Plasmid p725 contains the wild-type SRAl DNA in a two-micron, YEp24 vector ...... transformed by wild-type and temperature-sensitive Kirsten sarcoma virus.
Copyright 0 1986 by the Genetics Society of America
SUPPRESSORS OF THE R A S ~MUTATION OF SACCHAROMYCES CEREVISIAE JOHN F. CANNON,* JACKSON B. GIBBSt
AND
KELLY TATCHELLZ
*Department of BiologylG5, University of Pennsylvania, Philadelphia, Pennsylvania 19104, and +Department of Virus and Cell Biology, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486 Manuscript received November 1 1, 1985 Revised copy accepted February 24, 1986 ABSTRACT
Saccharomyces cereuisiae contains two members of the ras gene family. Strains with disruptions of the RASP gene fail to grow eficiently on nonfermentable carbon sources. This growth defect can be suppressed by extragenic mutations called sru. We have isolated 79 independent suppressor m&?ations,68 of which have been assigned to one of five loci. Eleven additional dominant mutations have not been assigned to a specific locus. Some sral and SRA4 and all SRA3 mutations were RAS independent, allowing growth of yeast cells that lack a functional RAS gene. Mutations in sral, SRA3, SRA4 and sra6 are linked to his6, inol, met3 and ade6, respectively. Some sra mutants have pleitropic phenotypes that affect glycogen accumulation, sporulation, viability, respiratory capacity and suppression of two cell-division-cycle mutations, cdc25 and cdc35. The proposed functions of many of the suppressor genes are consistent with the model in which R A S activates adenylate cyclase.
R
AS genes were originally identified as the transforming genes in HARVEY and KIRSTEN sarcoma viruses (reviewed by ELLIS, LOWYand SCOLNICK 1982) and were subsequently identified as the transforming genes in a number of human tumors. T h e ras genes code for a group of closely related 21,000 dalton proteins, called p21, that bind and hydrolyze G T P and are found associated with the plasma membrane (reviewed by GIBBS,SICALand SCOLNICK 1985). Transforming genes, whether of viral or cellular origin, differ from their normal cellular homologues at a few positions (amino acids 12, 13, 59, 61 or 63). Concomitant with the transformation potential of these mutant p21 molecules is a decrease in their GTPase activity, implicating this activity with a central role in ras function (MCGRATHet al. 1984; SWEETet al. 1984; GIBBS et al. 1984). Three genes that share homology with the vertebrate ras genes have been found in Saccharomyces cerevisiae. T h e YP2 gene is located between the genes for actin and B-tubulin. It shares approximately 40% homology with ras in the DONATHand SANDER1983). T h e amino terminal 160 amino acids (GALLWITZ, RASI and RAS2 genes exhibit more than 60% homology over this same region (DEFEo-JONESet al. 1983; POWERSet al. 1984). Like their mammalian homoI
Genetics 113: 247-264 June, 1986.
