four genes responsible for a position effect on … genes responsible for a position effect on...

Copyright 0 1987 by the Genetics Society of America

Four Genes Responsible for a Position Effect on Expression From HML and HMR in Saccharomyces cerevisiae

Jasper Rine' and Ira Herskowitz2 Department ofBiology and Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403

Manuscript received September 16, 1986 Revised copy accepted January 30, 1987

ABSTRACT Mating type interconversion in Saccharomyces cerevisiae occurs by transposition of copies of the a or

ci mating type cassettes from inactive loci, HML and HMR, to an active locus, MAT. The lack of expression of the a and LY genes at the silent loci results from repression by trans-acting regulators encoded by SIR @lent information Regulator) genes. In this paper we present evidence for the existence of four SIR genes. Inactivation of any of these genes leads to expression of cassettes at both HML and HMR. Unusual complementation properties are observed for a number of sir mutations. Specifically, some recessive mutations in different genes fail to complement. The correspondence between SIRI , SIR2, SIR3, SIR4 and other genes with similar roles (MAR, CMT, STE8 and STEP) is presented.

ELL type in the unicellular eucaryote Saccharo- C myces cerevisiae is under control of a single genetic locus, the mating type locus (MAT) . The two mating types of yeast, a and a, are determined by the MATa and M A T a alleles, respectively (LINDEGREN and LINDEGREN 1943). The gene products that are encoded by both MATa and MATa are regulatory proteins UOHNSON and HERSKOWITZ 1985).

The genetic and physical structure of the mating type locus is known (SPRAGUE, RINE and HERSKOWITZ 198 1, a, b; ASTELL et al. 198 1 ; NASMYTH and TATCH- ELL 1980). MAT consists of a block of DNA approximately 2 kilobase pairs (kb) in length. The structural details of MATa and M A T a are indicated in Figure 1 [adapted from ASTELL et al. (1 98 1) and ABRAHAM et al. (1984)]. Two points are particularly relevant to this work: (1) the central regions of MATa and M A T a are nonhomologous; ( 2 ) both M A T a and MATa encode two transcripts, a1 and a2 in a celIs and a1 and a2 in a cells, that are transcribed from divergent promoters located in the regions of nonhomology between a and a.

Certain strains of yeast, namely those containing the HO gene (WINCE and ROBERTS 1949), are capable of switching from one cell type to another as fre- quently as once per generation (HICKS and HERSKO- WITZ 1976; STRATHERN and HERSKOWITZ 1979; RINE et al. 1981). When a cell switches from a to a, MATa is replaced by MATa; switching from a to a results from the opposite change. HMLa and HMRa are additional genes required for mating type intercon-

' Present address: Department of Biochemistry, University of California,

' Present address: Department of Biochemistry and Biophysics, University Berkeley, California 94720.

of California, San Francisco, California 94143.

Genetics 116: 9-22 (May, 1987)

version (HARASHIMA, NOGI and OSHIMA 1974). H M L a and HMRa are cryptic copies of M A T a and MATa sequences that are the source of cassettes of mating type information. These cassettes are trans- posed to MAT during mating type interconversion [reviewed in HERSKOWITZ and OSHIMA (1 98 l)]. The mechanism of mating type interconversion is a site specific gene conversion event between MAT and the cryptic copies of M A T a or MATa sequences at HML and HMR (STRATHERN et al. 1982; KOSTRIKEN et al. 1983).

In most strains of yeast, HML contains an unexpressed yet intact copy of M A T a (designated as the HMLa allele) and HMR contains an unexpressed copy of MATa information (the HMRa allele). However in some strains isolated from nature, or in others isolated in the laboratory, HML contains an unexpressed copy of MATa (the HMLa allele) and HMR contains a unexpressed copy of M A T a coding information (the H M R a allele) (SANTA MARIA and VIDAL 1970; BLAIR 1979).

Implicit in the pattern of expression of the genes at MAT, HML, and HMR is an indication of a novel mechanism of controlling expression of these sequences. Consider M A T a and H M L a , for example. Although their nucleotide sequence is identical, the a2 and a2 genes are expressed at MAT yet remain unexpressed at HML. Since the promoters of the a1 and a2 genes are roughly in the middle of the M A T a or H M L a locus, any sequences that would specify the differential expression of H M L a and MATa must act at a distance of a kilobase or more from the promoter (NASMYTH et al. 1981; NASMYTH 1982).

Two primary observations indicate that the differ-

10 J. Rine and 1. Herskowitz

E HML I MAT. E HMR I

a21 f' c-

a2 a1 MATa

FIGURE 1 .-Diagrammatic representation of mating type alleles of chromosome 111 as adapted from ASTELL et al. (1981). The horizontal arrows represent transcripts made from the MAT alleles. HML contains a cryptic copy of M A T a and H M R contains a cryptic copy of MATa. HML and H M R are flanked by E and I , sites required in czs for repression of these loci by the products of the SIR genes. Further details are provided In the text.

entia1 expression of MAT, HML and HMR is due to negative regulation of HML and H M R rather than positive activation of MAT. First, several recessive mutations have been identified that are unlinked to either HML or HMR yet result in constitutive expression of both loci. These mutations are referred to variously as sir, mar, and cmt (HERSKOWITZ et al. 1977; RINE et al. 1979; KLAR, FOCEL and MACLEOD 1979; HABER and GEORGE 1979). We shall use the SIR nomenclature for these mutations @lent Information - Regulator) for the sake of uniformity. Secondly, mutations adjacent to both HML and HMR result in the constitutive expression of the a and a sequences present at these loci (ABRAHAM et al. 1984; FELDMAN, HICKS and BROACH 1984). These mutations identify sites referred to as E and Z that are essential in cis for regulating the cryptic mating type genes and presumably define the site of action of one or more SIR gene product(s) (see Figure 1).

T h e genetic identification of mutations of the sir type has been accidental in that the first such mutations were uncovered in the course of other work (HERSKOWITZ et al. 1977; KLAR, FOGEL and MACLEOD 1979; HOPPER and HALL 1975; RINE et al. 1979). In order to correspondence among the existing mutations, we undertook a large scale, systematic identification of genes involved in controlling expression of the silent mating type loci. In addition, several mutations identified by others have phenotypes that over- lap those expected for a sir mutation. T h e relationship between these mutations and SIR genes has also been investigated. T h e observations presented in this paper establish that multiple gene products are required to prevent expression of the silent mating type genes of yeast.

MATERIALS AND METHODS

Media: YEPD (complete medium), SD (minimal medium), SM (dilution buffer), sporulation medium, and supplements

have been described (HICKS and HERSKOWITZ 1976). BBMB plates used in the a-factor halo assays are described in BLAIR (1 979). Cryptopleurine (Chemasea Pty., Sydney, Australia; and Dr. L. DOLBY, Chemistry Department, University of Oregon) was used at a final concentration of 1 mg/liter.

Mutagenesis: Mutagenesis was performed with ultraviolet irradiation from a germicidal UV lamp at approximately 170 ergs/"'. A 2-ml aliquot of 1 O6 cellsjml in sterile water was placed in a glass Petri plate and irradiated while shaking. One milliliter of the irradiated culture was diluted 1 : l O in YEPD broth and incubated in red tubes at 30" for 4 hr with aeration. Fifty-two independent cultures were mutagenized to a survival of 25% and plated onto YEPD plates at approximately 400 viable cells per plate for further screening. Half of the plates from each culture were incubated at 30" and the remaining plates were incubated at 23" or 33".

Mating type determination and mutant isolation: Strains with unknown mating types were replica plated onto SD plates spread with a lawn of approximately 10' cells of a mating type tester strain (either a strain 227 or a strain 70). Mating was scored as formation of prototrophic diploids. All mutants were single colony purified, pregrown at 23" or 33" , and tested for their ability to mate with an a lawn at 23" and 33" and with an a lawn at 23".

Monitoring a-factor sensitivity and production: a-Fac- tor sensitivity was measured by the a-factor confrontation assay (HICKS and HERSKOWITZ 1976). a-Factor production was monitored by the halo assay as modified by BLAIR (1 979), using a cells supersensitive to a-factor (strain XMB4- 12b) as the lawn.

Efficiency of mating determination: The strains to be tested were grown on YEPD plates, suspended in dilution buffer (SM), and titered by plating serial dilutions onto YEPD plates. Aliquots of the titered suspensions were plated onto YEPD plates along with IO8 cells of the appropriate mating type tester strain containing complementary auxotrophic markers. The plates were incubated overnight at 30" to allow mating to occur and were then replica-plated to minimal medium to select for diploids. Frequencies of reversion to prototrophy were accounted for in calculating the efficiency of mating. The efficiency of mating is expressed as the number of prototrophs formed divided by the number of cells plated. This efficiency is then normal- ized to the efficiency of a reference wild-type a or a strain. Unnormalized mating efficiencies of a and a strains were from 0.4 to 1.0.

