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Copyright 8 1988 by the Genetics Society of America

Cycloheximide-Resistant Temperature-Sensitive Lethal Mutations of Saccharomyces cerevisiae

John H. McCusker' and James E. Haber Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02254

Manuscript received April 3, 1987 Revised copy accepted February 22, 1988

ABSTRACT We describe the isolation and preliminary characterization of a set of pleiotropic mutations

resistant to the minimum inhibitory concentration of cycloheximide and screened for ts (temperature- sensitive) growth. These mutations fall into 22 complementation groups of cycloheximide resistant ts lethal mutations (crl). None of the crl mutations appears to be allelic with previously isolated mutations. Fifteen of the CRL loci have been mapped. At the nonpermissive temperature (37"), these mutants arrest late in the cell cycle after several cell divisions. Half of these mutants are also unable to grow at very low temperatures (5"). Although mutants from all of the 22 complementation groups exhibit similar temperature-sensitive phenotypes, an extragenic suppressor of the ts lethality of crl3 does not relieve the ts lethality of most other crl mutants. A second suppressor mutation allows crll0, cr112, and cr114 to grow at 37" but does not suppress the ts lethality of the remaining crl mutants. We also describe two new methods for the enrichment of auxotrophic mutations from a wild-type yeast strain.

C YCLOHEXIMIDE is a potent inhibitor of eu- caryotic protein synthesis. The addition of a

high level of cycloheximide to a growing culture of Saccharomyces cerevisiae results in an immediate halt in protein synthesis and growth. When much lower levels of cycloheximide are added to growing cultures, the resulting decreased growth rate is entirely accounted for by an increase in the length of the GI phase of the cell cycle (HARTWELL and UNCER 1977). This perturbation of the cell cycle has been inter- preted to support a model for the control of the cell cycle where a critical cell size must be reached before the cell cycle can be initiated. The minimum inhibitory concentration of cycloheximide completely in- hibits growth but does not completely inhibit protein synthesis (SHILO, RIDDLE and PARDEE 1979). When the minimum inhibitory concentration of cycloheximide is used to inhibit growth, most of the population is present as single unbudded cells (J. H. MCCUSKER, unpublished observation). Mutations which would allow the cell to grow under such conditions of protein synthesis limitation have not previously been described.

When high levels (10 pg/ml) of cycloheximide are used to select resistant mutants one recovers only lesions in the ribosomal protein locus c ~ h 2 , the structural gene for ribosomal protein L29. The use of low levels of cycloheximide to select resistant mutations

' Present address: Department of Biological Chemistry, California Col- lege of Medicine, University of California, Irvine, California 92717.

Genetics 119 303-315 Uune, 1988).

results in a much higher frequency of spontaneous mutations. Three classes of low level cycloheximide resistant mutations might be expected: (1) mutations which affect the cell cycle, allowing the cells to grow under conditions where protein synthesis is limited; (2) mutations which reduce the effect of cycloheximide on protein synthesis; and (3) mutations which affect the ability of cycloheximide to enter the cell. Most of the mutations previously isolated as being resistant to low levels of cycloheximide are alleles of the permeability mutations cyh3 (now named p d r l ) (RANK, ROBERTSON and PHILLIPS 1975). The high frequency of permeability mutations may have ob- scured other mutations which affect protein synthesis which would confer only low level resistance to cycloheximide.

We have used the minimum inhibitory concentration of cycloheximide to select resistant mutants which we then screened for temperature sensitive lethality. These cycloheximide resistant ts lethal (crl) mutations comprise 22 complementation groups that are not allelic with previously described mutations, including all of the cycloheximide resistance loci tested. None of the crl mutations confers resistance to low concentrations of other protein synthesis in- hibitors (MCCUSKER and HABER 1988), indicating that they are not mutations that allow initiation of the cell cycle under conditions where protein synthesis is limited. All of the crl mutations exhibit a wide range of phenotypes that may result from alterations in the fidelity of protein synthesis.

304 J. H. McCusker and J. E. Haber

MATERIALS AND METHODS

Strains: All experiments were performed in the Y55 genetic background of Saccharomyces cereuiriae using isogenic Y55 strains. The genotype of Y55 is HO gal3 MALI SUCI. The following loci were introduced into the Y55 genetic background by backcrossing at least four times, to minimize any possible background effects, prior to being used in any experiments: rad2, cdc7, G A W , mall, hir2, gcdl, ho. The loci introduced into Y55 were used exclusively for mapping purposes.

Isolation of auxotrophic mutations in a wild-type strain: Y55 is a prototrophic homothallic strain which we use because of its very efficient and synchronous sporulation. To take advantage of this genetic background for experiments we first isolated auxotrophic mutations. Be- cause Y55 is homothallic and therefore diploid for most of its vegetative life cycle, it is not possible to use most of the previously developed techniques for mutant enrichment because they depend on the presence of particular mutations in a strain or require a stable haploid strain. We have developed two new enrichment techniques for auxotrophic mutations. The first technique uses ethidium bromide to promote the loss of mitochrondrial DNA in growing cells. The second technique uses 5-fluorouracil, which has the dual effect of either killing growing cells or making them petite. By using these two techniques we have been able to isolate hundreds of different auxotrophic mutations in one genetic background. This has allowed us to do all of our experiments using completely isogenic strains.

Mutant enrichment using ethidium bromide: This technique takes advantage of the fact that ethidium bromide promotes the loss of mitochondrial DNA. In our experi- ence, growing cells lose their mitochondrial DNA much more rapidly than nongrowing cells. Diploid cells were treated with EMS (ethylmethane sulfonate) (SHERMAN, FINK and LAWRENCE 1974) and then sporulated. Spores obtained from mutagenized diploid cells were laced in minimal medium at an initial concentration of 10 cells/ml. We have found that the low initial cell density is critical. After 5 hr at 30" to allow prototrophic spores to germinate, 50 pg/ml ethidium bromide were added. Cells were incubated at 30" for 24 hr, pelleted, washed and plated onto YEPEG to allow only the p+ survivors to grow. After the p+ colonies had grown up, these plates were replica plated to minimal media to screen for auxotrophic colonies. We frequently found that 50% of the p+ survivors were auxotrophs. This technique works equally well with vegetative cells. This procedure is a modification of one developed by D. PERL- MAN (personal communication).