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J. F. CANNON, J. B. GIBBS AND K. TATCHELL
logues, the yeast RAS gene products bind and hydrolyze GTP and can be et al. 1984, 1985; TAimmunoprecipitated by anti-p21 antibodies (TEMELES MANOI et al. 1984, 1985). Yeast strains containing disruptions of either RASl or RAS2 are viable, but cells containing disruptions of both fail to grow (KATAOKAet al. 1984; TATCHELL et al. 1984). Such cells will grow, however, if they contain either the human et al. 1985), cellular (KATAOKAet al. 1985) o r viral ras genes (DEFEO-JONES implying that the amino acid homology extends to the functional level. Recent experiments suggest that the essential function of these genes in yeast is to activate adenylate cyclase. RAS2 disruption strains have reduced adenylate cyclase activity, whereas strains containing a dominant RAS missense mutation, analogous to the transforming mammalian gene, exhibit increased adenylate et al. 1985). Purified yeast and vertebrate ras proteins cyclase activity (TODA activate yeast adenylate cyclase in vitro (BROEKet al. 1985). Several phenotypes are associated with the ras2 disruption, including sporet al. 1985; TATCHELL, Roulation on rich media (hypersporulation) (TODA BINSON and BREITENBACH 1985), increased accumulation of the storage carbohydrates glycogen and trehalose and impaired ability to grow on nonferROBINSONand BREITENBACH 1985; mentable carbon sources (TATCHELL, FRAENKEL 1985). This last phenotype segregates meiotically with ras2 and can be eliminated by increased gene dosage of RASl; it can also be suppressed by extragenic suppressors. Initial characterization revealed that some of these extragenic suppressors allow a yeast cell to grow without a functional RAS gene (TATCHELL, ROBINSON and BREITENBACH 1985). In this work, extragenic suppressors of the ras2 disruption were extensively analyzed. Evidence obtained from these mutations corroborates the connection between RAS and yeast adenylate cyclase. MATERIALS AND METHODS
Yeast strains and plasmids: T h e strain EG81-40C is the product of three serial ROBINSONand BREITENBACH backcrosses to the S288C strain MCY3 17 (TATCHELL, 1985). T h e rasl-545 and ras2-530 alleles are URA3 and LEU2 disruptions of the two genes (TATCHELLet al. 1984). T h e hid-539 and lys2-801alleles are amber mutations; the ade2-101and trp5-48 alleles are ochre mutations. T h e cdc35-10 mutation in JC41024D was originally called tsm0185 (BOUTELET, PETITJEAN and HILGER1985). T h e cdc621 mutation in JC504-2D was from a strain provided by GERRYJOHNSTON (BEDARD, JOHNSTON and SINGER1981). T h e JC303 strains were derived from JC302-26B and, thus, are assumed to be isogenic. T h e plasmid YCp5O::HO contains a functional HO endonuclease in the URA3, centromere vector YCp50 and was kindly provided by ROBERTJENSEN. T h e strain DBY703/p725 contains the plasmid p725 integrated at SRAl. Plasmid p725 contains the wild-type SRAl DNA in a two-micron, YEp24 vector (L. ROBINSON, J. CANNONand K. TATCHELL, unpublished results). T h e genotypes of strains used in this work are listed in Table 1. Marker scoring and media: Strains were grown on rich media containing 2% Bactopeptone, 1% yeast extract and either 2% potassium acetate (YEPA), 2% (v/v) ethanol (YEPE), 3% (v/v) glycerol (YEPG) or 2% glucose (YEPD). Solid media contain 2% agar. Growth on acetate, ethanol or glycerol was generally scored after 48 hr at 37", except as noted otherwise. Nitrogen-free medium contained 0.17% yeast nitrogen base without amino acids or ammonium sulfate (Difco) and 2% glucose. Media used for sporulation
SUPPRESSORS OF RASZ
249
TABLE 1 Strains Strain
Genotype
