Copyright 0 1985 by the Genetics Society of America

MUTATIONS LEADING TO EXPRESSION OF T H E CRYPTIC HMRa LOCUS IN T H E YEAST SACCHAROMYCES

CEREVISIAE

YONA KASSIR AND GIORA SIMCHEN

Defartment of Genetics, The Hebrew University of Jerusalem, Jerusalem 9 1 904, Israel

Manuscript received May 18, 1984 Revised copy accepted October 19, 1984

ABSTRACT

Mutations leading to expression of the silent HMRa information in Saccha- romyces cereuisiae result in sporulation proficiency in mata l /MATa diploids. An example of such a mutation is sir5-2, a recessive mutation in the g w e SlR5. As expected, haploids carrying the sir5-2 mutation are nonmaters due to the simultaneous expression of HMRa and HMLa, resulting in the nonmating phe- notype of an a / a diploid. However, sir5-2/sir5-2 mata l /MATa diploids mate as a yet are capable of sporulation. The sir5-2 mutation is unlinked to s i r l -1 , yet the two mutations do not complement each other: mata l /MATa sir5-2/SIR5 S l R l / s i r l - l diploids are capable of sporulation. In this case, recessive mutations in two unlinked genes form a mutant phenotype, in spite of the presence of the normal wild-type alleles.-The PASI-1 mutation, Provider of a Sporulation function, is a dominant mutation tightly linked to HMRa. PASI-1 does not affect the mating ability of a strain, yet it allows diploids lacking a functional MATa locus to sporulate. It is proposed that PASI-I leads to partial expression of the otherwise cryptic a1 information at HMRa.

ATING type in the yeast Saccharomyces cerevisiae is controlled by two co- M dominant alleles of the mating-type locus (MAT). Cells with the a-mating type have the MATa allele, and cells with the a-mating type have the MATa allele. The mating of an a cell to an a cell leads to a third cell type known as an a/a diploid. .a/a cells have two characteristics that distinguish them from a/a and a/a cells: they are unable to mate (a/a and a/a cells mate as the respective haploids) and are capable of sporulation (ROMAN and SANDS 1953). We shall refer to this phenotype as nonmater Spa+. The a and a alleles encode regulators that control the expression of other loci concerned with mating and sporulation (MACKAY and MANNEY 1974; STRATHERN, HICKS and HERSKOWITZ 198 1 ; SPRAGUE, JENSEN and HERSKOWITZ 1983).

MATa and MATa are not typical alleles; they differ from each other by a unique region, Y, of about 700 base pairs (bp) (STRATHERN et al. 1980; NAS- MYTH and TATCHELL 1980; NASMYTH et al. 1981). Two transcripts that are transcribed in opposite directions have been shown to exist for each allele. Both a transcripts and the a1 transcript start at Y, whereas the a2 transcript seems to begin in X (NASMYTH et al. 1981; ASTELL et al. 1981; see also review by NASMYTH 1982).

Genetics 109: 481-492 March. 1985.

Y. KASSIR AND G. SIMCHEN

TABLE 1

Strain list

Strain Genotype/relevant markers Source or reference

Haploids 309

53 1

535

720 919 17-15

17-16

2009

5404-la 540 1 -4b

5 4 0 4 - 1 ~ 5 4 0 4 - 4 ~ 5404-6a

5404-8a 5404-47a

alf l-28 H70 H227 349-1 K394

XJ104-25a

5404-6b

5404-53b

XR197-6d

Y130 Y131 Y138

Diploids 540

540-a1 5401 5401-7

5401-8

5402 5404 5404-1 5404-2

MATa ade2-RB canl-11

MATa RME ade2 tyrl-2 ural his7-1

MATa RME ade2 his(7 or 6 ) tyrl-2

MATa adel MATa cdc6 his4 ural MAL2 gall SUC matal rme ade2 ura3 canl-11 leul cyh2-21