Mutant enrichment using 5-fluorouracil: Spores obtained from EMS-mutagenized diploid cells were placed in minimal media (containing 2.5 gAiter urea in place of (NH4)2S04 as a nitrogen source) for 5 hr at 30°C at an initial concentration of lo6 cells/ml. The use of urea in place of (NH4)$S04 allows much more efficient killing. The reason for this is not known but may be due to increased uptake of 5-fluorouracil or decreased pool size of uracil. After 5 hr of incubation to allow prototrophic spores to germinate, 300 pg/ml 5-fluorouracil were added and the cells were shifted to 37" for 24 hr. The shift to 37" allows much more efficient killing. When cells from a culture treated with 5-fluorouracil at 30" and 37" are examined microscopically they are very different in appearance. Cells from the 30" culture are uniformly very large and contain a single large bud. These cells appear very similar to cells blocked in S phase. Cells from the 37" culture are also uniform in appearance, containing single

E

buds. However, the cells are small and very shriveled in appearance.

Cells were then pelleted, washed twice and plated onto minimal medium (containing 26 gAiter lactic acid in place of dextrose as a carbon source) with one nutritional sup- plement (e.g., minimal lactate + methionine). The use of defined media containing a nonfermentable carbon source allows optimal enrichment. These plates were incubated at 30" for 10- 14 days and were then replica plated to minimal media to screen for auxotrophs. Prototrophic survivors formed smaller colonies than auxotrophic survivors. In reconstruction experiments only 1/104 prototrophs sur- vived while starving auxotrophs were unaffected. Between 1% and 10% of the survivors growing on minimal lactate + tryptophan were tryptophan auxotrophs and greater than 50% of the survivors growing on minimal-lactate + methionine were methionine auxotrophs. This technique works equally well with vegetative cells and may be appli- cable to other fungi.

Genetic analysis: Y55 is a homothallic diploid strain; such strains can be readily manipulated for complementation testing or crossing to other strains by sporulating the diploid on a KAc plate and mixing asci on a YEPD plate. Diploids carrying complementing auxotrophic markers can then be isolated on media lacking the auxotrophic requirements of both parents. Complementation tests between temperature-sensitive strains were performed by replica plating the YEPD plate to another YEPD plate that was then incubated at 37" or 5". Complementation tests with previously isolated temperature-sensitive mutations were carried out using strains obtained from the Yeast Genetic Stock Center at Berkeley.

Media: Minimal medium contained (per liter) 6.7 g yeast nitrogen base without amino acids, 20 g dextrose and 20 g agar. Synthetic complete medium and dropout media lacking one amino acid or base were made as described by SHERMAN, FINK and LAWRENCE (1974). All of these media were adjusted to pH 5.5. Sporulation medium (KAc) con- sisted of (per liter) 20 g potassium acetate, 0.5 g dextrose, 2.2 g yeast extract, 20 g agar and all of the amino acids and bases as added to synthetic complete growth medium.

The basic rich medium used for most purposes was YEPD (per liter): 10 g yeast extract, 20 g bactopeptone, 20 g dextrose and 20 g agar. Fermentation markers were scored on YEPGAL or YEPMAL: 10 g yeast extract, 20 g bactopeptone, 20 g agar and 900 ml H20, after autoclaving 100 ml of 20% galactose or maltose was added. Respiratory competence was scored using YEPEG (per liter): 10 g yeast extract, 20 bactopeptone, 10 g succinic acid, 26 g glycerol, 20 g agar. After autoclaving 27.5 ml of 95% ethanol was added. All rich media were adjusted to pH 5.5.

Cycloheximide resistance was determined using YEPD + 10 pg/ml cycloheximide (high level) and YEPD + 1 kg/ ml cycloheximide (low level). The ani1 mutation was scored as being able to grow on YEPD + 50 pg/ml anisomycin; cry1 strains were tested on YEPD + 10 pg/ml cryptopleurine. The cyh3 mutation was scored on YEPEG + 4 mg/ml chloramphenicol + 0.01 pg/ml antimycin A. All drugs were obtained from Sigma and were added to media after autoclaving.

Scoring fermentation markers: YEPGAL and YEPMAL plates were scored anaerobically by generating a Con atmosphere from crushed dry ice. A Nalgene vacuum chamber was used as an anaerobic incubator. Crushed dry ice (400 g) was placed in a container at the bottom of the vacuum chamber with several paper towels acting as insu- lation and a large weight was placed on top of the vacuum chamber lid. A piece of tubing was run from the lid into a

crl Mutants of Yeast 305

flask of water as an air trap. A large glass desiccator may also be used.

DAPI staining: This procedure was a personal communication from C. HOLM. Cells were scraped off a plate, resuspended in 1% saline, pelleted and then resuspended in 2 ml of 3: 1 methanol: acetic acid for 30 min or longer. Cells were pelleted, washed twice with 1% saline and resuspended in 0.1 pg/ml DAPI (4',6-diamidino-2-phenyl- indole; Sigma) + 0.1 % o-phenylenediamine (Sigma).

Isolation of drug-resistant mutants: Resistant mutants were isolated by plating spores of Y55 strains on the appropriate media. Spontaneous mutations in cyh3 and cyhlO were isolated as being able to grow on YEPD + 1 pg/ml cycloheximide. UV induced mutations in c r y l , cyh2 and ani1 were isolated as being able to grow on YEPD + 10 pg/ml cryptopleurine, 10 pg/ml cycloheximide and 50 pg/ml anisomycin, respectively.

RESULTS

Selection of crl mutations: The minimum inhibitory concentration of cycloheximide on YEPD for the Y55 genetic background was determined to be 1 kg/ ml. Strain Y55 adel was subcloned on YEPD. Single colonies were picked, inoculated into a 1 ml culture of YEPD, grown overnight, washed twice and were then transferred to 5 ml of 1% KAc to sporulate. After 48 hr approximately lo7 colony forming units (spores and cells) were plated on YEPD + 1 pg/ml cycloheximide. These plates were incubated for 10 to 14 days at 25" to select for spontaneous cycloheximide resistance mutations. A single YEPD + 1 pg/ ml cycloheximide plate was used for each of 35 independent selections. The frequency of cycloheximide resistant mutations was approximately lop5.

