Current Genetics (1981) 4:205 214 © Springer-Verlag 1981
New Mutations in the Yeast Saccharomyces cerevisiae Affecting Completion of "Start" D. P. Bedard, G. C. Johnston, and R. A. Singer Departments of Microbiology, Medicine and Biochemistry, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, B3H 4H7 Canada
Summary. Here we report the isolation of several new temperature-sensitive mutations which cause cells of the yeastS accharomyces cerevisiae to arrest in the G1 period of the cell cycle. Four different selection schemes were employed. The cell division cycle (cdc) mutations define five new complementation groups. At non-permissive temperatures, strains bearing these new cdc mutations arrested in G1 within one cell division cycle. By orderof-function mapping, cells of each population were found to be arrested at "start", the regulatory point in the G1 period of yeast. Mutations were grouped into two categories by the abilities of mutant strains to continue extensive macromolecular synthesis and to conjugate with cells of the opposite mating type. For strains with mutations in one category, shift to the non-permissive temperature caused an abrupt decrease in the rates of labelling of protein and RNA, and rendered cells unable to mate efficiently. For strains with mutations in the second category, cells continued to grow and mating ability was not significantly impaired. Each selection scheme was also designed to isolate mutations which specifically affect the ability of cells to reinitiate the cell cycle from stationary phase. This was done to test the hypothesis that stationary phase cells are in a unique developmental state referred to as GO. No mutations specific for resumption of growth from stationary phase were isolated. Key words: Cellcycle - G1 period
cdc genes
Introduction Cell division is an orderly sequence of events in which the genetic material is replicated and subsequently segre-
Offprint requests to. G. C. Johnston
gated along with cellular constituents into progeny cells. In eukaryotes the regulation of this complex process occurs within the G1 period, between the completion of nuclear division and the onset of DNA replication (S phase). One area of interest concerns the nature of this G1 regulation. For these studies the budding yeast Saccharomyces cerevisiae has proved a useful model system. In particular, many mutants defective in the yeast cell division cycle (cdc mutants) have been isolated and characterized by Hartwell and his colleagues (Hartwell et al. 1973, 1974). Mutations in one cdc gene (cde28) allowed Hartwell to define a major regulatory step in G1, which has been referred to as "start" (Hartwell et al. 1974). Cells arrested in G1 either by mutation or by the presence of one of the yeast mating pheromones are at "start". Conversely, the completion of "start" heralds the onset of a new cell division sequence. Thus the regulation of cell division is thought to occur at the "start" event. In addition to the arrest caused by cdc28 mutations or mating pheromones, another form of G1 arrest is represented by cells in stationary phase. Like most other eukaryotes, yeast cells when deprived of required nutrients complete cell divisions and arrest in G1 (Hartwell et al. 1974). Such Gl-arrested cells can be shown by orderof-function mapping experim'ents to be either at or prior to the "start" event. One fruitful approach to illuminate the regulation of cell division has been to isolate mutants conditionally defective in the ability to initiate new cell division sequences. Recently, Reed (1980) reported an elegant approach which resulted in the isolation of mutations in three new cdc genes, all of which, like mutations in cdc28, cause defects in the ability to progress from mitosis to the next S phase. All such mutants were shown to be defective in the ability to execute the "start" event, and were by order-of-function mapping criteria arrested O172-8083/81/0004/0205/$02.00
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D.P. Bedard et al. : New Mutations in the Yeast Saccharomyces cerevisiae
at "start". As Reed pointed out, "start" mutants may be grouped into two phenotypic classes. He defined Class I mutants as those mutants which arrest at "start" but continue to grow and also retain the ability to conjugate. (Conjugation is a property of growing ceils in the G1 period [Reid and Hartwell 1977].) In contrast, Class II mutants arrest at "start" with phenotypes similar to those of cells starved for some required nutrient. Presumably Class II mutants would be better described as stationary phase-like mutarlts. By virtue of Reed's selection scheme, only Class I mutants were selected in those experiments. Thus mutations in at least four Class I "start" genes have been previously described, which all affect a single regulatory step ("start") in the cell cycle. Here we have used a number of selection strategies to isolate new G1 mutants. Different rationales were employed to test the hypothesis that "start" is the sole point of regulation for initiation of the cell cycle. In this way, several new Class I and Class II type "start" mutations were isolated and characterized. We also wished to test the hypothesis that unique gene functions might exist for resumption of growth from stationary phase. It has been suggested (by analogy with proposals for quiescent mammalian cells) that stationary phase yeast cells are actually in a unique developmental state referred to as GO (Pinon 1978). The existence of such a unique developmental state has been inferred from observations that stationary phase cells are by several criteria in a physiological state different from that of cells arrested at "start" by mutation or mating pheromones (for review see Bedard et el. 1981). However, no direct evidence has been reported verifying the existence of such a unique development state. If indeed GO represents a unique gene-mediated developmental state, as well as a unique physiological state, then GO mutants should exist. Some such mutants should be conditionally defective for resumption of growth from stationary phase, b u t should not be impaired once cell cycles have been initiated. Mutations conferring this GO phenotype were sought by four different procedures; none were found.