Efficiency of sporulation determination: Sporulated cultures were suspended in sterile water and aliquots observed in a Petroff-Hauser counter at X200-300 magnification. Efficiency of sporulation is expressed as a percentage of total cells that have formed 2, 3 or 4 spored asci.

Low frequency mating: To obtain low frequency mating between cells of the same mating type or between cells with mutations affecting mating, IO7- IO cells of each strain were mixed on a YEPD plate and incubated overnight at 30". Because the mating partners carried multiple complementary auxotrophic mutations, diploids were selected by replica plating the mating plates to minimal medium. Independent matings were assured by using cultures grown from different single colonies.

Dominance tests: All mutations were tested for dominance by two methods.

Method 1. Mutants were mated to a matal strain (G40- lb), and the mating type of the diploids obtained by proto- troph selection was determined. Since matal is recessive to MATa (KASSIR and SIMCHEN 1976), dominant mutations conferring the a phenotype form diploids with a mating phenotype when mated to matal strains. If the mutation

S. cerevisiae SIR Genes 11

causing the a phenotype is recessive, the diploid will have the a mating phenotype.

Method 2. Mutants were mated to a MATa RMEI strain (227) and the ability of these diploids to sporulate was determined. This test measures the expression of a2 function by virtue of its role in sporulation (STRATHERN, HICKS and HERSKOWITZ 198 1) and is more sensitive for monitoring a2 than is Method 1. The REMI allele is present in these diploids in order to prevent mating-type-independent sporulation caused by the rmel mutation which exists in some laboratory strains (RINE, SPRAGUE and HERSKOWITZ 1981). Mutations that were demonstrated to be recessive by both criteria were assigned to complementation groups as described below.

Complementation tests: S IRl alleles. In order to determine which mutants contained mutations in the SIRl gene, all strains bearing recessive mutations were mated to a MATa sir l -1 RMEI strain (XR29-1Oc) to form diploids with the following genotype:

x- HMLa matal HMRa rmel-1 mutant)

sirl-I HMLa MATa HMRa RMEI (XR29-1OC)

If the x- mutation is in S I R I , the diploid will lack functional SIRl gene product and the cryptic mating type genes will be expressed. In this case, a1 function is provided by both the MATa locus and by HMRa, and the a2 function will be provided by both HMLa and HMRa. Hence the diploid will be able to sporulate (STRATHERN, HICKS and HERSKO- WITZ 1981). If the x- mutation is not in S I R I , the diploid will possess at least one wild-type copy of each SIR gene and the HML and HMR loci are expected to be unexpressed. Hence the diploid will not sporulate. The criterion for complementation was established at 1 % sporulation: 5 1 % sporulation indicates complementation; >1% sporulation indicates failure to complement. The mutations that complemented sirl-1 were tested for their ability to complement mutations in other SIR genes.

SIR2 alleles. The mutation marl-I (KLAR, FOGEL and MACLEOD 1979) allows the expression of HMLa and HMRa. Since marl-I and sarl-1 complement, MAR1 defines a second gene that we designate SIR2. sir2-1 mutants are unable to mate since simultaneous expression of HMLa and HMRa in a haploid results in the nonmating phenotype of an a / a diploid. A mutation at HMLa (hmla-) restores the a mating phenotype to a sir2-1 strains (A. J. S. KLAR, personal communication). A MATa hmla- HMRa sir2-1 strain (KM2b- 36c) was used in the complementation tests. The theory of the complementation test is as described for SIRl alleles.

SIR3 and SIR4 alleles. Data presented in this paper indicate that STE8 and STE9 (HARTWELL 1980) define two additional SIR genes, SIR3 and SIR4, respectively. The alleles of STE8 and STE9 used in the complementation tests are designated sir3-8 (strain 520) and sir4-9 (strain 519). Strains 519 and 520 were mated at 23" to the mutants described below and the diploids were tested for the ability to sporulate at 30". This temperature is sufficiently restrictive to obtain unambiguous sporulation results and avoids the reduced sporulation efficiency exhibited by some strains at 34".

Monitoring segregation of sir mutations: The defining mutations in SIR2, SIR3 and SIR4 allow full expression of HMLa and HMRa and therefore confer a non-mating phenotype to otherwise wild-type a and a cells. Thus, segregation of these mutations can be easily monitored by a mating assay. Unlike mutations in SIR2, SIR3 or SIR4, the sirl-1 mutation allows only partial expression of HMLa and HMRa. The effects of this leaky expression cannot be de-

tected on wildtype a and a cells by the conventional mating type assay. Therefore to monitor segregation of sirl-1 in crosses, segregants were mated to a matal sirl-1 strain ( X J 1 16-22c), diploids selected and the resulting diploids were tested for the ability to sporulate. If a segregant contains sir l -I , the diploid formed with XJ116-22c will express the cryptic mating type genes and provide a1 and a2 functions thus allowing the diploid to sporulate.

Nomenclature: All mutants isolated in this study are referred to by a hyphenated number such as 6-8b. The number to the left of the hyphen refers to the clone in which the mutant was found. Thusall mutants with different clone numbers are independent. The characters to the right of the hyphen indicate separate mutants from the same clone.

Strains and crosses: The strains and crosses used in this work are described in Tables 1 and 2.

RESULTS

T h e analysis of mutations that allow expression of the silent mating type locus information of H M L and H M R will be presented in two parts. First, we show that two mutations, ste8 and ste9, define two new SIR genes, SIR3 and SIR4. Second, we describe the isolation of new sir mutants and place these and other sir mutations into complementation groups.

Are STE8 and STE9 SIR genes? A number of temperature-sensitive mutations were isolated on the basis of their ability to cause a cells to be resistant to growth arrest by a-factor (HARTWELL 1980). Muta- tions in many of the genes so identified blocked the mating ability of both a cells and a cells at the non- permissive temperature. Hence these mutations are referred to as ste for sterile. Two mutations were particularly interesting since, at the restrictive temperature, haploid cells carrying mutations in these genes took on the characteristic polar budding pattern of a / a diploid cells (FREIFELDER 1960; HICKS, STRATHERN and HERSKOWITZ 1977; HARTWELL 1980). Since resistance to a-factor and a polar budding pattern are two characteristics of the original sir mutant (HERSKOWITZ et al. 1977; RINE et al. 1979), ste8 and ste9 mutants were studied to determine whether they were in fact sir mutants.

STE8 If STE8 is a SIR gene and thus regulates the expression of the cryptic mating type information at HMLa and HMRa, then the nonmating phenotype of ste8 mutants should depend upon the mating type information at H M L and HMR. Specifically, M A T a ste8 mutants should be nonmaters when they contain at least one cryptic a gene, and MATa ste8 mutants should be nonmaters when they contain at least one cryptic a gene. If STE8 is not a SIR gene, then diploid STE8/ste8 heterozygotes should yield two maters and two nonmaters in each tetrad regardless of the alleles at M A T , H M L and HMR. Furthermore, if STE8 is unlinked to M A T , the number of segregants that mate as a cells should be equal to the number of segregants that mate as a cells.

12 J. Rine and I . Herskowitz

TABLE 1

Strain list

Strain Genotype Source or reference

7 0 227 412 413 519 520 D135a

DC6 G40- 1 b G58-2b G 5 8 - 1 2 ~ J R Y l 3 l JRY49 1

R l O l A E l l XHB71-39d

KM2B-36c

XJ 1 1 6 - 2 2 ~ XMB4-12b XR28-lc XR29- 1 0c

XR 160- 1 2b XR213-2a-15b

XR156-45d

XR261-1 Id XR270-1 Ib XR272-7b XR273-45d XR274-5a XR320-12a

XR320-18a XR32 1-2 la XR323-3d

MATa thr3-I0 MATa lysl-I MATa leu2-I ura2-9, 15, 30, his4-260, 39 MATa leu2-I ura2-9, 15, 30, his4-260, 39, canl MATa cryl-3 ste9-I his4-58am trplam ade2-loc tyrloc lys2oc RMEI MATa cryl-3 ste8-5a his4-58am trplam ade2-loc tyrloc lys2oc R M E l MATaIMATa ade2/ade2 adel/+ tyrl/+ ural /+ his7/+ lys2/+ gall/+ cyh2/+

canl/+ leul/+ cmtlcmt MATa his4 leu2 canl gal2 matal ade6 leul trp5 ura3 rmel canl MATa cryl-3 rmel leu1 trp5 his6 ade6 canl MATa sirl-1 leu2 adeb MATa rad52-I leu2 trpl met10 ura4 his? ade4 matal-5 sir3-45 ade6 his4 leul trp5 gal2 canl met1 MATa hmla- HMRa ade6 marl-I RMEl MATa cryl-1 his4am trplam 1ys2oc tyrloc ade2 leul ura? rmel MATa HMLa HMRa ade6 ural met