A256-99A" DBY7031p725 EG8 1-40Cb JC302-26B JC302-26D JC303-X JC304-X JC305-4.1 JC3 10-2 1 D JC32 3- 1 5 D JC337-2B JC365-5A JC375-17B JC375-18C JC377-16C JC377-10B
Mata leu2-3 trp5 lys9 met10 adel pet9 Mata ura3-52 trpl A his3 [SRAl::URA3] Mata leu2-3,112 ras2-530 lys2-801 his4-539 Mata leu2-3,112 ras2-530 ura3-52 his4-539 Mata leu2-3,112 rad-530 ade2-101 lys2-801 his4-539 Mata leu2-3,112 ras2-530 ura3-52 his4-539 sra Mata leu2-3,112 rad-530 ade2-101 lys2-801 his4-539 sra Mata leu2-3,112 ras2-530 his4-539 SRA4-1 Mata leu2-3,112 rad-530 trp5 lys7 met3 Mata leu2-3,112 rad-530 his4-539 cdc25-1 tyrl lys2 Mata leu2-3,112 ura3-52 rasl-545 canl cyh2 lys2-801 Mata leu2-3,112 rad-530 trp2 inol met3 Mata leu2-3,112 rasl-545 rad-530 lys2-801 SRA3-3 Mata leu2-3,112 ura3-52 his4-539 canl SRA3-3 Mata leu2-3,112 ura3-52 lys2-801 canl SRA4-6 Mata leu2-3,112 ura3-52 rasl-545 rad-530 his4-539 lys2-801 canl SRA4-6 Mata leu2-3,112 hid-1 1,15 ura3-52 trpl-1 cdc35-IO Mata leu2-3,112 rad-530 ura3-52 lys2-801 Mata leu2-3,112 ras2-530 his4-539 cdc6 arg3 inol Mata leu2-3,112 rad-530 ade6 leu1 trp5 his4-539 sra6-15 Mata leu2-3,112 rad-530 his6 ade2-101 Mata leu2-3,112 ras2-530 sral-3 lysll trp5-48 inol Mata Eeu2-3,112 rad-530 cdc62-1 Mata ade2-101 his4-539 Mata his4-539 trpl ras2-530 ura3-52 lys2-801 sral-18 Mata rasl-545 ras2-530 trpl ura3-52 lys2-801 sral-18 Mata his4-917 ura3-52
JC410-24D JC423-18D JC459-18D JC470-16A JC494-18B JC500-55A JC504-2D MCY3 17' RX1 3-1 7A Rx23-3A SR 140-6A
Some of these strains have been previously described: (GABERet al. 1983); * (TATCHELL, and BREITENBACH 1985). ' This strain was a gift from MARIAN CARLSON. ROBINSON and scoring other markers have been described (TATCHELL et al. 1981; GABERet al. 1983). The viability of sral mutants could be increased by sporulating them on YEPA. Phloxine B (Sigma) was included in YEPD medium at 20 mg/liter to stain inviable cells (FANTES1981). Glycogen accumulation was scored in situ either by inverting a plate over I:! crystals in a chromatography tank or by flooding the plate with 0.4% 12, 0.2% KI (CHESTER1968). Either method stained the colonies brown to varying degrees depending on the amounts of glycogen accumulated. The reduction of tetrazolium dyes was performed by overlaying the colonies with 50 mM Tris-HCI, pH 7.4, 1.5% agar containing 0.1% triphenyltetrazolium chloride, followed by incubation at 30" for 1 hr. Selection of sra mutants: The parental strain for all sra mutants was JC302-26B. Approximately lo6 cells from cultures grown overnight in YEPD were spread onto acetate, ethanol or glycerol plates, followed by incubation at 25 or 37" for 4 days. One colony from each plate was then purified on the medium and at the temperature at which it was selected. The overnight cultures were from individual JC302-26B colonies to ensure the independence of the sra mutants. The resulting mutants were called JC303-X, where X is the isolation number. C o m p l e m e n t a t i o n analysis: JC303 strains were mated to JC302-26D and the diploids were purified by prototrophic selection. Tetrads were dissected following sporulation. Meiotic progeny from such crosses that contain the sra mutation and the same genetic
250
J. F. CANNON, J. B. GIBBS AND K . TATCHELL