A total of 2604 cycloheximide-resistant colonies were picked and purified by patching onto YEPD + 1 pg/ml cycloheximide at 25". Of the cycloheximide- resistant mutations selected in this fashion, greater than 90% were estimated to be in cyh3 ( p d r l ) , as evidenced by their cross-resistance to chloramphenicol and antimycin A in YEPEG (SAUNDERS and RANK 1982). As a further demonstration that most mutants were in cyh3,20 cycloheximide resistant mutants were picked at random and crossed with a leul strain ( q h 3 is tightly linked to leul ). Nineteen were tightly linked to leul and one was allelic with cyhl0. None of the q h 3 mutations were ts for growth.

All of the Cyh' colonies were screened for temperature sensitivity by replica plating to YEPD at 37" and incubating for 1 day. These plates were then re- replica plated to YEPD at 37" and scored for lack of growth after 1 day. All ts colonies were purified by subcloning on YEPD at 25" and were retested for ts lethality. A total of 186 cycloheximide resistant ts lethal (crl) colonies (approximately 7% of total cycloheximide-resistant colonies) were found. Since approximately 90% of the cycloheximide resistant mutations were in qh3, this means that greater than

TABLE 1

Number of alleles and range of cycloheximide resistance of the crl loci

ml locus No. of alleles Range of cycloheximide

resistance (kg/rnl)

1 7 1-5 3 16 2.5 4 2 2.5 5 7 1-2.5 6 12 1-2.5 7 2 2.5 8 1 2.5 9 17

10 11 2 2.5 12 4 13 1

1-2.5 1

14 3 15 3

1-2.5 1

16 1 1 17 4 18 4

1-2.5 1-5

19 1 1 20 2 21 2 2.5 22 1 5 23 1 2.5

1-2.5 13 1

1-2.5

A total of 106 different crl mutations were isolated in 22 complementation groups. The absence of cr12 is due to an early mistake in complementation testing. The level of cycloheximide resistance of all of the crl mutations was determined by their ability to grow on YEPD containing 1,2.5,5 or 10 p,g/ml of cycloheximide at 25". A range of resistance indicates variation between different alleles within a complementation group.

70% of the CYH3 + cycloheximide resistance mutations were ts.

All of the 186 crl mutations confer recessive ts growth. We were therefore able to place all of the mutations in complementation groups on the basis of their temperature sensitivity (see MATERIALS AND

METHODS). To ensure that all of the mutations were independent only a single mutation from a given complementation group was saved from each selection. The results of this are shown in Table 1; 106 different crl mutations are distributed among 22 crl complementation groups. Eighty mutants were dis- carded because more than one mutant from a given complementation group was isolated from a given selection, i e . , they were not necessarily independent isolates. Six crl loci are represented by one allele and five loci by two alleles. Given these results, we calculate from a Poisson distribution that there are approximately 60 genes which can mutate to confer a crl phenotype. If we subtract the 16 alleles of cr13 (which all appear to be identical, see below) from the total number of crl mutations, we calculate that there are approximately 50 CRL genes. Thus we may have isolated mutations in only a fraction of the genes which can mutate to confer a crl phenotype.


FIGLKL 1,-.4 c r l l Y / + diploid \vas dissecteci and the segregants were replica plated to YEPD + 1 pg/ml cycloheximide, incubated at 25' (A) and to YEPD, incubated at 37" (B).

Some complementation groups contain many more alleles than others. Over half of the crl mutations (58/106) are found in four complementation groups (0-13, 6 , 9, IO). We wanted to determine if these alleles resulted from different mutations in these genes. Each 0 - 1 3 , 6 , 9 and 10 allele (carrying an adel auxotrophic mutation) was crossed with a CRL +

strain carrying complementing auxotrophic markers to make derivatives carrying different auxotrophic mutations. Crosses were performed in every combi- nation between all the crl alleles within each complementation group. No intragenic complementation for temperature sensitivity was observed for any combi- nation of alleles in crl3, 6 , 9 or 10 . T o determine if different alleles within the same gene could recombine, diploids were selected and purified using complementing auxotrophic mutations, replica plated to YEPD at 25" (2 days), replica plated to sporulation media at 25" (3 days), replica plated to YEPD at 37" (1 day) and then re-replica plated to YEPD at 37" (1 day) to score for Ts+ intragenic recombinants. There was no intragenic recombination observed between any of the cr13 alleles. Since we could recover intragenic revertant mutations of cr13 (see below), the crl3 mutations are not large deletions or insertions. This indicates that the same allele must have been isolated in 16 independent selections and suggests that there is a mutational hotspot within C R W . We did recover Crl+ intragenic recombinants from many pairs of alleles from 0-16, 9 and IO. Intragenic recombination (data not shown) and differences in osmotic sensitivity or osmotic remediability indicate that at least half of the alleles of cr16, cr19 and crllO are different (data not shown).

Cosegregation of cycloheximide resistance and ts lethality: One member from each crl complementation group was crossed with a Crl+ strain to determine if ts growth and cycloheximide resistance cosegre-

gated. Figure 1 shows tetrads from a crll9 heteroz- ygote, tested for both cycloheximide resistance and ts lethality. In every tetrad cycloheximide resistance and ts lethality cosegregate, demonstrating that both phenotypes were the result of the same mutation. This is true for each of the 22 crl loci.

The crl mutations are not allelic with previously isolated mutations: We wished to determine if any of the crl mutations were allelic with previously isolated mutations. The first approach which was used was complementation testing with known ts mutations. All of the crl mutations were crossed with 26 cdc (cdcl, 2, 3, 4 , 5, 6, 7, 8, 9, 12, 13, 14, 15, 16, 18, 19, 20, 21 , 24 , 25, 27 , 28 , 29, 30, 31, 35) , 8 mu ( m a 3 , 4 , 5, 6, 7 , 8, 10, l l ) , 2 pt (prt2, 3 ) and 3 tsm ( t sml , 5 , 1 3 4 ) mutations and the resulting diploids tested for ts growth. All of the crl mutations comple- mented all of these ts mutations.