I matal sirl-1 ade6 ura3 MATa sstl-I ilv3 arg9 ural killer+ MATa cryl-3 his4 leu2 lys2-I arg4-I7 MATa cryl-3 sirl-1 ade6 arg4-17 leu2 trpl R M E l M A T a HMLa H M R a HO leul ade5 ura3 ura4 rmel canl-11 matal HMLa H M R a ade2 ura3 leul canl-11 cyh2-21 rmel MATa cryl-3 HMLa HMRa ade6 his4 leu2 met MATa sir3-8 his4-58am leu2 trpl ura MATO his4am lys2oc trplam tyrloc ade6 ade2oc MATa cryl-3 cmtl ade6 leu2 his4 arg MATa sir?-L?ts his4 ade2-1 lys2 tyrl trpl M A T a sir4-9ts his4 adeb (ade2 ?) lys2 trpl MATa sir4-351 cryl-I his4am lys2oc trplam tyrloc ura3 leul ade2 rmel canl-11

MATa sir4-?51 cryl-1 hislam lys2oc trplam tyrloc ura3 ade2 cyh2 rmel MATa sirl-717 cryl-3 his4am lys2oc tyrloc trplam ade2 leul ura3 canl-11 cyh2-21 MATa cryl-1 his4am tyrloc cyh2-21 ade2 leul ura? sir2-540

cyh2-21

F. SHERMAN

G. FINK G. FINK L. HARTWELL L. HARTWELL A. HOPPER

J. STRATHERN

A. KLAR

RINE et al. (1979)

The following analysis was carried out to determine whether the phenotype of a ste8 mutant depends upon the alleles of HML and HMR. A M A T a HMLa H M R a strain (XR156-45d) was mated to a MATa HMLa HMRa ste8 strain (520) and segregants from the diploid (XR261) were analyzed (Table 3). A striking observation from Table 3A is that most of the tetrads have more than two segregants able to mate at the restrictive temperature. Therefore, the nonmating phenotype of ste8 is suppressed in some segregants. It is unlikely that a suppressor of ste8 was selected in the formation of the diploid since the diploid was formed efficiently in a cell-to-cell mating. A more plausible explanation is that some combination of alleles contained in XR261 suppresses the ste8 mating defect. ste8 is unlinked to MAT, HML or HMR. If the suppressor is the H M R a allele, suppression should be observed only for MATO ste8 segregants, not MATa ste8 segregants. Since STE8 is unlinked to MAT or HMR, the majority of the M A T a segregants should be capable of mating as a because, among M A T a segregants, only those that are both HMRa ste8 will be

sterile. Approximately one-half of the MATa segregants will be capable of mating as a because all MATa ste8 segregants will be sterile. The ratio of mating- proficient segregants from XR261 was 66a:89a, similar to the 58a:93a expected from the above considerations, given the 39% recombination between MAT and H M R (MORTIMER and SCHILD 1980). More im- portantly, the observed tetrad frequencies approximate that expected if the ste8 phenotype depends upon the allele at HMR, and is in striking disagree- ment with the frequencies expected if the phenotype is independent of the HMR allele. From the genotype of the diploid, we infer that these ste8 segregants that retain the ability to mate as a contain both HMLa and HMRa. This inference is borne out by the isolation of additional sir mutations in the STE8 (SIR3) complementation group as described below.

Similarly, a MATa HMLa HMRa strain (XR2 13-2a- 15b) was mated to a M A T a HMLa HMRa ste8 strain (XR273-45d) to form diploid XR289. As observed for XR261, this cross exhibited an excess of mating- proficient segregants, in this case an excess of a segre-

S. cerevasiae SIR Genes 13

TABLE 3 TABLE 2

List of crosses

Cross Parents"

XR257 17-13 X G58-2b XR261 520 X XR156-45d XR266 519 X XR156-45d XR270 519 X XR261-11d XR272 D-135a-la X XR29-10C XR273 520 X G58-12~ XR274 519 X G58-12~ XR275 52-9 X XR272-7b

XR284 D-135a-la X XR273-45d XR288 XR274-5a X XR213-2a-15b XR299 XR273-45d X XR213-2a-15b

XR276 49-1A X XR272-7b

XR304 5-8 X XR28-lc XR305 17-2 X XR28-lc XR306 4-7 X XR28-lc XR320 7-14 X RlOlAEll XR321 7-17 X RlOlAEll XR323 5-40 X RlOlAEll XR328 XR323-3b X DC6 XR400 520 X DC6 XR402 DC6 X XR320-12a XR403 5 19 X XR274-5a XWRl JRY491 X JRYl3l

a 17-13, 52-9, 49-1A, 5-8, 17-2, 4-7, 7-14, 7-17, 5-40 are all sir mutants isogenic with strain XR160-12b.

gants (Table 3B). Again, it is unlikely that a suppressor of the nonmating phenotype of ste8 was selected during the mating since XR289 was formed efficiently by a cell-to-cell mating. As noted for XR261, the tetrad frequencies observed from XR289 approximate those expected if the ste8 phenotype depends upon the allele at HML. Taken together, the results of XR261 and XR289 suggest that the nonmating phenotype of ste8 is relieved in a strains when both HML and HMR contain a mating type information, and is relieved in a strains when both HML and HMR contain a mating type information. Since the mating defect of ste8 is dependent upon the allele at HML and HMR, we define STE8 as a SIR gene.

As described below, a large number of sir mutants were isolated and several of the mutations in them complement both sirl-1 and sir2-1 but do not complement ste8. Therefore, STE8 defines a new SIR gene which is designated herein as SIR3. STE9: The analysis of STE9 parallels that of STE8.

The mating phenotype of MATa HMLa HMRa ste9 strains was investigated by mating a MATa HMLa HMRa ste9 strain (XR274-5a) with a MATa HMLa HMRa strain (XR213-2-15b). Analysis of this cross (XR288, Table 4A) revealed many tetrads with more than two mating proficient segregants. In addition, the excess of segregants able to mate as a cells and the distribution of tetrad types indicate that the mating defect caused by ste9 in a strains is dependent upon the presence of HML.

Dependence of mating defect of sted segregants on HML and HMR alleles

A. Effect of HMR" on phenotypes of ste8 segregants from XR26 1,

ste8 HMLa a HMRa

+ HMLa a HMRa

Expected distribution (%) if ste8 phenotype is:

lnde end Dependent' ent O P H A on HMR

Observed

Tetrad tvDes allele allele Percent No. of tetrads

2a:Oa:2nm 16.7 16.9 9.9 4 la:la:2nm 66.7 44.7 26.0 18 Oa:2a:2nm 16.7 0.9 0.5 2 2a: la: l nm 0 22.0 12.8 24 1 a:2a: lnm 0 11.0 6.4 9 2a:2a:Onm 0 5.0 2.9 - 1

58 total

B. Effect of HMLa on phenotypes of ste8 segregants from XR289.

ste8 HMLa a HMRa

+ HMLa a HMRa

Expected distribution (%) if ste8 phenotype is: Observed

Independ- Dependent' ent of HML on HML

Tetrad types allele allele Percent No. of tetrads

2a:Oa:2nm 16.7 2.0 0.0 0 la:la:2nm 66.7 12.3 34 34 Oa:2a:2nm 16.7 32.9 16 16 2a:la: l nm 0 10.3 17 17 1 a:2a: lnm 0 37.9 29 29

3 2~2a :Onm 0 4.0 3 - 99 total

"nm, nonmater; a, mates as a; a, mates as a. *Based upon MAT vs. HMR recombination of 80 P.D.:15-

"Based upon MAT vs. HML recombination of 116P.D.:54 N.P.D.:179T (MORTIMER and SCHILD 1980).

N.P.D.:272T (MORTIMER and SCHILD 1980).