markers as JC302-26D (Table 1) were saved and called JC304-X, where X is the same isolation number as the original JC303 strain. JC303 X JC304 diploids were plated on acetate, ethanol and glycerol media. Complementation between two recessive sra mutations is indicated by a failure of the diploid to utilize nonfermentable carbon sources. Identification of sru amber mutations: Approximately 1O6 cells of the JC303 strains were placed on histidine-free medium. The growth of the histidine prototrophs was scored on ethanol medium. Coreversion of the histidine and sra phenotypes indicated putative sra amber mutations (his4-539 is an amber mutation). Corevertants were crossed to JC302-26D and gave a marker segregation consistent with a SUP amber sra amber genotype. Adenylate cyclase assay: Cultures were grown at 30" in YEPD to a density of A600 = 0.5- 1.O. Cells were harvested, prepared by Glusulase (Dupont) digestion (CASPERSON et al. 1983) and homogenized in buffer containing 50 mM NaMES, pH 6.0, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride (TODA et al. 1985). A crude particulate fraction was prepared by centrifugation at 100,000 X g for 30 min at 4". Adenylate cyclase assays were performed at 30" for 30 min, using either 10 mM MgC12 or 0.5 mM MnCI2 (CASPERSON et al. 1983). Protein concentrations were determined by the Coomassie dye method (Bio-Rad) using bovine serum albumin as standard. Phosphodiesterase assay: Reactions (100 pl) contained 50 mM NaMES, pH 6.0, 2 mM dithiothreitol, 0.1 mM EGTA, 0.1 mg/ml bovine serum albumin, 0.5 mM MnC12, 0.5 p~ ['HICAMP (Amersham, 30,000 cpm/pmol), and 15 pg of crude particulate protein. After incubation at 30" for 10 min, the reaction product was quantitated as described (THOMPSON et al. 1979). Protein kinase assay: Cultures were grown at 30" in YEPD to a density of A600 = 1.5. Cells were harvested and lysed by glass bead disruption in a buffer containing 50 mM NaHEPES, pH 7.5, 10 mM EDTA, 100 mM NaF, 1 mM dithiothreitol, 5% gycerol, 50 p~ tosyl-L-lysine chloromethyl ketone, and 50 p~ phenylmethylsulfonyl fluoride (TEMELES et al. 1984). Protein (100-150 pg in 25 pl of the above buffer) was added to 25 PI of 50 mM NaHEPES, pH 7.5, 20 mM MgC12, 8 FM [Y-~'P]ATP(ICN, 50,000 cpmjpmol) and 4 mg/ml protamine sulfate. When present, cyclic AMP was at 50 p ~ . After 10 min at 30", reactions were terminated with 1 ml ice-cold 10% trichloroacetic acid, and "P-labeled protein was collected on Millipore filters and counted by liquid scintillation. RESULTS
Isolation of sru mutants: When cells of strain JC302-26B, which contain a ras2-530 mutation, a r e plated o n acetate, ethanol or glycerol carbon source media at 37", they bud five t o six times and then arrest unbudded in GI. Similar behavior is observed for cells growing in liquid media. At lower temperatures (25"), the number of cell divisions before arrest is slightly greater on acetate a n d ethanol. O n glycerol at 25", complete arrest is never achieved, although the growth rate is severely reduced. Revertants capable of growth o n these nonfermentable carbon sources appeared at a frequency of approximately of the growth arrested cells. After 4 days these revertants yield colonies in a lawn of nongrowing cells. O n glycerol medium a t 25", revertants were evident as faster growing papillations o n a thick lawn. Seventy-nine independent, spontaneous revertants were obtained from JC302-26B. These strains, called JC303-X, harbor sra (for suppressor of U S ) mutations that allow them t o grow on nonfermentable carbon sources. In all cases the ability of ras2-530 cells t o grow on nonfermentable carbon sources segregated 2:2 in
25 1
SUPPRESSORS OF RAS2
TABLE 2 Isolation summary
Medium
Temperature (")
Acetate Ethanol Glycerol
25 25 25
lb 0
Acetate Ethanol Glycerol
37 37 37
0 5 1 14
Totals a
sra 1
7
SRA3
SRA4
sra5
sm6
SRAX"
2
1
1
0 1
0 0
8 0
4. 5 1
9 3
0
2
4
0 2 4
0 2 11
0 0 1
16
9
7
22
11
0 1 1
2
These uncharacterized dominant mutations may not be linked. The number of alleles recovered at each locus at the temperature and on the medium stated.