The second approach used was mapping of the crl mutations. The linkage of crl mutants to other chro- mosomal markers was determined by tetrad analysis after crossing each crl mutant to a series of strains carrying a large number of mutations (adel, ade2, ade4, ade6, adei, ade8, argl, arg4, arg6, canl, cryl, cyh2, cyhl0, his3, his4, his5, his6, his7, ho, h o d , hom3, leul, led?, lysl , lys2, lys5, lys9, MALI , met2, met3, met4, met5, metl3, metl4, thr4, trpl, trp4, trp5, tyrl, ural, ura2, ura3, ura4). A total of 15 crl mutations have been mapped. The tetrad data are shown in Table 2 and the map positions in Figure 2.

The map positions of four crl mutations suggested the possibility of allelism with other loci affecting protein synthesis or cell growth; however a more detailed analysis has shown that none of these crl mutants are actually allelic with the other loci. The most striking is the very tight linkage (<2 cM) between crll3 and gcd l . However, the two mutations complement and a replicating plasmid carrying GCDl (obtained from D. Hill) fails to complement cr l l3 . CRLl3 complementing activity is present on a DNA fragment immediately adjacent to GCDl (obtained from D. HILL); analysis of the C R L l 3 gene will be reported elsewhere.

Similarly, the crll7 mutation maps near both tcml, the structural gene for ribosomal protein gene L3 and ani l , a mutation in another gene conferring anisomycin resistance but not trichodermin resistance u. H. MCCUSKER, unpublished data). We have trans- formed a crll7 strain with plasmid pTCM (FRIED and WARNER 1981) carrying the TCMl gene, and have shown that crll7 is not allelic with tcml (J. H. McCvs- KER and J. ROSENBERC, unpublished observation). The crll7 mutation could be allelic to ani l .

The close linkage between crl9 and tyrl (1 cM, based on 237 tetrads) places this mutation near the omnipotent suppressor SUP46. However, crl9 does not appear to be allelic with SUP46, as SUP46 lies