Course. Strains SR653-1 (cdc28-4)~ SR661-2 (cdc36-16), SR672-3 (cdc37-1) and SR665-1 (cdc39-1) were obtained from S. Reed (1980), and strains BR205-2A (cdc25-1), H185-3-4 (cdc28-1), El7 (cdc33-1) and BR214-4A (cdc35-1) were obtained from Hartwell. To determine centromere linkage, strain X2928-3D-1C (MATe adel gall trpl ura3 his6 leul metl4) was employed and was obtained from the Yeast Genetic Stock Center, Berkeley, California. The diploid strain AG1-7 has been described (Johnston et al. 1977b). Mutant strains isolated or constructed for this study were the following: ID-1 (MATe adel leul cdc60-1); ID-2 (MATa aro cdc61); GD-2-1 (MATe his6 ural cdc61-1); SG44-1 (MATe his6 ural cdc63-1); initial isolates of the cdc4 survival procedure were $7 (MATe leu2 cdc62-1), $24 (MATe cdc62-1), $33 (MATe cdc62-1), $44 (MATe ural cdc63-1), and S104 (MATe leu2 cdc64-1). Several of the above strains were crossed with strains GR2 or GR1 (MATe his6 ural) to obtain the following temperature-sensitive segregants of the opposite mating type for complementation studies: XS33 (MATe cdc62), XD-2 (MATe cdc61), XD-1 (MATa cdc60 ), XS24 (MATe cdc6 2), XS44 (MATe cdc63) and XS104 (MATe cdc64). Nutritional requirements for these latter strains were not determined.
Media The enriched liquid and solid media (YM-1 and YEPD respectively) have been described by Hartwell (1967). The liquid minimal medium (YNB) and solid synthetic medium have also been previously described (Johnston et al. 1977a). The mating pheromone e-factor was prepared according to the method of Biicking-Throm et al. (1973).
Mutagenesis For mutagenesis (Fink 1970), cells from 10 ml portions of stationary phase cultures were collected by centrifugation, washed once with sterile H20 and suspended in 10 ml of sterile H20; 50 t~l of ethylmethanesulphonate (EMS) (Sigma Chemical Corp.) was then added and incubation was continued at room temperature for periods of time, determined in preliminary experiments, which resulted in 50% survival, or 10% survivalfor the non-selective screening procedure. Mutagenesis was terminated by the addition of 5 volumes of 5% sodium thiosulphate. Cells were then washed 3 times by centrifugation, each time with 5 volumes of 5% sodium thiosulphate. To ensure the isolation of strains resuiting from independent mutational events, one drop portions of the mutagenized and washed cell suspensions were added to tubes containing fresh YM-1 medium and grown at room temperature to stationary phase. These populations of cells were used to start each isolation procedure.
Materials and Methods Strains
Isolation Procedures
The following strains were employed in the selection procedures reported here. Strain 18032 (MATe adel ade2 leu2 ural his7 lys2 tyrl gall cdc7-3) was obtained from L. H. Hartwell; strain GJ301-47 (MATe his7 ural cdc4-3 cdc7-4) was constructed by crosses between strain GR2 (Johnston et al. 1977b), and cdc mutant strains H135-1-1 (cdc4) and H201-14-4 (cdc7), also obtained from Hartwell; strain MC-6A (MATe/nol-3 ino4-8) was provided by S. Henry; strain A4840A (MATe ade2 his5), strain 4116E (MATe leu2) and strain D273-11A (MATe adel hisl) were provided through the Cold Spring Harbor Yeast Genetics
For each procedure the initial step involved exposure of stationary phase Gl-arrested (or GO) cells to the non-permissive temperature under conditions that would favour the survival of cells blocked in G1 (or GO). The use of stationary phase cells as starting populations was a feature to allow isolation of both G1 and GO mutants. Inositol-less Death Procedure. Stationary phase cells from 500 independent, mutagenized cultures of strain MC-6A, an inositol auxotroph, were placed at the non-permissive temperature
D. P. Bedard et al.: New Mutations in the Yeast Saeeharomyces eerevisiae
207
(36 °C) for 60 min. These prewarmed cells were then diluted into prewarmed YNB medium without inositol and incubated at 36 °C for a period (30 h, as determined by preliminary experiment) sufficient to allow extensive cell death. Survivors were plated on YEPD medium and the resultant colonies were tested for temperature sensitivity by replica-plating. One temperature-sensitive clone was isolated from each independent culture. For each of the 500 independently derived, temperature-sensitive clones the terminal phenotype was determined in two ways. In one method, stationary phase ceils were suspended in fresh YM-1 medium at 36 °C and incubated overnight before examination;in the other method, exponential phase cultures growing at 23 °C were shifted to 36 °C and examined after further incubation.