The influence of the HMR allele on the phenotype of a ste9 strains was examined in a cross between a MATa HMLa H M R a strain (XR156-45d) and a MATa HMLa HMR a ste9 strain (519), forming diploid XR266 (Table 4B). In a total of 25 tetrads, no a segregants were capable of mating. Slightly more than half of the a segregants were able to mate. A consistent explanation for the results from this diploid is that ste9 became homozygous. All four segregants from a single tetrad of XR266 were found to be sir4 in a complementation test with newly derived sir mutants described below. Thus all a segregants would be nonmaters as they would contain at least one expressed a locus. If STE9 is a SIR gene, the a segregants would not be able to mate unless they inherited HMRa. Since HMRa in this cross is coupled with and linked to MATa, one would expect to find a slight abundance

14 J. Rine and I. Herskowitz

TABLE 4

Dependence of mating defect of ste9 segregants on HML and HMR alleles

A. Effect of HMLa on phenotypes of ste9 segregants from XR288.

ste9 HMLa 01 HMRa

+ HMLa a HMRa


Independ- Dependent ent of HML on HML

Tetrad tvms allele allele Percent No. of tetrads

2a:Oa:2nm 16.7 2.0 6.2 4 1a:la:2nm 66.7 12.3 33.8 22 Oa:2a:2nm 16.7 32.9 9.2 6 2a: 1a:lnm 0 10.3 13.8 9 1 a:2a: 1 nm 0 37.9 18.5 12 2~2a:Onm 0 4.0 18.5 - 12

65 total

B. Effect of HMRa on phenotypes of ste9 segregants from XR266.

~~

ste9 HMLa a HMRa

ste9 HMLa a HMRa


Independ- Dependent ent of HMR on HMR

Tetrad types allele allele Percent No. of tetrads

1 a:Oa:3nm 0 62 72 18 2a:Oa:2nm 0 26 20 5

2 Oa:Oa:4nm 100 12 8 - 25 total

HMRa SIR "a" mating type - HMLa mats 1

OFF

MUTAG ENESl S

HMLa mata 1 HMRa "a" mating type

ON ON ON FIGURE 2.--Scheme for isolation of sir mutants. The top strain

contains cryptic a cassettes at both HML and HMR in addition to the matal mutation. matal has no effect on the mating type of the top strain, but unlike MATa, matal is recessive to MATa (KASSIR and SIMCHEN 1976). Therefore, any mutation leading to expression of either HMLa or HMRa will result in a change from a to a in the mating phenotype of the strain.

of segregants with the a mating type as was found. Complementation tests described below indicate

that among the sir mutants that complement ski-I, sir2-I, and sirj-8, several fail to complement ste9.

TABLE 5

Phenotypic classes of representative sir1 mutants ~ ~~~

Sporulation efficiency' after mating with

a-Factor Mating Mating produc- Mata MATa No. of

Class" as ab as a tion s i r l - l SIR1 mutants

- ++ ++ - 15" 4 2 9

A ++ B ++ ++ + C ++

- - - ++ - - - D +/++ ++/+ - ++

~ ~ ~ ~~~~

a Class A includes mutants 2-5, 6-1, 13-3A, 14-1 3A, 15-1 1,24-2, 26-2, 31-10A, 34-8, 35-1, 42-12, 43-11, 45-10A, 46-2. Class B includes 1-8, 18-2, 23-1 1, 27-12B. Class C includes 8-5B, 16-10. Class D includes 4-13, 7-8, 10-2A, 21-3, 21-5, 22-5A, 25-llB, 30- 3, 33-8A. The relevant genotype of the parental strain (XR160- 126) is HMLa matal HMRa.

*In this table and subsequent tables mating efficiency that IS scored as +++ indicates a confluent patch of growth on mating type tester lawns; ++ indicates that individual papillae are discern- ible over the entire patch; + indicates that papillae over the patch are well separated.

'-,no sporulation; +, ~ 2 0 % sporulation; ++, 220% sporulation.

Therefore STE9 defines a new SIR gene; this temperature sensitive allele is designated sir4-9.

Isolation of sir mutants: As noted above, sir mutants are associated with a mating defect. In order to isolate additional sir mutations and simultaneously avoid isolating additional ste mutations unrelated to SIR, a special strain was constructed as shown in Figure 2. This strain has only cryptic a genes at both HML and HMR as well as the a l - 1 mutation at MAT. Unlike MATa, mata l -1 is recessive to M A T a such that mata l - I IMATa diploids have the a mating type (KAS- SIR and SIMCHEN 1976). Therefore, a mutation isolated in this strain that leads to expression of either HML or HMR or both should result in a mutant with the mating behavior of an a cell. Reconstruction experiments confirmed that sir3-8 and sir#-9 in H M L a matal -1 H M R a strains exhibited the a mating type at the restrictive temperature. The rmel mutation cir- cumvents the need for MATa and M A T a functions and allows diploids to sporulate regardless of mating type (KASSIR and SIMCHEN 1976; RINE et al. 198 1).

Fifty-two independent cultures of the parent strain (XR160-12b) were mutagenized and plated onto YEPD plates; half of the plates from each clone were grown at 30" and the remaining half were incubated at 23" or 33". Once grown, the colonies were tested for their ability to mate with both an a and an a mating type tester lawn. Among 675,000 colonies screened, 296 colonies were able to mate with an a mating type tester. Each clone had from one to 16 mutants and the overall mutant frequency was 4 x

All mutants were capable of growth at 23", 30" or 36" regardless of the temperature at which they were isolated. Therefore, it is unlikely that these regulators of HML and HMR expression have an essential role in the cell.


TABLE 6

Quantitative complementation tests of representative sirl mutations

Sporulation efficiency (%) after mating with' a-Factor produc-

Mutant Mating as a Mating as a tion 23" a a sirl-1 a sir.2-1 a sir3-8 a sir4-9

6-1 ++ - +++ <o. 1 85 <o. 1 CO. 1 CO. 1 7-8 +/++ +/++ N T CO. 1 77 0.2 CO. 1 co.1

43-1 I ++ - +++ co.1 90 CO. 1 <o. 1 CO. 1

"The diploids formed between the mutants 6-1, 7-8, or 43-1 and the a strains were transferred to spore plates and the efficiency of sporulation determined after 5 days. The strains used in the sporulation tests of Tables 6 , 7, 8, 9 and 10 were: a SIR, 227; a sir2-1, KM2B- 36c; a sir3-8, 520; a sir4-9, 5 19. NT, not tested.

TABLE 7

Complementation tests of representative SIR2 mutations

Sporulation efficiency (%) after mating with a-Factor pro- No. of

Class" Mating as a Mating as a duction a a sirl-1 a sir2-1 a sir3-8 a sir4-9 mutants

A +++ - +++ CO. 1 CO. 1 280 CO. 1 <0.1 5 B +++ +++ - <o. 1 <o. 1 2 1 1 CO. 1 <o. 1 3 C +++ + - CO. 1 CO. 1 21 ,53 CO. 1 co.1 2

"Class A includes mutants 18-IA, 30-1, 33-8B, 37-3, 45-10B. Class B includes mutants 1-9B, 6-13A, 47-6. Class C includes mutants 28-7 and 39-7. These mutants mate as a cells at 23" and as (Y cells at 34".

TABLE 8


Sporulation efficiency (%) after mating with a-Factor No. of

Class" Mating as a Mating as a production a a sirl-1 a sir2-1 a sir3-8 a sir4-9 mutants

A ++ - CO. 1 CO. 1 CO. 1 3, 3 <o. 1 2 B ++ - +++ CO. 1 <o. 1 1.0, 2.0 18,66 CO. 1 2 C +++ - +++ CO. 1 0.2-0.4 0.2-2.0 5-68 <o. 1 5 D +++ - + to +++ 0.2 0.1-5.0 0.8-2.0 11-31 <o. 1 3 E +++ +++ 10 .2 0.4, 0.6 1 .o 58 ,61 0.2 2 F +++ +++ - CO. 1 <0.1, 5.0 0.3, 2.0 2.0, 82 CO. 1-4.0 2

-

-

"Class A includes mutants 1-7B and 27-12A. Class B includes mutants 17-13 and 33-4. Class C includes mutants 1-9A, 13-7, 15-4B, 32- 13. Class D includes mutants 6-1 1 , 13-9, 33-7. Class E includes mutants 11-4, 13-5. Class F includes mutants 22-7, 50-1 1.

SIRl: Among all mutants mated to a sirl-1 strain (XRZg-lOc), a total of 73 were unable to complement sirl-1 as indicated by the ability of the diploids to sporulate. Table 5 describes 30 independent SIRl mutations that are representative of the phenotypes associated with these mutants. Most mate only as a. However, some of the mutants were able to mate with both a cells and with a cells and do not form a-factor halos. This ability of a colony to mate with both an a lawn and an a lawn is termed the bimating phenotype. Therefore, it is possible that the SIR1 mutations in these bimating mutants allow lower leve1 expression of the cryptic a mating type genes. As a working hypothesis, those mutants that exhibit a bimating phenotype or lack of a-factor production are considered to bear leaky sirl mutations. Both of these general classes of sirI mutants gave efficient sporulation with the sirl-1 tester, indicating noncomplementation of the sirl-1 mutation. A quantitative analysis of the complementation tests for some SIRl mutants is presented in Table 6.