TABLE 4

Genetic mapping crl mutations ~~ ~ ~ ~~ ~~

No. of tetrads

P N T FDS SDS mutant arm Gene pair ( 3 ) ( W (cM)

~~~~

CTl Chromosome %SDS/2 XS

crll 6R

crW

cr14

cr17

crl9

7L

5R

15R

2R

crll0 7R

crll I 15L crll2 7R

cr113 15R

crll 5 8R

crll6 4R

crll7 15R crll8 4L

hisZ-cr11 hkZ-ga17 crll -gal7 trpl-gal7 hisZ-trpl crl I 4rpl cen6-his2 cendcrll cen2-gal7 cen4-trpl

~ t l 3 - c y h 2 metl3-crl3 cyh2-crl3

crl3-trp5 ~ d 3 - ~ y h 3 flW-bul t$5-~yh3 trpfi-leul

cyh2-trp5

~ y h 3 - l e ~ l ura3-cr14 ura3-hod 0-14-horn3 urd-cen5 crl4-cen5

anil-crl7 ani1 -oh2 0-17-de2

lys2-cr19

crl9-tyrl crl9-his7 tyrl-his7 cdc28-cr19

crll0-ade6

argl -crlll

rad2-crll2 rad2-MALI crll 2-MALI

ade2-his3 ade2-~113 ade9-his3 ade9-0-113

ade2-gcdl

crll3-gcdl

arg4-crll5 arg4-cen8 crll5-cen8

lys2-tyrl

his3-0113

his3-g~dl

A 8 - t r p l ade8-crll6 crll6-trp4 crll7-ani1 cdc7-cr118 crll8-trpl

158 1

70 0 31 4 39 2 20 5 47 1 41 1 34 3 77 0 69 0 89 0 60 3 17 2 35 1

32 0 28 0 40 0

74 10 75 8

232 0 48 14 48 14 73 0

29 5 37 0

148 0 85 4 79 5 44 11 21 19

112 0 32 4 98 4 15 13 43 1 88 0 37 0

40 4 68 1 75 1

215 0 263 0 267 0

52

23 61 54 67 42 51 59 15 23 8

54 54 37

37 20

7

152 153

5 175 175

12 89 17

13 72 79

163 87 20 97

192 66 45

0 8

62 36 36 2 7 2

13.7 121 88 149 62 149 62 151 59 198 14

12.4 44.3 34.7 52.7 26.7 30.6 40.1

8.1 12.5 4.1

30.8 45.2 29.4

80 16 65 31

26.8 20.8

7.4 44.9 42.6

1 .o 54.6 54.6

7.1 48.4

15.7

4.0 29.8 33.4

52.5 Unlinked

7.6 45.5 36.7

Unlinked 28.6 0

8.9 34 12 25 22

40.6 20.0 18.7 0.9 1.3 0.4

13.5

12.8 1.8

13.4 1.8

12.2 47.2 35.7 58.8 26.9 31.2 42.0

7.9 12.3

31.4 48.4 29.9

8.3 16.1

27.1 20.7 7.2

48.0 45.1

61.8 61.8 6.9

52.6 15.5

30.3 34.3 58.5

7.4 48.8 38.0

29.0

13.0 23.4

8.7

42.6 19.9 18.6

(continued un p . 308)


TABLE 2-Continued

No. of tetrads CT1 Chromosome

Gene pair P N T FDS SDS mutant arm ( W ( W XP %SDSlP X I

( W

~dc7-trpl 260 0 9 trpl -gal3 268 0 0

1.7 0

crD1 10 crD1 -met3 149 0 12 3.7 met3-cenl0 149 12 3.7 crD1 -cenlO 161 0 0

crD2 13R crUZ-ade4 45 0 15 12.5 12.3

P , parental ditype; N , nonparental ditype; T , tetratype. FDS, first division segregation; SDS, second division segregation. %SDS/2 = (100(SDS)/(FDS + SDS))/2. xp = (3(N) + 1/2(T))/(P + N + T ) (PERKINS 1949). x, = (80.7(xp) - (0.883(x2))/83.3 - x,,) (MA and MORTIMER 1983). cM = centimorgan. x, correction was not done for xp < 5 cM or for %SDS. The centromere to gene recombination distances for crll were determined using the formula of WHITEHOUSE (1950). crZ10 is not linked to leu1 and is therefore distal to ade6 (data not shown). ani1 lies approximately 20 cM distal to tcml ; therefore crZl7 is about 20 cM distal to tcml. The order, relative to the centromere, of crll3- gcdl , crl17-ani1 and crl22-ade4 was not determined. The order cenl5-crlll -argl has been determined (data not shown).

approximately 6 cM from tyrl (MORTIMER and SCHILD 1985).

Finally, the map position of cr118 is very near lts4, a cold sensitive mutation (SINGH and MANNEY 1974). It is interesting to note that lts4 and cr118 show similar terminal phenotypes at their restrictive temperatures (J. H. MCCUSKER, unpublished observation, lts4 data not shown). Since cr118 has a cold-sensitive growth defect (see below) we were able to show that cr118 and Zts4 complement at 5". The map positions of the other Its mutations do not correspond to any of the crl mutations.

Level of cycloheximide resistance and growth in the presence of cycloheximide: The level of cycloheximide resistance was determined for all of the crl mutations by their ability to grow on YEPD + 1 pg/ ml, 2.5 pg/ml, 5 kg/ml and 10 pg/ml of cycloheximide (Table 1). All of the crl mutations show low level resistance; none can grow on YEPD + 10 pg/ml cycloheximide. Cycloheximide resistance was recessive in every case and ts lethality was not suppressed by cycloheximide.

Prototrophic derivatives of a single allele from each of the 22 crl loci were constructed to determine the growth rates of the crl mutants relative to the wild type in the presence and absence of cycloheximide (Table 3). All of the crl mutations show a decreased growth rate relative to the wild type in the absence of cycloheximide. (This growth difference was also apparent on plates when a crl/+ heterozygous diploid was dissected. ts growth and small colony size, indicating a slow growth rate and not respiratory defi- ciency, show 2 : 2 cosegregation). The cycloheximide resistance of the crl mutations is also clearly apparent. Irl the presence of cycloheximide, all of the crl mutations show a much faster growth rate than the wild type control. Almost all of the crl mutations show some inhibition of growth by cycloheximide.

An interesting phenomenon was noted when the

growth rates of the crl mutations were monitored. The growth curves of the crl mutations in the presence and absence of cycloheximide were similar until the cells began to enter stationary phase. In the absence of cycloheximide all of the crl mutations showed a normal growth curve as they entered stationary phase. In the presence of cycloheximide all of the crl mutations showed a much more pronounced reduction in growth rate at the end of logarithmic growth. An example of this is shown in Figure 3. The reason for this sharp leveling off of the growth rate is not known; however, it appears to reflect an increased cycloheximide sensitivity of both wild-type and cycloheximide-resistant strains (crl, cyh2, cyh3 or cyhl0) when grown on a nonfermentable carbon source (YEPEG) rather than on YEPD (data not shown).

Characterization of the ts growth phenotype: The ts lethal phenotype was examined using one allele from each of the crl loci. crl mutants were grown on a YEPD plate at 25" for 2-3 days to stationary phase and then replica plated to a fresh YEPD plate and incubated at the non-permissive temperature of 37". All the crl mutations show essentially the same phenotype: more than 80% of the cells are arrested with large buds and nearly all of these have a single nucleus, usually stuck in the neck between the mother and the daughter cell (Figure 4). When a log phase liquid culture is shifted to the nonpermissive temperature most of the crl mutants grew for several generations before arresting. Thus the crl mutations do not fit the criterion for cdc mutations. Moreover, in liquid culture, the arrested cells did not exhibit a uniform arrest phenotype (data not shown). The reason for the difference between the behavior of these cells on YEPD plates and in liquid culture is not understood.

We also asked if the presence of cycloheximide in the medium would allow the crl mutants to grow at

crl Mutants of Yeast

I

adel

i IOcM

II

cyhl cyhl0

lys2

SUP95 /YrI -lcr/91 ‘SUP96

his7

m

his9

leu2

cry1

thr9

Ip P

io

, cdc7

. trpl ‘9013

-m

ham2 SUP35

gcnz

p G - 1 trp4

ode8

ran I

uro3 7cn4

ham3 org6

his2

100121

C

” !

I

1

“ I

‘4

rde5

ys5

net13

:yh2

cr/Jl

trp5 , CYh3 -= Ipncr/l

+I

ode6

Icr//zl rod2

MALI

or99

H:

.- his5

-- his6

)

-- 1ysI

i

ur02

0r93

Jc./z/l met3

H

wol

met19 gcn3

HII xp.

t

0de4

met2

met9

1vj9

argl

tcm I [ c r / / l l

/

\ - ani1

cyhl

0de2

0de9 ’ his3

! - gcdl ;-m