Non-selective Screen. Mutagenized cells of strain GR2 were spread
Mating Procedure. The mating procedure was adapted from the
Measurement of Cellular Parameters
method described by Hartwell (1973). Stationary phase ceils from 280 independent, mutagenized cultures of strain 18032 (cdeT) were incubated at the non-permissive temperature (37 oC) for 30 rain and diluted into fresh YM-1 medium prewarmed to 37 °C. After 12 h incubation, when more than 99.9% of the cells had become arrested at the cde7 step and were refractory to mating (as determined in preliminary experiments), each culture was mixed with an equal number of cells of the opposite mating type (strain A4840A) bearing complementary auxotrophic markers. These cell mixtures were collected on filters (Milfipore Corp., Bedford, Mass.), and the filters were placed on YEPD plates and incubated for 3 h at 37 °C. Conjugation was interrupted after 3 h by suspending the cells in ice-cold 1.0 M Sorbitol and subjecting the cell suspension to sonic oscillation for 5 s with a Branson Sonifier (Heat Systems - Ultrasonics, Inc., Plainview, N.Y.). Diploid clones were selected on minimal medium (SD) plates containing adenine. One diploid clone was picked from each independent culture and sporulated; haploid clones were then recovered by the random spore technique. For complementation analysis, sixteen spore clones from each sporulated diploid were mated on solid medium with edc7 mutant tester strains. Eighty of the 280 derived diploid strains segregated haploid clones bearing temperature-sensitive mutations which complemented the ede7 mutation. The terminal phenotype of each new temperature-sensitive clone was determined in liquid cultures as above.
edc4 Survival Procedure. Stationary phase cells from one mutagenized culture of strain GJ301-47 (ede4 ede7) were used. The ede7 mutation was included to decrease reversion frequencies of the temperature-sensitive phenotype. These cells were preincubated at 37 °C for 30 min, diluted into fresh prewarmedr(37 °C) YM-1 medium and incubated for 24 h. (Preliminary experiments indicated that by 24 h cell viability was decreased by a factor of 103.) The surviving cells were suspended in fresh medium and grown to stationary phase at the permissive temperature. This procedure was repeated once and the survivors were plated on YEPD medium to give 100-200 colonies per plate. To test for enhanced survival at the non-permissive temperature, colonies were replica-plated, the replicas incubated at 37 °C for 3 days, and then replica-plated to 23 °C. For the majority of colonies, there was no indication of viability even after 48 h incubation at 23 °C. However, 120 colonies displayed only a negligible loss of viability during the 3 day, 37 °C incubation. These 120 clones were mated to the temperature-insensitive strain 4116E and sporulated. Thirty-two haploid clones from each sporulated diploid strain were tested for complementation using tester strains bearing ede4 and cde7 mutations. One hundred eighteen temperature-sensitive clones with new mutations that complemented both the cdc4 and cdc7 mutations were isolated. The terminal phenotype of each new temperature-sensitive clone was determined in liquid cultures as above.
on YEPD medium at dilutions to produce single colonies. After an initial i5 h incubation at 23 °C the plates were shifted to 37 °C. The approximately 2 x 105 individual colonies that appeared at 37 °C were replica-plated to uracil-free medium to induce entry of cells into stationary phase. Cells were incubated on uracil-free medium for 3 days (a protocol shown in preliminary experiments to yield a high proportion of cells in G1 [or GOD. The replicas were then preincubated at 37 °C for 2 h and replica-plated to two prewaxmed 37 °C YEPD plates. One plate was incubated at 37 °C and the other was incubated at 23 °C. Colonies were scored for growth at each temperature.
Methods for determination of cell concentration have been described previously (Hartwel11970). Cell nuclei were stained using Giemsa as described by Robinow (1975). DNA content was determined by the method of Lab arca and Paigen (1980), or in one case (strain ID-1) by the micro-method of Roth and Silva-Lopez as described by Haber and Halvorson (1975).
Order-of-function Mapping The rationale for this method was detailed by Hereford and Hartwell (1974). Exponential phase cultures were shifted to the non-permissive temperature and after a period sufficient to arrest the cells at the temperature-sensitive block (generally 4 h), the cultures were split and returned to the permissive temperature; to one portion a-factor was added. At hourly intervals after the shift to the permissive temperature, 0.5 ml samples were removed from the control and c~.factor-treated cultures to determine cell concentration and proportion of unbudded cells. In the reciprocal shift experiment, exponential phase cells were treated with enough c~-factor preparation to arrest cells for 5 h. Two hours after the addition of c~-factor, the treated cultures were shifted to the non-permissive temperature and incubated for 15 min, and the ceils were then collected by centrifugation and suspended in fresh, prewarmed YM-1 medium in two portions. One portion of each culture was l e f t a t 37 °C, and the other portion was returned to 23 °C. Samples for cell concentration and bud morphology determinations were removed at hourly intervals.
Measurement of Protein and RNA Synthesis of protein and RNA was estimated by the incorporation of radioactive precursors (New England Nuclear Corp.) into trichloroacetic acid-precipitable material as described by Johnston et al. (1977b).