The mutations that complemented sirl-1 were tested for complementation with other sir mutations by mating the mutants to an a sir2-1 strain (KM2b- 36c), an a sir3-8 strain (520), and an a sir4-9 strain ( 5 19) and the diploids were checked for the ability to sporulate.

SIR2: Thirteen mutations were identified in the SIR2 complementation group and the phenotypes resulting from 10 representative SIR2 mutations are presented in Table 7. The mutations in the SIR2 complementation group efficiently complement the defining alleles in SIRl, SIR3, and SIR4 as indicated in the table.

SIR3: Thirty-one mutants were identified whose mutations failed to complement the sir3-8 mutation. Data for 16 representative mutants are given in Table 8. As with sirl and sir2 mutations, some sir3 mutations cause the mutants to mate as a cells and to produce a-factor, whereas other mutations result in the bimating phenotype and no a-factor production. These

16 J. Rine and I . Herskowitz

TABLE 9


Sporulation efficiency (%) after mating with

Class Mating as a a-Factor pro-

Mating as a duction a a s i r l - 1 a sir2-1 a sir3-8 a si74-9

A (47-2) B (2-14B) C (1 8-5) D (1-15B) D (4-5) D (1 6-7) D (28-13A) D (33-1 1A) D (37-1 1B) D (45-1A) E (6-8) E (9-1 2) E (1 3-2) F (1-5B) F (1-1 1) F (1-12) F (28-1)

++ ++ +++ +++ +++ +++ ++ +++ +++ +++ +++ +++ ++

+++ +++ +++ +++

+++ ++ +++ +++

- N T +++ +++ +++ +++ +++ +++ +++

N T N T +++

- N T N T +++

- - - -

- - - - - - -

-

- -

<o. 1 <o. 1

0.3 <o. 1 <o. 1 <o. 1 <o. 1 <o. 1 <o. 1 <o. 1 <o. 1 <o. 1 C O . 1 <o. 1 <o. 1 <o. 1 <o. 1

0.7 7.1 2.7 6.2 3.5 2.4 5.0 3.7 4.0 3.9 0.8 0.1 7.4

11.6 5.6

19.5 10.1

<o. 1 3.4

10.1 2.4 1.6 1.3 1.8 2.8 3.2 1.4 2.1 0.2 9.0 4.6 2.1 8.0 4.7

<o. 1 0.3

<0.8 <o. 1 <o. 1 <o. 1 <o. 1 <o. 1 <o. 1 <o. 1 <o. 1 <o. 1 <o. 1 <0.1 <o. 1 <o. 1 <o. 1

16.0 28.0 63.0 22.9 35.0 12.0 23.0 31.0 58.0 17.0 38.0 25.0 34.0 29.5 13.3 33.0 19.0

"Class A contains mutant 47-2. Class B contains mutant 2-14B. Class C contains mutant 18-5. Class D can be generalized as sir- mutants with the efficiency of complementation being sir?-8 > sir2-l > s i r l - l > sir4-9. Class E can be generalized as sir- mutants with the efficiency of complementation being sir?-8 > sirl-Z > sir2-Z > sir4-9. Class F contains mutants that are tentatively assigned to the SIR4 gene rather than SIR1 or SIR2 due to the relative strength of the complementation with these test alleles.

mutations were placed in the SIR? complementation group because they fail to complement the defining sir3 mutation. Interestingly, some mutations that are most deficient in SIR? complementing ability for sporulation are able to complement sirl and sir2 mutations only poorly. Mutants 6-11 and 50-11 are extreme examples of this pleiotropic phenotype and fail to complement sirl-I .

SIR4 Twenty-four mutations from 17 independent clones were assigned to the SIR4 complementation group. Data for 17 representative mutants are presented in Table 9. All sir4 mutations are clearly recessive to wild type. As was seen in the previous section for sir3 mutations, some sir4 mutations complement sirl and sir2 mutants only weakly. As above, mutations were assigned to the sir4 complementation group based upon the relative levels of complementation of the defining sir mutations. As can be seen in Table 9, the distribution of complementing ability among mutations in this group is quite variable. The only gen- eralization that emerges is that mutations in the SIR4 complementation group usually complement s i r58 efficiently, and often fail to complement sirl-1 or sir2- 1.

Others: Seventeen mutants could not be assigned to any of the four complementation groups and 12 are characterized in Table 10. A common characteristic of these mutants is that they fail to produce a- factor and many are bimaters. This observation sug- gests that these mutants may be very leaky alleles of one of the four SIR genes and retain enough activity to complement each of the four SIR mutations in the

tester strains. In fact, 10 of the mutants show some evidence of incomplete complementation of one of the defining SIR alleles (frequency of asci is greater than 0.001 but less than 0.010). Other possibilities are discussed below. It is of interest to note that all mutants with less leaky phenotypes can be assigned to one of the four complementation groups described here.

Organization of the SZR genes: In order to determine whether the S I R genes are clustered or are distinct genes, the map positions of the S I R genes relative to each other and to the centromere were investigated.

Centromere linkage: The data presented in Table 11 indicate that S I R I , S IR3 and SIR4 are not linked to a centromere or to trpl . In contrast, the data of KLAR, FOGEL and MACLEOD (1 979) indicate that SIR2 is linked to t rp l . Therefore S I R I , S IR3 and SIR4 cannot be tightly linked to SIR2.

Linkage of SZRI vs. SIR3: A diploid formed by mating an a sir3-8 strain (520) with an a-sirl-I strain (G58-12c) was used to determine whether S I R l and SIR3 are linked. The segregation of sirl-1 in tetrads from this diploid (XR273) was determined as described in MATERIALS AND METHODS, and the segregation of sir3-8 was determined by testing the mating ability of segregants at both 23" and at 34". Tetrad analysis of XR273 indicates that S I R l and S I R 3 are unlinked (9 PD: 44 T: 12 NPD). The segregation pattern of sir3-8 and sirl-1 indicates that segregants containing both sirl-1 and sir3-8 are unable to mate at either the permissive or restrictive temperature: in

S . cerevisiae SIR Genes 17

TABLE 10

Complementation tests of representative unclassified mutations ~ ~~

a-Factor produc-

Sporulation efficiency (%) after mating with

Mating as a Mating as a tion at No. of Class" 23" 23" 23" a a sirl-1 a sir2-1 a sir3-8 a sir4-9 mutants

A ++ ++ - CO. 1 <o. 1 <o. 1 <0.1 CO. 1 5 B ++ ++ - <o. 1 0.2, 0.3 CO. 1 <o. 1 CO. 1 2 C ++ ++ - CO. 1 CO. 1 CO. 1 0.7 <o. 1 1 D ++ ++ - co.1 0.5, 1.0 co.1 CO. 1 0.8, 0.9 2 E ++ ++ - CO. 1 CO. 1 CO. 1 <0.1 0.2, 0.4 2

"Class A contains mutants 8-1B, 10-9, 11-5, 26-5 and 51-7. Class B contains mutants 37-5 and 45-14. Class C contains mutant 1-4. Class D contains mutants 12-13A and 33-10B. Class E contains mutants 30-13 and 31-10B.

TABLE 11

Mapping and allelism of SIR genes

Diploid

XR273 XR274 XR270 XR272 XR275 XR284 XWRl

SIR genes

sir2 vs. trpIb sirl-1 vs. trpl sir3-8 vs. trpl sir4-9 vs. trpl sirl-1 vs. sir3-8 sirl-1 vs. sir4-9 sir3-8 vs. sir4-9 cmt vs. sirl-1 cmt vs. sir3-52 sir3-45 vs. sir3-8 sir3-45 vs. ura4

~~ ~ ~

Tetrad types"

PD T NPD Interpretation

25 4

12 10 9

14 8 3

43 47 10

30 15 48 38 44 31 46 13 2 0 8

0 5

12 9

12 12 10 0 0 0 1

SIR2 linked to TRPl SIRl not centromere linked SIR3 not centromere linked SIR4 not centromere linked SIRl + SIR3 not linked SIR1 + SIR4 not linked SIR3 + SIR4 not linked CMT + SIRl not linked CMT + SIR3 tightly linked sir3-45 + sir3-8 tightly linked SIR3 is linked to URA4

"Tetrad types for each cross are described in text. Data from KLAR, FOGEL and MACLEOD (1979).

tetrads with two temperature-sensitive maters, the other two segregants contain sirl-1; and in tetrads with two segregants unable to mate at either temperature, the other two segregants contain neither sirl-1 nor sir3-8.

Linkage of SIR1 vs. SZR4: The linkage of SIRl and SIR4 was examined in a cross (XR274) of an a sir4-9 strain (519) with an CY sirl-1 strain (G58-12c). The results of XR274 (Table 11) indicate that SIRl and SIR4 are unlinked. Furthermore, the pattern of segregation of XR274, analogous to that of XR273, indicates that the sirl-1 sir4-9 double mutant is a nonmater at both the restrictive and permissive temperature.