crosses where the sra mutation was heterozygous and the ras2-530 mutation was homozygous. This behavior is consistent with the hypothesis that the sra mutations are single lesions of the nuclear genome. The number of sru loci: Diploids heterozygous for each sra mutation were constructed by mating the JC303 strains to JC302-26D. Some, but not all, of these diploids grew on nonfermentable carbon sources, which indicates that there were both dominant and recessive sra mutations. The recessive mutations were placed into complementation groups as described above. Complementation analysis indicated three complementation groups called sral, sra5 and sra6. The dominant sra mutations were grouped based on linkage to an outside marker. Dominant SRA3 and SRA4 mutations were thus defined by their linkage to in01 and met3, respectively. Eleven additional dominant sra mutations, which are not linked to SRA3 or SRA4, were also found, and these were not analyzed further. Linked dominant sra mutations were assumed to be allelic because of their similar phenotypes, although allelism was not rigorously proven. Table 2 summarizes the isolation of the 79 sra mutants. A striking feature of these data is that only mutations at the SRA3 locus were isolated on acetate at 37", although this is not the only medium on which such mutations were isolated. Phenotypic characterization of sru mutants: T h e sra mutants were screened to determine if they contained amber sra alleles. T w o amber alleles were found for both sral and sra5 (mutations: sral-6, sral-12, sra5-2 and sra5-5). No amber mutations were found among the 23 sra6 and nine SRA4 mutants screened. T h e presence of nonsense mutations among the SRA3 mutations was not tested. T h e sra mutants were tested for growth on acetate, ethanol and glycerol media at 30 and 37", respectively. Figure 1B shows that all the sra mutants have the ability to grow on ethanol at 30", whereas the growth of the JC30226B parental strain is greatly attenuated on this carbon source. This growth difference is amplified at 37" (Figure IC). In general, all sra mutants grew well on all three carbon sources; however, only SRA3 mutants grew well on
252
J. F. CANNON, J. B. GIB= AND K. TATCHELL
FIGURE1.-Phenotypic characterization of sra mutants. A, Representative sra mutants uC303 strains) were gridded on a master plate and then replica-plated to diagnostic media. Numbers within the circles are allele numbers. Included are three colonies of the JC302-26Bparental strain (checked circles). B, YEPE plate incubated at 30" for 2 days. C,YEPE plate incubated at 37" for 2 days. D, YEPG plate incubated at 30" for 2 days and then overlayed with triphenyltetrarolium chloride (MATERIALS AND METHODS). The dark colonies were dark red; light colonies were pink. E, YEPE plate overlayed with triphenyltetrazolium chloride. F, YEPD plate incubated for 2 days at SO" and then iodine vapor stained. The dark colonies were black to brown; light colonies were yellow. G,YEPD-phloxine B plate incubated for 2 days at SO0. H, YEPD-phloxine B plate incubated at 37" for 2 days. The dark colonies were dark red, the light colonies were white. I, nitrogen starvation survival. After 4 days on nitrogen-free medium, the colonies were replicaplated to YEPD and were incubated for 24 hr. The JC303-68 (sral-12) colony has a papillation that has reverted many of the sral-I2 phenotypes. This is most clearly seen on the 37" phloxine B plate.