A ssay of Mating Ability Exponential phase 23 °C cultures were divided into two portions; one portion was preincubated at 35 °C for 3 h, while the other portion was incubated at 23 °C. From each portion, 2 x 106 cells were then mixed with an equal number of cells of the opposite mating type, and collected on filters; these filters were placed on YEPD medium to allow conjugation. Cells preincubated at 35 °C were mated at 35 °C; cells incubated at 23 °C were mated at both 23 °C and 35 °C. After a further 3 h incubation the cell mixtures were recovered from the filters and spread on medium selective for diploid cells.
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D.P. Bedard et al.. New Mutations in the Yeast Saccharomyces cerevisiae
Table 1. Mutant strains harboring new Gl-arresting temperaturesensitive mutations Enrichment procedure Inositol-less death
cdc7 Mating
cdc4 Survival
ID-1 ID-2
M7-7A
$7 $24 $33 $44 S104
Table 2. Segregation of mutant alleles Strain
XD 1 (cdc60) GD-2-1 (cdc61) $7 (cdc62) $44 (cdc63) S104 (cdc64) M7-7A
Asci with temperaturesensitive: temperatureinsensitive-spores 2:2
1:3
0:4
47 15 34 37 29 3
0 0 0 0 0 4
0 0 0 0 0 9
% Second division segregation a
69 NDb 63 69 59 -
a New mutant strains were mated with strain X2928-3D-1C, asci from the sporulated diploid strains were dissected, and spore clones were tested for temperature sensitivity and for nutritional requirements. The percentage of second division segreagation during meiosis, indicating centromere linkage, was determined by analyzing the segregation patterns of the temperature-sensitive mutations relative to a mutation in the centromere-linked trpl gene of strain X2928-3D-1C (Mortimer and Hawthorne 1966) b Not done
Genetic Methods Standard yeast genetic methods used here have been described by Mortimer and Hawthorne (1969).
Results Isolation and Genetic Analysis This paper describes the isolation of several new G 1-arrest mutants (Table 1). Each mutant isolation procedure employed in this study was designed to enrich for mutants conditionally defective in cell cycle initiation. In each protocol the starting populations were stationary phase cultures, and each mutant isolation procedure was based on a different set of assumptions.
The inositol-less death procedure was based on the assumption that some mutant cells arrested in G1 (or GO) would be unable to grow at the temperature-induced block and thus would be protected from inositol-less death (Henry et al. 1975). From survivors of inositol starvation, 500 independently derived temperature-sensitive clones were examined, of which two arrested in G1 at the non-permissive temperature. The cdc4 survival procedure was based on the assumption that additional mutations which arrest cdc4 cells in G1 (or G0) would prevent such arrested cells from reaching the cdc4 block, and thereby protect the addditionally mutated cells from the loss of viability that normally occurs at that cell cycle block (unpublished data). From 120 clones that retained viability at the non-permissive temperature , 5 Gl-arrest mutants were isolated, with mutations in 3 complementation groups (see below). The cclc7 mating procedure was based on the assumption that cdc7 cells blocked in G1 (or GO) by additional mutations would consequently retain the ability to conjugate. T h e design of this procedure was influenced by the phenotype exhibited by the original Gl-arrest mutations, which defined the cdc28 gene (Reid and Hartwell 1977). Cells bearing a Cdc28 mutation retain conjugational competence at the temperature-induced block, and show a mating ability at least 100-fold greater than that of cells blocked at other points in the cell cycle. Therefore, in this procedure, populations of cells bearing a cdc7 mutation, generally unable to conjugate because of prior arrest at the cdc7 block, were screened for cells which had acquired the ability to mate at the non-permissive temperature. From 280 independent clones identified by successful conjugation, 80 new temperature-sensitive mutants were isolated. From these, one G1 arrest mutant was identified. Each new mutation was crossed into other genetic backgrounds for further study. The construction of diploid strains, each heterozygous for one of these new conditional mutations, showed that each mutation was recessive to its wild-type allele. With the exception of the mutations in strain M7-7A, each of the recessive mutations segregated as a single nuclear gene (Table 2). The results of complementation tests between the newly-isolated mutations, and between these new mutations and previously characterized Gl-arrest cdc mutations (Hartwell et al. 1973; Reed 1980), are summarized in Table 3. Because mutations in strains $7, $24 and $33 did not complement one another, and were not independently isolated, only mutant strain $7 was analyzed further. None of these mutations tested were centromere4inked, as evidenced by the high proportions of meiotic second division segregation of the temperature-sensitive mutation from a centromere-linked marker (Table 2). In those cases examined, the r/rotations were not linked to each other (Table 4). Thus the mutations isolated by the various procedures described here define five newly
D. P. Bedard et al.: New Mutations in the Yeast Saccharomyces cerevisiae
209
Table 3. Complementation tests between various edc mutant strains MA Ta o
Strain
ede Allele
F-
2 iB
m
m
i
90
/
i
i
n
i
,,¢
60
..-~
~ /4r
New Mutations in the Yeast Saccharomyces cerevisiae Affecting Completion of "Start" D. P. Bedard, G. C. Johnston, and R. A. Singer Departments of Microbiology, Medicine and Biochemistry, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, B3H 4H7 Canada