Linkage of SIR3 vs. SZR4: The linkage of SIR3 and SIR4 was determined by mating an a sir4-9 strain (5 19) with an CY sir3-8 strain (XR261-1 ld) forming the diploid XR270. Tetrads from XR270 were classified as PD, T, or NPD on the basis of the number of segregants per tetrad that were able to mate at 34": tetrads with no maters at 34" are PD; tetrads with one mater at 34" are T; and tetrads with 2 maters at 34" are NPD. The data from XR270 (Table 11) indicate that SIR3 and SIR4 are unlinked. In most

tetratype asci, there is one segregant which mates at both low and high temperature and another segregant which mates at neither temperature. These latter segregants are presumably the sir3-8 sir4-9 double mutants.

Linkage of SIR3 and URA4: In several independent crosses in which both sir3 and ura4 were heterozy- gous, both markers became unexpectedly homozygous, suggesting that both markers were on the same chromosome arm. The map position of SIR3 was determined in cross X W R l , and demonstrates that SIR3 is linked to URA4 which is known to map on the right arm of chromosome XII. Based upon these mapping experiments, we conclude that the SIR genes represent four distinct loci. These genes must encode distinct diffusible products and cannot represent independent domains of a multifunctional protein.

Characterization of a SIR-like gene: On the basis of phenotype differences between the sirl-1 allele and cmtl , it was suggested that cmtl is a mutation in a gene other than SIRl that regulates HML and HMR (HABER and GEORGE 1979). Complementation tests were performed to determine whether cmtl affects a known SIR gene. Two different a cmtl strains were mated to


four matal HMLa H M R a mutants, each containing a mutation in a different sir gene. The complementation test is identical to those described earlier: complementation is indicated by the inability of a diploid to sporulate and noncomplementation by the ability to sporulate. Both a cmt strains were able to complement sir1 (mutant 9-12B), sir2 (mutant 6-8), and sir4 (mutant 37-3); but neither a cmtl strain was able to complement sir3 (mutant 52-9).

The allelism of CMT and SIR3 was determined by analyzing the diploid formed between an a cmt strain (XR272-7b) and matal HMLa HMRa sir3 (mutant 52-9). If cmtl and sir3 are allelic, each a segregant should contain at least one cryptic a gene, which is expressed due to the sir or cmtl mutation, and therefore will be a nonmater. If cmtl and sir3 are not allelic, then a SIR recombinants should be recovered which would be able to mate with an a tester strain. Among 45 tetrads from XR275, only two a segregants with the a mating type were recovered. The two a’s were probably due to conversion of one of the two mutations to a wild-type allele or to intragenic recombination forming a wild-type allele. Segregants with the a mating phenotype were recovered from XR275 which, when mated to a MATa strain, were unable to promote sporulation. Presumably, the segregants with the a phenotype have the genotype matal HMLa HMRa and contain either the cmtl or the sir3 mutation. Therefore, we conclude that cmtl is a mutation in the SIR3 gene.

At least one SIR gene encodes a protein: The existence of nonsense suppressible alleles of a locus is presumptive evidence that the locus encodes a protein product. Several different sir mutants were crossed to an a strain containing several amber- and ochre-suppressible auxotrophic mutations (RlOl-AE1 1) and sir recombinants containing these mutations were identified. Nonsense suppressors were introduced into these strains by selection for simultaneous reversion of either two amber alleles or two ochre alleles. Two different segregants (XR320-12a, -1 sa) containing the sir#-351 allele were found to gain mating ability in the presence of ochre suppressors but to remain nonmaters in the presence of amber suppressors. Other alleles of various SIR genes were unaffected by either class of suppressors. Therefore nonsense suppression per se has no effect on the sir- phenotype. Both XR320-12a and XR320-18a, each containing an ochre suppressor, were mated to a strain containing the same amber and ochre suppressible auxotrophic markers (XR270-4b). Tetrad analysis reveals that the ochre suppressor was capable of restoring mating ability to both a and a strains containing the sir#-351 allele. Furthermore, this ability cosegregated with suppression of ochre auxotrophic mutations. T o confirm that sir4351 is in fact an allele of SIR#, a cross was

TABLE 12

Characterization of a nonsense allele of SIR4

Strain Relevant genotype“ Mating efficiency

412 a SIR4 1 .o XR320-12a a sir4-351 sup 1 . 1 x io-’ XR320-12a-1 a sir4-351 SUPoc 0.1 XR320-12a-2 a sir4-351 SUPam 3.6 x 10-7 XR323-3d a sir2- sup 1.4 x IO-’

~ ~~

“sup indicates a strain with no nonsense suppressors. SUPoc indicates a strain with an ochre suppressor. SUPam indicates a strain with an amber suppressor.

performed by mating an a sir#-9 strain to an a sir#- 351 by prototrophic selection. In each of 25 tetrads, there were no SIR4 recombinants. Therefore sir4-351 is a bona jde SIR4 allele.

The quantitative effect of an ochre suppressor on the sir#-351 nonmating phenotype (shown in Table 12) indicates a 106-fold enhancement of mating e%- ciency in the presence of ochre suppressors. Nonsense suppression in yeast is typically no better than 10% efficient (GILMORE, STEWART and SHERMAN 197 1). We infer that, in strains containing sir#-351 and SUPoc, less than the normal amount of SIR4 protein is made. Therefore at low concentrations of SIR#, the mating efficiency of strains is not proportional to the amount of SIR protein.

Analysis of dominant mutations: Twenty-three independent mutants with a dominant a phenotype were recovered from the screen described above. Eight independent mutants were mated to a MATa strain (XR28-lc) carrying the cryl-3 mutation, a marker tightly linked to the mating type locus, and the diploids were subjected to tetrad analysis. The diploids formed by mutants 5-8 and 17-2 XR304 and XR305) yielded 2a:2a segregants in each of 14 and 20 tetrads, respectively. The a segregants carried the cryl-3 allele and the a segregants carried the CRY1 allele. Therefore, both dominant a’s resulted from a switch of the MAT allele from matal to MATa. The six remaining mutants were probably diploids since, when crossed to XR28-1 c they displayed the poor spore viability characteristic of triploids (RINE 1979).

DISCUSSION

Four genes regulate expression of cryptic mating type loci: Complementation tests were performed on the sir mutations isolated in a matal HMLa HMRa strain in order to determine how many SIR genes are involved in regulating expression of the cryptic mating type loci of S. cerevisiae. All of the mutations that cause a strong sir- phenotype can be assigned to one of four complementation groups. Since there are at least 10 independent mutations in each complementation group, it is likely that most nonessential genes directly involved in negative regulation of HML and HMR


have been identified. One point regarding the isolation of sir mutants merits discussion. The isolation of the mutants described in this paper required only that the a genes at HML or HMR (or both) be expressed. There was no requirement that cryptic a genes be expressed in sir mutants. Nevertheless, genetic analysis indicates that all mutations that were isolated due to their failure to regulate silent a genes also fail to regulate silent a genes. Thus, all sir mutations result in the expression of both HMLa and HMRa. We have not eliminated the possibility that there may be a SIR gene specifically dedicated to regulation of HMLa. However, the existence of such a gene appears unlikely since in a screen capable of uncovering genes specifically dedicated to regulation of HMRa, no such mutations were found.

In general, complementation tests are definitive only when the diploid used in the test has been sub- sequently analyzed to insure that the mutations did not revert or recombine to produce a wild-type gene. Only a small fraction of the complementation tests reported here have been subjected to tetrad analysis. Therefore the assignment of mutations to complementation groups must be considered tentative. How- ever, several considerations support the general valid- ity of the complementation groups presented. First, the assignment of a mutation to a given complementation group is based upon both the ability of that mutation to complement the defining alleles of other complementation groups and the inability of the mutation to complement the defining allele of the group to which the mutation is assigned. Second, the way in which the complementation test is performed insures that the result is based upon many individual tests: a large colony (patch) of the mutant to be tested (-lo6 cells) is replicated onto an a lawn containing an allele of one of the SIR genes. The resulting diploid colony is thus formed from hundreds of individual zygotes. Therefore, a low frequency event such as reversion or mitotic recombination will affect only a small fraction of the cells in the diploid colony. Because of the unusual complementation results with some mutations in the SIR4 complementation group, it is most likely that if any mutations have been misidentified, they will be from this group. A previous report of a fifth SIR gene (KASSIR and SIMCHEN 1985) has recently been withdrawn (see “Corrigendum,” this issue).