acetate at 37", and none of the JC303 strains grew well on acetate at 30". In addition, some s r d mutants did not grow on ethanol at 37" (Figure 1C). Tetrazolium dye reduction: Tetrazolium dyes are reduced to colored, insoluble formazan compounds by respiring microorganisms (OGUR,ST. JOHN and NAGAI 1957). Wild-type (RASZ RAS2) yeast reduce triphenyltetrazolium
253
SUPPRESSORS OF RASP
TABLE 3
Phenotypic summary of sra mutants Locus
SRA+ sral sRA3 sRA4 sra5 sra6
Glycogen accumulation" Sporulationb
< or = > or < < <
lo%) on acetate and glycerol media (TATCHELL, ROBINSON and BREITENBACH 1985). All diploids heterozygous for the sra mutations exhibited the hypersporulation trait typical of ras2-530. Diploids homozygous for the sra mutations and homozygous for ras2-530 were made by transforming the haploid JC303 strains with a YCp5O::HO plasmid. This plasmid contains the wild-type HO gene that enables mating-type switching (JENSEN, SPRACUE and HERSKOWITZ 1983). Once mating types have switched, diploids are quickly formed. The sporulation of the transformed JC303 strains, which were mixtures of haploids and diploids, was scored as above. Several sral and two SRA4 alleles exhibited a recessive Spo- phenotype. This was confirmed by construction of JC303 X JC304 diploids, which were homozygous for the appropriate sra mutations. In all cases the results were identical to those obtained via the HO plasmid. One of the Spo- sral alleles was the amber allele sral-12; the other sral amber allele was sporulation proficient. The SRA4-5 and SRAI-6 mutants, which were Spo-, showed the greatest tetrazolium reduction and the least iodine staining of any of the SRA4 mutants (Figure 1F). Aside from the above exceptions, all of the sra mutants sporulate in a homozygous state and display the hypersporulation phenotype in ras2-530 strains. Viability of sru mutants: The RAS2""' missense mutation decreases the et al. 1985). This same viability of strains under starvation conditions (TODA phenotype was found for the sral-1 mutation (TATCHELL, ROBINSON and BREITENBACH 1985). The viability of the mutants isolated in this work was assayed
SUPPRESSORS OF RASZ
255
in two ways. T h e first was by staining dead cells with phloxine B on YEPD medium (FANTES1981). Figures l G and 1H show the phloxine B staining of colonies grown at 30 or 37", respectively. At 30" some of the sral mutant colonies are uniformly stained. An examination of the cells from such a colony reveals that the staining intensity of a colony reflects the percentage of dead, stained cells it contains. At 30" the sral-13 strain contains about 10% dead, unbudded cells. T h e darkest-stained colonies are those of the sral-13 and sral14 strains at 37". Fifty percent of the cells in these colonies are dead, arrested early in the cell cycle (buds no larger than 20% the size of their mother). T h e JC302-26B parental strain exhibits some temperature-induced inviability. Five percent of the cells in the 37" colony are stained; all are unbudded. In contrast to the above uniformly stained colonies, some colonies were unstained at their edges but were stained in the center (Figure 1H). T h e dead, stained cells from the center of these colonies are found with buds of all sizes. Such random arrest has been reported for some starvations (PRINGLEand HARTWELL 1982). Some sral and most sra5 mutants show this phenomenon (Figure 1H). Inviability was also assayed by replica-plating colonies to nitrogen-free medium, incubating at 30" for several days (usually four), then replica-plating back to rich medium to score survival. Figure 11 shows that only three of the sral mutants show great inviability when starved for nitrogen (sral-12, sralI? and sral-14 mutants). After longer starvation times the sral-9 and sral-10 strains also exhibit sensitivity to nitrogen starvation (data not shown). None of the alleles of other sra loci affected viability in this assay. T