FIGURE 2.-Map positions of 15 crl mutants are indicated on a map modified from that of MORTIMER and SCHILD (1985).


TABLE 3

Growth rates in the presence and absence of cycloheximide

Doubling time (hr)

Wl YEPD YEPD + 1 pg/ml cycloheximide

Crl +

1 3 4 5 6 7 8 9

10 1 1 12 13 14 15 16 17 18 19 20 21 22 23

2.5 3.3 2.9 3.3 4.0 3.3 2.7 3.0 3.1 2.8 4.0 4.0 2.6 2.9 2.9 2.5 3.5 4.1 2.8 2.9 3.3 5.1 3.3

>30.0 3.6 3.6 3.6 4.5 3.3 4.0 3.1 3.5 3.3 4.4 4.2 3.4 3.4 4.1 4.0 4.1 4.4 3.4 3.2 3.6 5.7 3.7

The doubling times of prototrophic CRL+ and isogenic crl strains was determined. Overnight YEPD cultures were diluted 1 : 50 into each of two Klett tubes. After 2 hr of growth at 25", cycloheximide was added to a concentration of 1 pg/ml and further hourly time points were taken. The doubling time of the wild-type control in the presence of cycloheximide is in excess of 30 hr. This doubling time is a minimum estimate, as we did not check for the appearance of spontaneous mutations to cycloheximide resistance within the population.

37". All of the crl mutants failed to grow on YEPD + 1 pg/ml cycloheximide at 37"C, ie., the crl mutants are not cycloheximide dependent at 37".

Many crl mutants are also cold sensitive: One allele from each of the 22 crl loci was tested for its ability to grow at temperatures below the permissive temperature of 25". All of the crl mutations grew at 18", but most grew slowly or failed to grow at 5" (Table 4). The cold sensitive (cs) growth phenotype displayed by the crl mutations on YEPD plates at 5" is not uniform for all of the crl mutations, unlike the ts phenotype at 37". Of 11 mutants that fail to grow at 5", only cr15 and crZ20 display a similar cell cycle arrest phenotype at both the high and the low restrictive temperature; both arrest as large budded cells. The other crl mutants arrest either at all points throughout the cell cycle (crl6, 14 ,21 ,22 and 23) , as normal sized unbudded cells (cr113, 16 and 18), or as large unbudded cells (crZ3) at the low non-permissive temperature.

Effect of the crZ mutations on sporulation: Most of the crl mutations cause aberrant sporulation at 25", the permissive temperature for growth. Prototrophic

1000 -

0 - c c 0 E IO

In L

O I

IO 20 30 40 50 Hours

+ Cycloheximide FIGURE 3."Growth of a wild-type strain in YEPD (0) and in

YEPD + 1 pg/ml cycloheximide (0) and a crl5 strain in YEPD (A) and in YEPD + 1 pg/ml cycloheximide (A).

homothallic crl strains were constructed by tetrad dissection and their sporulation efficiency was compared with that of Y55 (the homothallic prototrophic parent strain for all of this work) at 25°C. The results are shown in Table 4. It is obvious that many of the crl mutations significantly reduce the ability to sporulate, the production of four spored asci being affected most severely. In addition most of the crl mutations cause the production of spores which are either aberrant in shape or have poorly defined spore walls. These defects are recessive and are not due to an inability to respire since all of the crl mutants are p + . The sporulation defects caused by the crl mutations seem to occur late in meiosis and therefore are not due to an inability to initiate meiosis. This con- clusion is bolstered by DAPI staining of crl diploids sporulated at 25". The majority of the cells show no evidence of ascus formation but are tetranucleate (data not shown).

The ability of crl homozygous diploids to produce viable spores and to undergo recombination was


FIc.rKt 4.--:\rrest o f t w o c d mutants at 97"' -1'wo strains. r7 /3 (A) and cr113 (B) were grown on plates overnight at 37" and stained with DAPI. Most of the cells appear to be blocked prior to mitosis, with a single nucleus near the neck of a large, budded cell.

compared with crll+ heterozygous diploids. The small number of four-spored asci produced by most of the crl homozygotes was sufficient to permit analysis of recombination (with the exception of crl22 and 23) . Many of the crl mutants were extremely difficult to dissect because the spores within the ascus were very difficult to separate. Several of the crl homozygous diploids show evidence of small reduc- tions in spore viability (data not shown). All of the crl homozygous diploids tested show evidence of recombination between his6 and lysl (two markers approximately 50 cM apart on chromosome ZX). The numbers are too small to determine if any of the crl mutations have a quantitative effect on recombination, but none is Rec-.

The crZ mutations have many other phenotypes in common: In addition to their cycloheximide resistance, and inability to grow at 37" and (in half the cases) at 5", the crl mutants also have a number of other striking phenotypes. They are unable to arrest at the GI stage of the cell cycle under some starvation conditions and they are hypersensitive to amino acid analogs and to 3-aminotriazole. In addition they are

TABLE 4

Additional phenotypes of crl mutants

Sporulation efficiency at 25''

Percent Total

crl locus 4-spored

Growth at 5'" asci percent

sporulation

Crl+ + 76.6 87.8 1 - 1.6 10.9 3 - 67..5 83.1 4 + 9.5 30.5 5 - 0.5 7.7 6 - 9.3 43.4 7 + 18.5 45.6 8 SD 7.1 42.2 9 + 38.7 70.8

10 -c 25.0 51.8 1 1 '' 16.1 44.0 12 t 33.2 69.6 13 - 26.9 39.9 14 - 40.8 69.3 I5 + 9.4 38.8 16 - 52.5 67.9 17 ? 2.7 13.8 18 - 29.0 47.9 19 t 53.1 80.5 20 - 14.0 48.2 21 - 16.4 50.9 22 - 10.8 40.3 23 - 3.7 11.7

One allele from each of the crl loci were grown on a YEPD plate at 25" and then replica plated to another YEPD plate incubated at 5' for approximately two weeks. This plate was scored then re-replica plated to another YEPD plate which was incubated for 2 weeks at 5" and scored. SD = not determined.

Prototrophic diploid strains homozygous for a crl mutation were pregrown on YEPD at 25" for 3 days, replica plated to KAc and sporulated for 4 days at 25". A CRL' strain treated in an identical fashion was included as a control. A minimum of 500 cells were counted to determine sporulation efficiency. Many of the wl mutants produced cells containing spores which were misshapen or poorly defined. These were counted as sporulated cells.

sensitive to the aminoglycoside hygromycin B that causes translational misreading. The mutants also suppress at least one nonsense mutation. All of these phenotypes are described in detail in the accompa- nying paper (MCCUSKER and HABER 1988).

Suppressors of crZ3 mutations: In an attempt to define how the crl mutations confer ts lethality we decided to isolate mutations which suppress the ts lethality of one crl locus and to then determine if this suppressor would also relieve the ts lethality of other crl mutants.

The crl3 complementation group contains 16 independently isolated mutations all of which fail to recombine with each other. One interpretation of this result would be that there was a mutational hot spot in the cr13 gene or that there was a high probability of deletion formation at the cr13 locus as has been previously described for the sup4-qcl region (ROTHSTEIN 1979). The fact that ts lethal pe t18 mu-