Summary. Here we report the isolation of several new temperature-sensitive mutations which cause cells of the yeastS accharomyces cerevisiae to arrest in the G1 period of the cell cycle. Four different selection schemes were employed. The cell division cycle (cdc) mutations define five new complementation groups. At non-permissive temperatures, strains bearing these new cdc mutations arrested in G1 within one cell division cycle. By orderof-function mapping, cells of each population were found to be arrested at "start", the regulatory point in the G1 period of yeast. Mutations were grouped into two categories by the abilities of mutant strains to continue extensive macromolecular synthesis and to conjugate with cells of the opposite mating type. For strains with mutations in one category, shift to the non-permissive temperature caused an abrupt decrease in the rates of labelling of protein and RNA, and rendered cells unable to mate efficiently. For strains with mutations in the second category, cells continued to grow and mating ability was not significantly impaired. Each selection scheme was also designed to isolate mutations which specifically affect the ability of cells to reinitiate the cell cycle from stationary phase. This was done to test the hypothesis that stationary phase cells are in a unique developmental state referred to as GO. No mutations specific for resumption of growth from stationary phase were isolated. Key words: Cellcycle - G1 period
cdc genes
Introduction Cell division is an orderly sequence of events in which the genetic material is replicated and subsequently segre-
Offprint requests to. G. C. Johnston
gated along with cellular constituents into progeny cells. In eukaryotes the regulation of this complex process occurs within the G1 period, between the completion of nuclear division and the onset of DNA replication (S phase). One area of interest concerns the nature of this G1 regulation. For these studies the budding yeast Saccharomyces cerevisiae has proved a useful model system. In particular, many mutants defective in the yeast cell division cycle (cdc mutants) have been isolated and characterized by Hartwell and his colleagues (Hartwell et al. 1973, 1974). Mutations in one cdc gene (cde28) allowed Hartwell to define a major regulatory step in G1, which has been referred to as "start" (Hartwell et al. 1974). Cells arrested in G1 either by mutation or by the presence of one of the yeast mating pheromones are at "start". Conversely, the completion of "start" heralds the onset of a new cell division sequence. Thus the regulation of cell division is thought to occur at the "start" event. In addition to the arrest caused by cdc28 mutations or mating pheromones, another form of G1 arrest is represented by cells in stationary phase. Like most other eukaryotes, yeast cells when deprived of required nutrients complete cell divisions and arrest in G1 (Hartwell et al. 1974). Such Gl-arrested cells can be shown by orderof-function mapping experim'ents to be either at or prior to the "start" event. One fruitful approach to illuminate the regulation of cell division has been to isolate mutants conditionally defective in the ability to initiate new cell division sequences. Recently, Reed (1980) reported an elegant approach which resulted in the isolation of mutations in three new cdc genes, all of which, like mutations in cdc28, cause defects in the ability to progress from mitosis to the next S phase. All such mutants were shown to be defective in the ability to execute the "start" event, and were by order-of-function mapping criteria arrested O172-8083/81/0004/0205/$02.00
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D.P. Bedard et al. : New Mutations in the Yeast Saccharomyces cerevisiae
at "start". As Reed pointed out, "start" mutants may be grouped into two phenotypic classes. He defined Class I mutants as those mutants which arrest at "start" but continue to grow and also retain the ability to conjugate. (Conjugation is a property of growing ceils in the G1 period [Reid and Hartwell 1977].) In contrast, Class II mutants arrest at "start" with phenotypes similar to those of cells starved for some required nutrient. Presumably Class II mutants would be better described as stationary phase-like mutarlts. By virtue of Reed's selection scheme, only Class I mutants were selected in those experiments. Thus mutations in at least four Class I "start" genes have been previously described, which all affect a single regulatory step ("start") in the cell cycle. Here we have used a number of selection strategies to isolate new G1 mutants. Different rationales were employed to test the hypothesis that "start" is the sole point of regulation for initiation of the cell cycle. In this way, several new Class I and Class II type "start" mutations were isolated and characterized. We also wished to test the hypothesis that unique gene functions might exist for resumption of growth from stationary phase. It has been suggested (by analogy with proposals for quiescent mammalian cells) that stationary phase yeast cells are actually in a unique developmental state referred to as GO (Pinon 1978). The existence of such a unique developmental state has been inferred from observations that stationary phase cells are by several criteria in a physiological state different from that of cells arrested at "start" by mutation or mating pheromones (for review see Bedard et el. 1981). However, no direct evidence has been reported verifying the existence of such a unique development state. If indeed GO represents a unique gene-mediated developmental state, as well as a unique physiological state, then GO mutants should exist. Some such mutants should be conditionally defective for resumption of growth from stationary phase, b u t should not be impaired once cell cycles have been initiated. Mutations conferring this GO phenotype were sought by four different procedures; none were found.
