. A. Benjamin, Inc., New York.

13. WILLIAMSON, D. H. 1966. Nuclear events in syn-chronously dividing yeast cultures, p. 81-101.In I. L. Cameron and G. M. Paiula [ed.], Cellsynchrony. Studies in biosynthetic regulation.Academic Press, Inc., New York.

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