tations are deletions,suggested the possibility that the mutations in cr13 could be deletions (ToH-E and SAHASHI 1985). The isolation of Ts+ revertants of cr13 would serve the dual purpose of allowing us to distinguish between a deletion and a mutational hotspot and providing us with either intra- or extragenic suppressors of 0-13.

T s + revertants of crl3-1 and crl3-2 were isolated by plating homozygous diploids on YEPD at 37". A diploid was used to allow the isolation of suppressors which were dominant in terms of their ability to suppress crl3 but conferred recessive lethality. A total of 17 spontaneous Ts+ revertants were isolated. These Ts+ revertants were dissected and the viability and segregation of ts lethality was determined; none were recessive lethal suppressors. In several cases the T s + revertants were found to be homozygous T s + / Ts + indicating that recombination had occurred. This indicates that the use of a diploid did not preclude the isolation of recessive suppressors.

In each case a single T s + segregant was then crossed with a Crl+ strain. The resulting diploid was then sporulated and dissected and the segregants were tested for ts lethality, cycloheximide resistance, hygromycin B sensitivity and amino acid analog sensitivity. Out of 17 Ts+ revertants tested in this fashion 15 proved to be complete revertants to wild type. This result combined with the fact that there is no recombination between any of the 16 independently isolated crl3 mutations suggests that there is a mutational hotspot in the cr13 gene and that the crl3 phenotype is due to a point mutation, not a deletion. The characterization of the other two T s + revertants is described below.

An intragenic suppressor of crW: An intragenic suppressor of cr13 was isolated, designated crW-2-8. This mutation is a dominant suppressor of the ts lethality of 0-13, as a cr13lcr13-2-8 diploid is Ts+. When a 1x13-2-8ICRL + diploid is dissected 4 Ts+ : 0 Ts- segregation is observed. There were no ts segregants recovered in several hundred tetrads, indicating very tight linkage of the original mutant allele and its second site suppressor mutation. How- ever, the other phenotypes of cr13 continued to show 2 : 2 segregation cycloheximide resistance (but the cycloheximide resistance is weaker than the original cr13-2), cold-sensitive lethality, hygromycin B sensitivity, and amino acid analog sensitivity. In addition crl3-2-8 reduces the suppression of met3-2 (a UAA mutation which is suppressed by all of the crl mutations and by hygromycin B; cf. Figure 3, MCCUSKER and HABER 1988).

An extragenic suppressor of crW: The second suppressor of cr13 proved to be an extragenic suppressor which we have named SCLl-1 (Suppressor of Crl ts Lethality). SCLl-1 is a dominant suppressor of the ts lethality of 013. We have mapped SCLl-1 to the left

TABLE 5

Genetic mapping of SCLI-1

Chromosome arm 7L and Gene pair: P N T xp ( W xs (cM)

trp5-cyh3 284 1 63 9.9 9.7 trp5-leul 276 1 74 11.4 11.2 cyh3-leu1 341 0 10 1.4 trp5-SCLI-1 273 1 73 11.4 11.2

SCLI-I-leu1 350 0 1 0.1

trp5-leu1 100 0 31 11.8 11.6 trp5-SCLI-I 100 0 30 11.5 11.3 SCLl - I -leu1 130 0 1 0.4

trp5-SCLI-1 663 2 206 12.5 12.3 trp5-leu1 660 2 225 13.3 13.1 trp5-pmal 642 2 243 14.4 14.2 SCLl-1-leu1 860 0 13 0.7 leul -pml 869 0 20 1.1 SCLI-I-pmal 841 0 32 1.8

~yh3-SCLI-I 338 0 9 1.3

P , parental ditype. N , nonparental ditype; T , tetratype. xp = (3(N) + 1/2(T))/(P + N + T ) (PERKINS 1949). xe = (80.7(xp) - (0.883(x;))/(83.3 - xp) (MA and MORTIMER 1983). cM = centimorgan. xe correction was not done for xp < 5 cM. This table presents the mapping data from three different crosses. The gene order is trp5-cyh3-SCLI-I-leuI -pmal-cen7. Since SCLI-I and pmul have different map positions they are in different genes.

arm of chromosome 7, less than one centimorgan distal to leul (Table 5 ) . This map position does not correspond to any previously mapped mutation (MORTIMER and SCHILD 1985). Four factor crosses involving trp5, SCLl-1, leul and F a 1 have shown that SCLl-1 is not allelic with the plasma membrane ATPase (pmal) (Table 5 ) . We felt that it was important to show this because we have recently shown that mutations in pmal confer a hygromycin B resistance phenotype (MCCUSKER, PERLIN and HABER 1987). The SCLl-1 mutation does not appear to have any strong phenotype in the absence of cr13 (it is not cold sensitive, high temperature sensitive or more or less resistant to cycloheximide) although there is a slight increased sensitivity to hygromycin B and anisomycin. SCLl-1 suppresses the ts lethality of all of the alleles of 0-13, a result that is is not surprising if all of the cr13 mutants are identical. SCLl-1 does not interact in any obvious way with cr13-2-8 (data not shown).

The effect of SCLl-1 on the other phenotypes of cr13 was determined. SCLl-1 has no effect on the cold sensitivity, hygromycin B hypersensitivity or amino acid analog sensitivity of cr13. The SCLl-1 mutation does decrease the cycloheximide resistance of 0-13, but does not restore wild type sensitivity. In addition the SCLl-1 mutation reduces the suppression of metl3-2 by cr13 (cf. Figure 3, MCCUSKER and HABER 1988).

We have also examined effect of SCLl-1 on mu-


tations in the other crl loci. A strain with the genotype met13-2 leul SCLl-1 was crossed with ura3 derivatives of all of the other crl loci. The leul marker allowed us to follow the segregation of the tightly linked SCLl-1. Only crll8 and cr119 show any suppression of ts lethality and in these two cases the suppression is only partial: SCLl-1 cr118 and SCLl-1 cr119 double mutant strains grow at 34" (the single mutants do not grow at this temperature) but do not grow at 37". Most other crl mutants are either unaffected by SCLl- 1 . In a few cases, SCLl-1 crl double mutants show increased ts lethality and increased suppression of the nonsense mutation, metl3-2. SCLl-1 does not interact in any obvious fashion with three different alleles of cyh2 or with cry1 or anil mutations (data not shown).

A suppressor of crZl0, crZ12 and crZ14: All of the work we have described was carried out entirely in the Y55 strain background. We were interested to know if the crl mutants exhibited similar behavior in other commonly used strains. One allele from each of the 22 crl loci was crossed with strain A701 (MATa lys2) that was derived from the commonly used S288c genetic background. In each case, the cycloheximide resistance of the crl mutant failed to segregate 2: 2. The small fraction of cycloheximide resistant segregants suggests that there must be more than two genes in the S288c genetic background capable of suppressing the cycloheximide resistance of the crl mutations. This interference with scoring the cycloheximide resistance of crl most likely results from alterations in permeability of low concentrations of the drug rather than in a suppression of the crl mutants themselves, because for 19 of the 22 crl loci only the cycloheximide resistance phenotype ap- peared to be affected. Temperature-sensitive growth segregated 2 : 2; furthermore, two other phenotypes of the crl mutants, hygromycin B hypersensitivity and amino acid analog hypersensitivity, were unaffected and could be scored as easily as in the original Y55 background.