All four SIR genes are unlinked to each other and therefore must define independent gene products. SIR2 maps on chromosome IV (KLAR, FOGEL and MACLEOD 1979). SIR3 has been mapped to chromosome XII near URA4. [These conclusions have been confirmed and extended by IVY, HICKS and KLAR (1985).]

The SIR genes regulate both cryptic a and cryptic (Y information: The sir2-1, sir3-8 and sir4-9 mutations

all result in the nonmating phenotype in both a and a cells. In fact, all three were initially characterized as nonspecific ste mutations. The analysis of KLAR, Fo- GEL and MACLEOD (1 979) for sir2-1, and the analysis here for sir3-8 and sir4-9 indicate that the nonmating phenotype of these mutations is due to the simultaneous expression of the a mating type genes at HMRa and the a mating type genes at HMLa, which results in the nonmating phenotype of an a/a diploid. The only SIRl mutation that has been analyzed in detail is sirl-1 (RINE et al. 1979). This mutation does allow expression of both HMLa and HMRa, yet it does not result in the nonmating phenotype characteristic of mutations in the other SIR genes. Furthermore, in combination with either sir3-8 or sir4-9, sirl-1 behaves synergistically to produce a phenotype more extreme than that caused by either mutation alone. To date, no SIRl mutations have been identified that express HMLa and HMRa at levels sufficient to result in a nonmating phenotype.

Unusual complementation behavior: Several observations in the complementation analysis of sir mutations raise an interesting question: how can two recessive mutations in different genes fail to complement each other? As an example of this problem, compare mutations in SIR3 with mutations in the SIR2 complementation group. The majority of the sir2 mutants have been quantitatively analyzed and result in unambiguous complementation of the defining alleles in the other SIR genes. Furthermore, sir2 mutations are clearly recessive to wild type. In contrast, most alleles of SIR3 do not efficiently complement sir2-1, barely complement sirl-1 (using 5 1 % sparula- tion as the criterion for complementation), yet efficiently complement si7-4-9. Although some sir3 mutations, such as those in class D, may not be completely recessive, most of the sir3 mutations are clearly recessive to wild type.

The complementation behavior of sir4 mutations is an exaggerated version of the SIR3 complementation results. sir4 mutations are recessive to wild type and efficiently complement sir3-8. However, the complementation of sir2-1 and sirl-1 by sir4 mutations (e.g., class C) is inefficient. In fact, an independently derived sir4 allele has recently been described that fails to complement sir1 or sir4 mutations (KASSIR and SIMCHEN 1985).

Although there is, as yet, no demonstration that the complementation behavior of each sir3 and sir4 mutation segregates as a single mutation, it is unlikely that most of the sir3 and sir4 mutants resulted from double or triple mutations. Furthermore, the phenotypes cannot be due to the polar effects of a mutation in one gene on other SIR genes since the SIR genes are not clustered. Furthermore, plasmids containing individual SIR genes appear to complement these


mutations, indicating that the strains do not contain multiple SIR mutations (J. RINE, unpublished data).

An explanation that is consistent with the erratic complementation behavior is that the gene products of at least some of the SIR genes interact in a complex. Thus, since most mutations cause an altered polypeptide (missense) rather than no polypeptide (nonsense), the altered polypeptide may participate in forming a protein complex, yet may not functionally interact with the other products of the locus. By this model, the recessive nature of the mutations must lie in their ability to be outcompeted by sufficient quantities of wild-type SIR gene products.

There are several other cases of lack of complementation between recessive mutations in yeast and other organisms. For example, recessive mutations in EAMZ or EAM2 are suppressors of choZ mutations in yeast, yet eamZ and eam2 mutations do not complement (ATKINSON 1985). Similarly, tup7 (pho80) mutations fail to complement recessive mutations in four other TUP genes (BISON and THORNER 1982). A particularly informative example of noncomplementation of recessive mutations comes from studies of mutations that fail to complement certain @2 tubulin alleles in Drosophila melanogaster. Based upon genetic criteria, some of these mutations may be in the genes for proteins that interact with @ tubulin (FULLER 1986).

SIR mutations have been identified by several phenotypes: Our results indicate that the ste8 and ste9 mutants, isolated in a strains as a-factor resistant mutants, are mutations in SIR3 and SIR4. By a similar selection, J. MCCULLOUGH (personal communication) has isolated additional ste mutants that, by complementation analysis, appear to be defective in SIR genes. The cmtZ mutation, originally identified by its ability to allow an ala diploid to sporulate (HOPPER and HALL 1975), is a mutation in SIR3. Similarly, the sca mutation, a natural variant in strains that allows a/a diploids to sporulate (GERLACH 1974), is also an allele of SIR3 (J. MARGOLSKEE and I . HERSKOWITZ, unpublished observation). In short, mutations in SIR genes have been found among mutations originally identified by their mating, sporulation, or a-factor resistance phenotypes, and may be common among mutations affecting other processes under mating type control. A summary of the mutations in the SIR genes and other names under which these mutations have been classified is presented in Table 13.

Models of SIR regulation: The results presented here demonstrate that the combined action of four different gene products is required to regulate the expression of the silent mating type genes of yeast. Evidence that the gene products are proteins is provided by the nonsense allele of sir4 presented here and the sequence of the SIR2 and SIR3 gene (SHORE, SQUIRE and NASMYTH 1984). The available evidence

TABLE 13

Distribution of sir alleles and assignment of other genes to SIR complementation groups

Other genes in

No. of in- same com- dependent plementa-

Gene mutations tion group References ~~

SIR1 39 HERSKOWITZ et al. (1977)

SIR2 10 MAR1 KLAR, FOGEL and MACLEOD

SIR3 17 STE8 HARTWELL ( 1 980)

RINE et al. (1 979)

(1979)

cmt SCA GERLACH (1 974) MAR2

HABER and GEORGE ( 1 979)

A. J. S. KLAR (personal communication)

SIR4 12 STE9 HARTWELL (1 980)

indicates that this regulation affects the steady-state level of transcripts from HML and HMR, presumably by blocking transcription initiation (NASMYTH et al. 1981; KLAR et al. 1981). The requirement for multiple trans-acting regulators is compatible with two general classes of models. One class of models includes regulatory cascades in which one protein influences the activity or synthesis of another which, in turn, influences another until eventually the last protein is synthesized or activated. Examples of transcriptional cascades are abundant in the biology of template bacteriophage (SUSSKIND and YOUDERIAN 1983), yet are unlikely to explain the role of four SIR genes since mutations in any one SIR gene have no effect on the steady state level of mRNA encoded by the other three genes (IVY, KLAR and HICKS 1986). As a sec- ondary possibility the regulatory cascade could be analogous to the activation of enzyme activity by cycles of phosphorylation and dephosphorylation (STADT- MAN, CHOCK and RHEE 198 1).

A second class of models involves the formation of a complex or structure assembled from the products of different SIR genes. The protein-protein interac- tions in such a complex might be very sensitive to alterations in any one component of the complex. It is somewhat easier to rationalize the intricate complementation patterns with models invoking formation of a complex than with models limited to regulatory cascades.

We thank LEE HARTWELL and AMAR KLAR for strains and unpublished results that were essential for this work, and BUFF BLAIR, GEORGE SPRAGUE, ROB JENSEN and DOUGLASS FORBES for numerous comments and suggestions during the course of this work. In addition, we thank ALICE WIELAND for help in mapping SIR3, ELIZABETH JONES for numerous helpful comments on the manuscript, and PEGGY MCCUTCHAN-SMITH for preparation of the manuscript. This work was supported by U . S. Public Health Service Research grant AI-13462.


LITERATURE CITED

ABRAHAM, J., K. A. NASMYTH, J. N. STRATHERN, A. J. S. KLAR and J. B. HICKS, 1984 Regulation of mating-type information in yeast: negative control requiring sequences both 5’ and 3‘ to the regulated region. J. Mol. Biol. 176: 307-331.

ASTELL, C. R., AHLSTROM-JONASSON, M. SMITH, K. TATCHELL, K. A. NASMYTH and B. D. HALL, 1981 The sequence of the DNAs coding for the mating type loci of Saccharomyces cerevisiae. Cell 27: 15-24.

ATKINSON, K. D., 1985 Two recessive suppressors of Saccharomy- ces cermisiae chol that are unlinked fall in the same complementation group. Genetics 111: 1-6.

Mutations in the PH080 gene confer permeability to 5’-mononucleotides in Saccharo- myces cereuisiae. Genetics 102: 341-359.

BLAIR, L. C., 1979 Genetic analysis of mating type switching in yeast. Ph.D. dissertation, University of Oregon, Eugene, Ore- gon.

Identification of sites required for repression of a silent mating type locus in yeast. J. Mol. Biol. 178: 815-834.