Course. Strains SR653-1 (cdc28-4)~ SR661-2 (cdc36-16), SR672-3 (cdc37-1) and SR665-1 (cdc39-1) were obtained from S. Reed (1980), and strains BR205-2A (cdc25-1), H185-3-4 (cdc28-1), El7 (cdc33-1) and BR214-4A (cdc35-1) were obtained from Hartwell. To determine centromere linkage, strain X2928-3D-1C (MATe adel gall trpl ura3 his6 leul metl4) was employed and was obtained from the Yeast Genetic Stock Center, Berkeley, California. The diploid strain AG1-7 has been described (Johnston et al. 1977b). Mutant strains isolated or constructed for this study were the following: ID-1 (MATe adel leul cdc60-1); ID-2 (MATa aro cdc61); GD-2-1 (MATe his6 ural cdc61-1); SG44-1 (MATe his6 ural cdc63-1); initial isolates of the cdc4 survival procedure were $7 (MATe leu2 cdc62-1), $24 (MATe cdc62-1), $33 (MATe cdc62-1), $44 (MATe ural cdc63-1), and S104 (MATe leu2 cdc64-1). Several of the above strains were crossed with strains GR2 or GR1 (MATe his6 ural) to obtain the following temperature-sensitive segregants of the opposite mating type for complementation studies: XS33 (MATe cdc62), XD-2 (MATe cdc61), XD-1 (MATa cdc60 ), XS24 (MATe cdc6 2), XS44 (MATe cdc63) and XS104 (MATe cdc64). Nutritional requirements for these latter strains were not determined.
Media The enriched liquid and solid media (YM-1 and YEPD respectively) have been described by Hartwell (1967). The liquid minimal medium (YNB) and solid synthetic medium have also been previously described (Johnston et al. 1977a). The mating pheromone e-factor was prepared according to the method of Biicking-Throm et al. (1973).
Mutagenesis For mutagenesis (Fink 1970), cells from 10 ml portions of stationary phase cultures were collected by centrifugation, washed once with sterile H20 and suspended in 10 ml of sterile H20; 50 t~l of ethylmethanesulphonate (EMS) (Sigma Chemical Corp.) was then added and incubation was continued at room temperature for periods of time, determined in preliminary experiments, which resulted in 50% survival, or 10% survivalfor the non-selective screening procedure. Mutagenesis was terminated by the addition of 5 volumes of 5% sodium thiosulphate. Cells were then washed 3 times by centrifugation, each time with 5 volumes of 5% sodium thiosulphate. To ensure the isolation of strains resuiting from independent mutational events, one drop portions of the mutagenized and washed cell suspensions were added to tubes containing fresh YM-1 medium and grown at room temperature to stationary phase. These populations of cells were used to start each isolation procedure.
Materials and Methods Strains
Isolation Procedures
The following strains were employed in the selection procedures reported here. Strain 18032 (MATe adel ade2 leu2 ural his7 lys2 tyrl gall cdc7-3) was obtained from L. H. Hartwell; strain GJ301-47 (MATe his7 ural cdc4-3 cdc7-4) was constructed by crosses between strain GR2 (Johnston et al. 1977b), and cdc mutant strains H135-1-1 (cdc4) and H201-14-4 (cdc7), also obtained from Hartwell; strain MC-6A (MATe/nol-3 ino4-8) was provided by S. Henry; strain A4840A (MATe ade2 his5), strain 4116E (MATe leu2) and strain D273-11A (MATe adel hisl) were provided through the Cold Spring Harbor Yeast Genetics
For each procedure the initial step involved exposure of stationary phase Gl-arrested (or GO) cells to the non-permissive temperature under conditions that would favour the survival of cells blocked in G1 (or GO). The use of stationary phase cells as starting populations was a feature to allow isolation of both G1 and GO mutants. Inositol-less Death Procedure. Stationary phase cells from 500 independent, mutagenized cultures of strain MC-6A, an inositol auxotroph, were placed at the non-permissive temperature
D. P. Bedard et al.: New Mutations in the Yeast Saeeharomyces eerevisiae
207
(36 °C) for 60 min. These prewarmed cells were then diluted into prewarmed YNB medium without inositol and incubated at 36 °C for a period (30 h, as determined by preliminary experiment) sufficient to allow extensive cell death. Survivors were plated on YEPD medium and the resultant colonies were tested for temperature sensitivity by replica-plating. One temperature-sensitive clone was isolated from each independent culture. For each of the 500 independently derived, temperature-sensitive clones the terminal phenotype was determined in two ways. In one method, stationary phase ceils were suspended in fresh YM-1 medium at 36 °C and incubated overnight before examination;in the other method, exponential phase cultures growing at 23 °C were shifted to 36 °C and examined after further incubation.