Three of the crl mutations (crllO, 12 and 1 4 ) failed to show 2:2 segregation for ts lethality in these crosses, implying that there was a suppressor of ts lethality as well in strain S288C. Since both crll0 and 12 map on the right arm of chromosome 7 we considered the possibility that strain A701 carried a second copy of all or part of chromosome 7 . We found that four chromosome 7 markers (leul, ade6, rad2 and MAL1 ) all showed 2: 2 segregation when when Y55 strains carrying these markers were crossed with A701. In addition we found that cr114 is not linked to either crll0 or cr112. The suppressor is similar to SCLl-I in that it fails to suppress the hygromycin B sensitivity and amino acid analog sensitivity of the three crl mutants. We conclude that there is a suppressor of temperature sensitivity of

crllO, 12 and 14 in strain A701. Diploids homozygous for crll0 and heterozygous for this suppressor fail to grow at 37", suggesting that this suppressor is recessive. We have designated it scL2. scL2 cannot be allelic with SCLl because unlike SCLl the scL2 mutation is not centromere linked (data not shown).

DISCUSSION

The crl mutations appear to be a previously undescribed class of mutations. They are not allelic with any of the 39 previously isolated ts mutations we have tested. In addition 15 of the 22 crl loci have been mapped. Although two of the crl mutants mapped to positions that suggested possible allelism with previously mapped mutations (lts4 and cr118, gcdl and crll3) both pairs of mutations have been shown to be not allelic. The CRLl3 gene has been cloned by its proximity to GCDl u. H. MCCUSKER, D. HILL and J. E. HABER, unpublished data). In addition the crll7 mutation has been shown to map very near anil , a previously undescribed drug resistance mutation which confers resistance to anisomycin and hypersensitivity to trichothecine antibiotics u. MCCUSKER, unpublished observation). We have not determined if crll7 is allelic with anil .

Why have mutations such as the crl mutations not been described previously? First, we have used the minimum inhibitory concentration of cycloheximide to isolate the crl mutations. Most previous work has involved the use of much higher concentrations of cycloheximide to avoid the isolation of permeability mutants. We estimate that approximately 90% of the spontaneous cycloheximide resistance mutations which we selected were in cyh3. The use of higher concentrations of cycloheximide would prevent the selection of crl mutations and the presence of so many cyh3 mutations would obscure the presence of crl mutations.

Second, there is the question of genetic background. The presence of one or more genetic differences in strain S288c compared to Y55 would have precluded the isolation of the crl mutations in that background, even though the temperature-sensitive phenotype of 19 of the 22 crl complementation groups is faithfully expressed when crossed with a strain from the S288c background. Third, although crl mutants arrest at 37" as large budded cells, the crl mutations do not fit the criteria for cell division cycle (cdc) mutations; arrest occurred only after several rounds of cell division. Furthermore, a log phase liquid population of a crl mutant strain does not arrest as a uniform population when shifted to the non-permissive temperature. Given this result, it is not surprising that none of the crl mutations are allelic, either by complementation testing or by genetic mapping, with previously isolated cdc mutations.


It is possible that the crl mutations are allelic with previously described I t s (low temperature sensitive) mutations (SINGH and MANNEY 1974) or streptomycin and neomycin hypersensitive mutations (BAYLISS and VINOPAL 1971; BAYLISS and INGRAHAM 1974). The I t s mutations show altered sensitivity to cycloheximide; no mention was made of high temperature lethality. We have found that the lts4 mutant arrests (5") with a phenotype similar to that of the crl mutations (37") u. H. MCCUSKER, unpublished observation). The crll8 mutation maps in the vicinity of lts4, but these two mutations complement at 5". None of the crl mutations can be allelic with ltsl or Its3 because they do not map in those regions. It is not known if any of the Its mutations have any effect on ribosome assembly or function. The streptomycin sensitive mutations (BAYLISS and INGRAHAM 1974; BAYLISS and VINOPAL 1971) have been shown to affect ribosome assembly and function at their non-permissive temperature. No description of the lethal phenotype is given; furthermore, no mapping data is available, and complementation tests with the crE mutations have not been done.

Many of the crl mutations also confer a cold- sensitive growth phenotype. We are not aware of any previous report of mutants exhibiting both high (37") and low ( S O ) temperature sensitivity, though most mutants have not been tested for such a 5"-sensitive phenotype. This result suggests that the practice of refrigerating strains for even short-term storage may not be wise unless the strain is known not to be cold sensitive.

How do the crl mutations confer cycloheximide resistance and ts lethality? One possibility is that the absence of a functional CRL gene product uncovers a basic temperature-sensitive process in yeast. Mu- tations in p e t l 8 , which are temperature-sensitive lethal, have been shown to be complete deletions (ToH-E and SAHASHI 1985). Apparently the wild-type gene product is required only at high temperatures. This may prove to be true for the crl mutations as well, although the pleiotropic effects, at 25", of crl mutants on cycloheximide resistance, hygromycin B hypersensitivity, suppression of a hygromycin B suppressible auxotrophic mutation and aberrant G1 arrest and starvation responses (MCCUSKER and HABER 1988) argue that the CRL genes are important at all temperatures.

The crl mutations have two other phenotypes in common which we feel are relevant to the cause of ts lethality: all of the crl mutations are hypersensitive to hygromycin B (an aminoglycoside antibiotic which stimulates translational misreading) and all of the crl mutations suppress a nonsense mutation which is suppressible by hygromycin B (MCCUSKER and HABER 1988). There are three additional observations which

we feel are relevant to the cause of the ts lethality: (1) two different suppressors of the ts growth defect of cr13 also reduce the suppression of met13-2 by cr13, (2) certain combinations of SCLl -I crl double mutants show increased ts lethality and increased suppression of met13-2, and (3) several hygromycin B suppressible nonsense mutations show considerably more growth at 37" than at 25" in a CRL,+ strain (in the absence of hygromycin B). We suggest that the ts lethal phenotype of the crl mutations is due to increased translational misreading, which becomes more severe (even for wild type cells) at the nonpermissive temperature.

We are grateful for the suggestions and comments of GERALD FINK, MICHAEL ROSBASH and MICHAEL WORMINGTON. J.H.M. was supported as a Trainee of U.S. Public Health Service Training Grant in Genetics GM70122. This research was supported by National Science Foundation grant DCB8409086 and by National Institutes of Health grant GM20056.

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