Bud position in Saccharomyces cereuisiae. J. Bacteriol. 80: 567-568.

Genetic analysis of spermatogenesis in Dro- sophila: the role of the testis-specific p tubulin and interacting genes in cellular morphogenesis. pp. 19-4 1 . In: Gametogenesis and the Early Embryo, Edited by J. G. GALL. Alan R. Liss, New York.

GERLACH, W. L., 1974 Sporulation in mating type homozygotes of Saccharomyces cereuisiae. Heredity 32: 241-249.

GILMORE, R. A., J. W. STEWART and F. SHERMAN, 1971 Amino acid replacements resulting from super-suppression of nonsense mutants of iso-1-cytochrome c from yeast. J. Mol. Biol. 61: 157-173.

A mutation that permits the expression of normally silent copies of mating type information in Saccharomyces cereuisiae. Genetics 93: 13-35.

HARASHIMA, S., Y. NOGI and Y. QSHIMA, 1974 The genetic system controlling homothallism in Saccharomyces yeasts. Genetics 77:

Mutants of S. cerevisiae unresponsive to cell division control by polypeptide mating hormones. J. Cell Biol. 85: 81 1-822.

Control of cell type in Saccharomyces cereuisiae: mating type and mating type interconversion. pp. 181-209. In: The Molecular Biology of the Yeast Saccharomyces: Li&e Cycle and Inheritance, Edited by J. N. STRATHERN, E. W. JONES and J. R. BROACH. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

HERSKOWITZ, I., J. N. STRATHERN, J. B. HICKS and J. RINE, 1977 Mating type interconversion in yeast and its relationship to development in higher eucaryotes. pp. 193-202. In: Proceed- ings to the 1977 ICN-UCLA Symposium: Molecular Approaches to Eucaryotic Genetic Systems, Edited by G. WILCOX, J. ABELSON and C. F. Fox. Academic Press, New York.

Interconversion of yeast mating types. I. Direct observation of the action of the homothallism (HO) gene. Genetics 83: 245-258.

HICKS, J. B., J. N. STRATHERN and I. HERSKOWITZ, 1977 Interconversion of yeast mating types. 111. Action of the homothallism (HO) gene in cells homozygous for the mating type locus. Genetics 85: 395-405.

HOPPER, A. K. and B. D. HALL, 1975 Mating type and sporulation in yeast. I. Mutations which alter mating-type control over sporulation. Genetics 80: 41-59.

Map positions of yeast genes S I R l , SIR3 and SlR4. Genetics 111: 735-744.

BISSON, L. AND J. T. THORNER, 1982

FELDMAN, J. B., J. B. HICKS and J. R. BROACH, 1984

FREIFELDER, D., 1960

FULLER, M. T., 1986

HABER, J. E. and J. P. GEORGE, 1979

639-650. HARTWELL, L. H., 1980

HERSKOWITZ, I. and Y. OSHIMA, 1981

HICKS, J. B. and I. HERSKOWITZ, 1976

IVY, J. M., J. B. HICKS and A. J. S. KLAR, 1985

IVY, J. M., A. J. S. KLAR and J. B. HICKS, 1986 Cloning and characterization of four SIR genes of Saccharomyces cermisiae. Mol. Cell. Biol. 6: 688-702.

JOHNSON, A. and I. HERSKOWITZ, 1985 A repressor (MATa2 product) and its operator control expression of a set of cell type specific genes in yeast. Cell 42: 237-247.

Regulation of mating and meiosis in yeast by the mating-type region. Genetics 82: 187- 206.

KASSIR, Y. and G. SIMCHEN, 1985 Mutations leading to expression of the cryptic HMRa locus in the yeast Saccharomyces cereuisiae. Genetics 109 481-492.

KLAR, A. J. S., S. FOGEL and K. MACLEOD, 1979 M A R l , a regulator of HMa and H M a loci in Saccharomyces cereuisiae. Genetics 92: 759-776.

KLAR, A. J. S., J. N. STRATHERN, J. R. BROACH and J. B. HICKS, 198 1 Regulation of transcription in expressed and unexpressed mating type cassettes of yeast. Nature 289 239-244.

KOSTRIKEN, R., J. N. STRATHERN, A. J. S. KLAR, J. B. HICKS and F. HEFFRON, 1983 A site-specific endonuclease essential for mating type switching in Saccharomyces cereuisiae. Cell 35: 167-174.

A new method for hybridizing yeast. Proc. Natl. Acad. Sci. USA 2 9 306-308.

Genetic map of Saccharo- myces cereukiae. Microbiol. Rev. 44: 5 19-57 1 .

The regulation of yeast mating type chro- matin structure by SIR: an action at a distance affecting both transcription and transposition. Cell 30 567-578.

NASMYTH, K. A. and K. TATCHELL, 1980 The structure of trans- posable yeast mating type loci. Cell 1 9 753-764.

NASMYUTH, K. A., K. TATCHELL, B. D. HALL, C. ASTELL and M. SMITH, 1980 Physical analysis of mating-type loci in Saccha- romyces cereuisiae. Cold Spring Harbor Symp. Quant. Biol. 45:

NASMYTH, K. A., K. TATCHELL, B. D. HALL, C. ASTELL and M. A position effect in the control of transcription

KASSIR, Y. and G. SIMCHEN, 1976

LINDEGREN, C. C. and G. LINDEGREN, 1943

MORTIMER, R. K. and D. SCHILD, 1980

NASMYTH, K. A., 1982

961-98 1 .

SMITH, 1981 at yeast mating type loci. Nature 289: 244-250.

RINE, J. 1979. Ph.D. thesis, University of Oregon, Eugene. RINE, J., G. F. SPRAGUE, JR. and I. HERSKOWITZ, 1981 rmel

mutation of Saccharomyces cereuisiae: map position and bypass of mating type locus control of sporulation. Mol. Cell. Biol. 1:

RINE, J., J. N. STRATHERN, J. B. HICKS and 1. HERSKOWITZ, 1979 A suppressor of mating type locus mutations in Saccha- romyces cerevisiae: evidence for and identification of cryptic mating type loci. Genetics 93: 877-901.

RINE, J., R. JENSEN, D. HAGEN, L. BLAIR and I. HERSKOWITZ, 1981 Pattern of switching and fate of the replaced cassette in yeast mating-type interconversion. Cold Spring Harbor Symp. Quant. Biol. 45: 951-960.

SANTA MARIA, J. and D. VIDAL, 1970 Segregacibn anormal del “mating type” en Saccharomyces. Inst. Nac. Invest. Agron. Conf. 30: 1-21.

SHORE, D., M. SQUIRE and K. A. NASMYTH, 1984 Characterization of two genes required for the position effect control of yeast mating type genes. EMBO J. 3: 2817-2823.

SPRAGUE, G. F., JR., J. RINE and I. HERSKOWITZ, 1981a Homology and nonhomology at the yeast mating type locus. Nature 289:

SPRAGUE, G. F., JR., J. RINE and I. HERSKOWITZ, 1981b Control of yeast cell type by the mating type locus. 11. Genetic interac- tions between M A T a and unlinked a-specified STE genes. J. Mol. Biol. 153: 323-335.

STADTMAN, E. R., P. B. CHOCK and S. G. RHEE, 1981 Interconvertible enzyme cycles in cellular regulation. Curr. Top. Cell Regul. 18: 79-94.

Asymmetry and directionality in production of new cell types during clonal

958-960.

250-252.

STRATHERN, J. N. and I. HERSKOWITZ, 1979


growth: the switching pattern of homothallic yeast. Cell 17:

Control of cell type in yeast by the mating type locus. The (ul-aZ hypothesis. J. Mol. Biol. 147: 357-372.

STRATHERN, J. N., A. J. S. KLAR, J. B. HICKS, J. A. ABRAHAM, J. M. IVY, K. A. NASMYTH and C. MCGILL, 1982 Homothallic switching of yeast mating type cassettes is initiated by a double- stranded cut in the MAT locus. Cell 31: 183-192.

371-381. STRATHERN, N., J. B. H ~ c ~ s a n d I. HERSKOWITZ, 1981

SUSSKIND, M. and P. YOUDERIAN, 1983 Bacteriophage P22 anti- repressor and its control. pp. 347-363. In: Lambda II, Edited by R. W. HENDRIX, J. W. ROBERTS, F. W. STAHL and R. A. WEISBERG. Cold Spring Harbor Laboratory, Cold Spring Har- bor, New York.

A gene for diploidization in yeasts. C. R. Trav. Lab. Carlsberg. Ser. Physiol. 28: 341-346.

Communicating editor: E. W. JONES

WINCE, 0. and C. ROBERTS, 1949

four genes responsible for a position effect on … genes responsible for a position effect on...

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