Non-selective Screen. Mutagenized cells of strain GR2 were spread
Mating Procedure. The mating procedure was adapted from the
Measurement of Cellular Parameters
method described by Hartwell (1973). Stationary phase ceils from 280 independent, mutagenized cultures of strain 18032 (cdeT) were incubated at the non-permissive temperature (37 oC) for 30 rain and diluted into fresh YM-1 medium prewarmed to 37 °C. After 12 h incubation, when more than 99.9% of the cells had become arrested at the cde7 step and were refractory to mating (as determined in preliminary experiments), each culture was mixed with an equal number of cells of the opposite mating type (strain A4840A) bearing complementary auxotrophic markers. These cell mixtures were collected on filters (Milfipore Corp., Bedford, Mass.), and the filters were placed on YEPD plates and incubated for 3 h at 37 °C. Conjugation was interrupted after 3 h by suspending the cells in ice-cold 1.0 M Sorbitol and subjecting the cell suspension to sonic oscillation for 5 s with a Branson Sonifier (Heat Systems - Ultrasonics, Inc., Plainview, N.Y.). Diploid clones were selected on minimal medium (SD) plates containing adenine. One diploid clone was picked from each independent culture and sporulated; haploid clones were then recovered by the random spore technique. For complementation analysis, sixteen spore clones from each sporulated diploid were mated on solid medium with edc7 mutant tester strains. Eighty of the 280 derived diploid strains segregated haploid clones bearing temperature-sensitive mutations which complemented the ede7 mutation. The terminal phenotype of each new temperature-sensitive clone was determined in liquid cultures as above.
edc4 Survival Procedure. Stationary phase cells from one mutagenized culture of strain GJ301-47 (ede4 ede7) were used. The ede7 mutation was included to decrease reversion frequencies of the temperature-sensitive phenotype. These cells were preincubated at 37 °C for 30 min, diluted into fresh prewarmedr(37 °C) YM-1 medium and incubated for 24 h. (Preliminary experiments indicated that by 24 h cell viability was decreased by a factor of 103.) The surviving cells were suspended in fresh medium and grown to stationary phase at the permissive temperature. This procedure was repeated once and the survivors were plated on YEPD medium to give 100-200 colonies per plate. To test for enhanced survival at the non-permissive temperature, colonies were replica-plated, the replicas incubated at 37 °C for 3 days, and then replica-plated to 23 °C. For the majority of colonies, there was no indication of viability even after 48 h incubation at 23 °C. However, 120 colonies displayed only a negligible loss of viability during the 3 day, 37 °C incubation. These 120 clones were mated to the temperature-insensitive strain 4116E and sporulated. Thirty-two haploid clones from each sporulated diploid strain were tested for complementation using tester strains bearing ede4 and cde7 mutations. One hundred eighteen temperature-sensitive clones with new mutations that complemented both the cdc4 and cdc7 mutations were isolated. The terminal phenotype of each new temperature-sensitive clone was determined in liquid cultures as above.
on YEPD medium at dilutions to produce single colonies. After an initial i5 h incubation at 23 °C the plates were shifted to 37 °C. The approximately 2 x 105 individual colonies that appeared at 37 °C were replica-plated to uracil-free medium to induce entry of cells into stationary phase. Cells were incubated on uracil-free medium for 3 days (a protocol shown in preliminary experiments to yield a high proportion of cells in G1 [or GOD. The replicas were then preincubated at 37 °C for 2 h and replica-plated to two prewaxmed 37 °C YEPD plates. One plate was incubated at 37 °C and the other was incubated at 23 °C. Colonies were scored for growth at each temperature.
Methods for determination of cell concentration have been described previously (Hartwel11970). Cell nuclei were stained using Giemsa as described by Robinow (1975). DNA content was determined by the method of Lab arca and Paigen (1980), or in one case (strain ID-1) by the micro-method of Roth and Silva-Lopez as described by Haber and Halvorson (1975).
Order-of-function Mapping The rationale for this method was detailed by Hereford and Hartwell (1974). Exponential phase cultures were shifted to the non-permissive temperature and after a period sufficient to arrest the cells at the temperature-sensitive block (generally 4 h), the cultures were split and returned to the permissive temperature; to one portion a-factor was added. At hourly intervals after the shift to the permissive temperature, 0.5 ml samples were removed from the control and c~.factor-treated cultures to determine cell concentration and proportion of unbudded cells. In the reciprocal shift experiment, exponential phase cells were treated with enough c~-factor preparation to arrest cells for 5 h. Two hours after the addition of c~-factor, the treated cultures were shifted to the non-permissive temperature and incubated for 15 min, and the ceils were then collected by centrifugation and suspended in fresh, prewarmed YM-1 medium in two portions. One portion of each culture was l e f t a t 37 °C, and the other portion was returned to 23 °C. Samples for cell concentration and bud morphology determinations were removed at hourly intervals.
Measurement of Protein and RNA Synthesis of protein and RNA was estimated by the incorporation of radioactive precursors (New England Nuclear Corp.) into trichloroacetic acid-precipitable material as described by Johnston et al. (1977b).