h e death of the sensitive sral mutants on nitrogen-free medium was dependent on the presence of glucose. If ethanol, acetate or glycerol was used as a carbon source, viability was significantly enhanced (data not shown). RAS independence: One way the sra mutations could suppress the ras2-530 mutation would be to bypass the requirement for RAS. If a rasl-545 ras2-530 cell remains viable, then the mutation responsible is said to provide RAS independence. This possibility was examined for all sral, SRA3, SRA4 and sra5 alleles and five alleles of sra6. T h e strain JC337-2B contains a URA3 disruption of RASl, called rasl-545 (TATCHELL et al. 1984). By sporulating JC337-2B X JC303 diploids, progeny could be obtained that had a Ura+ Leu+ phenotype and, thus, contained both rasl-545 and ras2-530. Four different growth patterns were found for the rasl-545 ras2-530 sra triple mutant spores. Complete RAS independence was characterized by a growth rate of the rasl-545 ras2530 sra cells that was comparable to that of RASl RAS2 cells. Complete RAS independence was observed for some alleles of both sral and SRA4. Partial RAS independence was found for all the SRA? alleles isolated; the rasl-545 ras2-530 SRA? cells had a significantly reduced growth rate compared to RASl RAS2 SRA3 cells. Some rasl-545 ras2-530 sra spores germinated and performed one to three divisions, after which all the cells arrested in an unbudded state (GI arrested). This growth pattern was found for some of the sra5 and SRA4 mutants. T h e genotype of these triple mutants was inferred from the phenotypes of the sibling spore clones. T h e last growth pattern was that of
256
J. F. CANNON, J. B. GIBBS AND K. TATCHELL
complete RAS dependence. In these cases the rasl-545 ras2-530 spore failed to bud. This outcome was noted for all spores that lacked an sra mutation or contained any of the sra6 alleles. Suppression of class I1 cdc start mutations: The hypersporulation phenotype of the ras2-530 mutation is also shared by cdc25 and cdc35 (SHILO, SIMCHEN and SHILO1978). Both of these cell-division-cycle mutants are classified as class 11, G,-arrest mutants. RAS function has been implicated in the G I phase of the cell cycle (KATAOKAet al. 1985). Since the sra mutants were isolated as suppressors of one member of this group of related genes, it was important to see if mutations in the other two genes were similarly suppressed. Several alleles of each sra gene were tested for their ability to suppress cdc25 o r cdc35. The selected JC303 strains were crossed to either JC323-15D (ras2530 cdc25-1) or JC4 10-24D (cdc35-10). Tentative evidence for the suppression of the cdc mutations was provided by the non-Mendelian segregation of the cdc temperature-sensitive phenotype. The results of these preliminary suppression tests are listed in Table 3. In all cases the ability to suppress the temperature-sensitive phenotype of cdc35-10 was independent of the RAS2 genotype. From the JC323-15D crosses it was clear that none of the sra mutations was linked to cdc25 (data not shown). Tight linkage of SRA4 and cdc35 prevented the determination of suppression for this combination. Complete analysis of the suppression of cdc mutations by sra mutations will be discussed elsewhere (L. ROBINSON,J. CANNON and K. TATCHELL, unpublished results). Complex phenotypes for sral mutants: Of all the sra mutations, alleles of the sral locus showed the greatest range of phenotypes. An additional characteristic of most of the sral and some of the SRA4 strains was the accumulation of a brown pigment. Spectroscopically, the brown pigment appears to be zinc porphyrin (J. MATTOON, personal communication). The pattern of porphorin accumulation in the various sral mutants was dependent on the carbon source. All of the phenotypes noted for sral alleles meiotically cosegregated with the sral mutation. Mapping sral, SRA3, SRA4 and sra6: T h e sra mutations were