A ssay of Mating Ability Exponential phase 23 °C cultures were divided into two portions; one portion was preincubated at 35 °C for 3 h, while the other portion was incubated at 23 °C. From each portion, 2 x 106 cells were then mixed with an equal number of cells of the opposite mating type, and collected on filters; these filters were placed on YEPD medium to allow conjugation. Cells preincubated at 35 °C were mated at 35 °C; cells incubated at 23 °C were mated at both 23 °C and 35 °C. After a further 3 h incubation the cell mixtures were recovered from the filters and spread on medium selective for diploid cells.
208
D.P. Bedard et al.. New Mutations in the Yeast Saccharomyces cerevisiae
Table 1. Mutant strains harboring new Gl-arresting temperaturesensitive mutations Enrichment procedure Inositol-less death
cdc7 Mating
cdc4 Survival
ID-1 ID-2
M7-7A
$7 $24 $33 $44 S104
Table 2. Segregation of mutant alleles Strain
XD 1 (cdc60) GD-2-1 (cdc61) $7 (cdc62) $44 (cdc63) S104 (cdc64) M7-7A
Asci with temperaturesensitive: temperatureinsensitive-spores 2:2
1:3
0:4
47 15 34 37 29 3
0 0 0 0 0 4
0 0 0 0 0 9
% Second division segregation a
69 NDb 63 69 59 -
a New mutant strains were mated with strain X2928-3D-1C, asci from the sporulated diploid strains were dissected, and spore clones were tested for temperature sensitivity and for nutritional requirements. The percentage of second division segreagation during meiosis, indicating centromere linkage, was determined by analyzing the segregation patterns of the temperature-sensitive mutations relative to a mutation in the centromere-linked trpl gene of strain X2928-3D-1C (Mortimer and Hawthorne 1966) b Not done
Genetic Methods Standard yeast genetic methods used here have been described by Mortimer and Hawthorne (1969).
Results Isolation and Genetic Analysis This paper describes the isolation of several new G 1-arrest mutants (Table 1). Each mutant isolation procedure employed in this study was designed to enrich for mutants conditionally defective in cell cycle initiation. In each protocol the starting populations were stationary phase cultures, and each mutant isolation procedure was based on a different set of assumptions.
The inositol-less death procedure was based on the assumption that some mutant cells arrested in G1 (or GO) would be unable to grow at the temperature-induced block and thus would be protected from inositol-less death (Henry et al. 1975). From survivors of inositol starvation, 500 independently derived temperature-sensitive clones were examined, of which two arrested in G1 at the non-permissive temperature. The cdc4 survival procedure was based on the assumption that additional mutations which arrest cdc4 cells in G1 (or G0) would prevent such arrested cells from reaching the cdc4 block, and thereby protect the addditionally mutated cells from the loss of viability that normally occurs at that cell cycle block (unpublished data). From 120 clones that retained viability at the non-permissive temperature , 5 Gl-arrest mutants were isolated, with mutations in 3 complementation groups (see below). The cclc7 mating procedure was based on the assumption that cdc7 cells blocked in G1 (or GO) by additional mutations would consequently retain the ability to conjugate. T h e design of this procedure was influenced by the phenotype exhibited by the original Gl-arrest mutations, which defined the cdc28 gene (Reid and Hartwell 1977). Cells bearing a Cdc28 mutation retain conjugational competence at the temperature-induced block, and show a mating ability at least 100-fold greater than that of cells blocked at other points in the cell cycle. Therefore, in this procedure, populations of cells bearing a cdc7 mutation, generally unable to conjugate because of prior arrest at the cdc7 block, were screened for cells which had acquired the ability to mate at the non-permissive temperature. From 280 independent clones identified by successful conjugation, 80 new temperature-sensitive mutants were isolated. From these, one G1 arrest mutant was identified. Each new mutation was crossed into other genetic backgrounds for further study. The construction of diploid strains, each heterozygous for one of these new conditional mutations, showed that each mutation was recessive to its wild-type allele. With the exception of the mutations in strain M7-7A, each of the recessive mutations segregated as a single nuclear gene (Table 2). The results of complementation tests between the newly-isolated mutations, and between these new mutations and previously characterized Gl-arrest cdc mutations (Hartwell et al. 1973; Reed 1980), are summarized in Table 3. Because mutations in strains $7, $24 and $33 did not complement one another, and were not independently isolated, only mutant strain $7 was analyzed further. None of these mutations tested were centromere4inked, as evidenced by the high proportions of meiotic second division segregation of the temperature-sensitive mutation from a centromere-linked marker (Table 2). In those cases examined, the r/rotations were not linked to each other (Table 4). Thus the mutations isolated by the various procedures described here define five newly
D. P. Bedard et al.: New Mutations in the Yeast Saccharomyces cerevisiae
209
Table 3. Complementation tests between various edc mutant strains MA Ta o
Strain
ede Allele
F-
2 iB
m
m
i
90
/
i
i
n
i
,,¢
60
..-~
~ /4r