genetically mapped to determine potential allelism with previously described loci. The wild-type SRAl gene was cloned by virtue of its ability to complement the sral phenotypes (L. ROBINSON,J. CANNONand K. TATCHELL, unpublished results). et al. 1979), was used to This gene, in a two-micron vector, YEP24 (BOTSTEIN destabilize the SRAl chromosome (FALCOet al. 1982). Data from these experiments indicated that chromosome I X was unstable in these strains. A threefactor cross involving his6, lysll and sral-3 localized sral between his6 and Zysl I , 9 cM from his6 (Table 4). The mapping of SRA3 and sra6 was by conventional tetrad analysis using markers distributed throughout the yeast genome. The markers used were a subset of those found in the mapping strains used to map frameshift suppressors (GABERet al. 1983). The mapping strains all had a Zeu2-3 frameshift mutation. The ras2-530 mutation was therefore easily introduced into these strains by crosses to JC302-26B. Progeny from several of these crosses were Leu' yet grew on ethanol, indicating the presence of sra mutations in some of
257
SUPPRESSORS OF RAS2
TABLE 4
Mapping data for sra loci Gene pair
sral-3 x l y s l l b sral-3 X his6 l y s l l X his6 SRA3-2 X inol“ SRA3-2 X cdc6 SRA3-2 X arg3 inol X cdc6 inol X arg3 SRA4-I X met? sra6-I5 X adeff sra6-I5 X leu1 leul X ade6 sra6-15 X cdc62 cdc62 X ade6
Segregation (PD:NPD:T)”
Map distance (cM)
27: 3:50 64: 0:15 26: 6:56 78: 0: 7 57: 0:18 19: 4:36 52: 0:22 20: 3:62 45: 0: 4 159: 0:15’ 82: 0:lO 26: 6:62 29: 2:64 62: 1: 9 45: 1 2 0
43 9 67 4 12 56 15 (21.8)d 59 (40.2)d 4 4 5 46 40 15 20
PD, parental ditypes; NPD, nonparental ditypes; T, tetratypes. The sral mapping data came from JC494-18B X JC500-55A. “ T h e SRA3-2 mapping data came from JC303-8.3 (SRA3-2) X JC459-18D. These are the published distances (DONAHUE and HENRY 1981). ‘ T h e SRA4-I mapping data are from JC310-21D X JC305-4.1. ’This is the met3 X SRA4 data totalled from all nine SRA4 alleles. gThe sra6-15 mapping data are from JC470-16A X JC423-18D and JC470-16A X JC504-2D. a
the mapping strains. In total, four of the ten Mata mapping strains used were found to harbor sra mutations. T h e sra mutation originally found in strain A256-99A was discovered to be a recessive allele of sral by complementation analysis. T h e SRA3-2 mutation, present in strain JC303-8.3, was found to be closely linked to inol (PD:NPD:T = 23:O:l). A four-factor cross was performed using a diploid heterozygous for SRA?-2, inol, cdc6 and arg3. T h e results of this cross are recorded in Table 4. T h e data are consistent with the order cdc6SRA3-inol-arg3, with SRA? 4 cM from inol. T h e sra6-15 mutation was used to map sra6. Linkage to ade6 (PD:NPD:T = 38:0:6) placed sra6 on chromosome VZZ near ade6. Data from a three-point cross involving sra6, leul and ade6 revealed that the order of these markers was leul-ade6-sra6, with 5 cM separating ade6 from sra6 (Table 4). A celldivision-cycle mutation, cdc62, was recently mapped to this region (HANICJOYCE 1985). A cross including cdc62, ade6 and sra6 indicated that cdc62 was linked but was not allelic with sra6 (Table 4),consistent with the order: ade6sra6-cdc62. T h e centromere linkage of SRA4 mutations was evident from the paucity of tetratypes observed with centromere-linked ura3. By testing linkage to other centromere markers, SRA4 was localized to the chromosome X centromere by its linkage to met3 (Table 4).
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J. F. CANNON, J. B. GIBBS AND K . TATCHELL
TABLE 5 Biochemical analysis of sra mutants -
Adenylate cyclase activity Strain
Relevant genotype
Mg2+
MnS+
SR140-6A JC302-26B Rxl3-17A Rx23-3A JC375-18C JC375-17B JC377-16C JC303-54 JC377-10B JC303-16 1C303-46
RASl RASP RASI ras2 R A S l ras2 sral-I8 rasl ras2 sral-18 R A S l RASP SRA?-? rasl ras2 SRA?-? RASl RAS2 SRA4-6 RASI ras2 SRA4-6 rasl ras2 SRA4-6 RASl ras2 sra5-? RASl ras2 sra6-I5
5.1 2.1 1.2