matal RME ade2 ura3 trpl canl-11 cyh2-21

MATa HMRa sir5-1 leul lys2 matal HMRa sir5-2 MATa PASI-I ade2 ura3 trpl matal PASI-1 ade2 hi47 or 6) tyrl-2 MATa PASI-1 ade2 his(7 or 6 ) MATa PASl-1 ade2 his(7 or 6 ) ura3 matal PAS1-I ade2 trpl matal PASI-1 leu2 matal PASI-I ade2 trpl matal PASl-1 his7 ade2 trpl urax metx matala2 rme ade5 leul leu2 lys2 try7 MATa ham3 MATa lysl-I MATa cdc24-3 ural tyrl-2 lys(2 or 4) hmIa2:LEU2 MATa HMRa cry1 MAL2 leu2

lys(2 or x) adel can1 thr4 trp(1 or 3) mata.2-1 sirl-1 RME his4 arg4 ade6 lys2 MATa cry1 HMRa sirl-1 ade6 leu(1 or 2)

MATa HMRa ade2 lys2 trp5 ha HMLa MATa HMRa ade2 trp5 lys2 HO HMLa MATa HMRa ade2 ade5 trpl

ura4

his5

matallMATa RMEIRME canl-1 IICANl

mata-IethallMATa matallMATa sir5-2/sir5-2 HMLa matal HMRaIHMLa MATa HMRa

sir5-21SIR5 sir1 -I /SIR 1 HMLa matal HMRaIHMLa MATa HMRa

sir5-llsir5-2 mata-IethallMATa matal PASl -1IMATa PAS 1 mala 1 PAS 1 - 1 IMATa PASl - 1 mala 1 PAS 1 - 1 /MATa PASl

~yh2-21/CYH2

KASSIR and SIMCHEN ( 1 976)

KASSIR and HERSKOWITZ ( 1 980)

KASSIR and HERSKOWITZ ( 1 980)

G. SIMCHEN N361-9a, G. KAWASAKI KASSIR and SIMCHEN

(1976) KASSIR and HERSKOWITZ

(1980) Y . KASSIR 540 1 5404 5404 5404 5404 5404 A. SHALOM 5404 5404 J . STRATHERN I . HERSKOWITZ I . HERSKOWITZ A. SHALOM A. KLAR and J . HICKS

J . STRATHERN J . R I N E

Y . KASSIR Y . KASSIR Y . KASSIR

17-16 X 535

540 540 5401-4b X XR197-6d

5401-4b X 2009

540 540 5404-1c X 5404-la 5404-1c X 309

EXPRESSION OF THE HMR LOCUS

TABLE I-Continued

483

Strain Genotype/relevant markers Source or reference

5404-3 matal PASl - I l M A T a PASl 5404-6b X 309 5404-5 5404-6 MATa PASIIMATa PASI-I 531 X 5404-6a 5404-7 MATa PASI-IIMATa PASl 5404-6-a1

matal a2 PAS I IMATa PAS I - I alfl-28 X 5404-4c

5404-1 0 matal PASI-I HMRaIMATa PASl HMRa 5404-53b X Y131

Tetraploid 5404-6-a1 MATa/MATa/MATa/MATa PASI-I /PAS1 - 1 1 UV induced in 5404-6

PASl/PASI

In addition to MAT, two other loci in yeast also harbor copies at MATa and MATa genes. These loci are known as HMR and HML and are located on the right and left arms of chromosome IZZ, respectively. In standard laboratory strains, a silent copy of the MATa sequence is present at HMR, and a silent copy of the MATa sequence is present at HML. However, all combinations of sites and information have been characterized and can be introduced into laboratory strains by genetic crosses (HERSKOWITZ and OSHIMA 198 1 ; NAS- MYTH 1982). Normally, only the a or a information at MAT is expressed, whereas the same information at HMR and HML is not expressed. Rather than serving as sites for expression of a and a genes, HMR and HML serve as a source of mating-type cassettes that are expressed following transposition to the MAT locus in the process of mating-type interconversion (reviewed in HERSKOWITZ and OSHIMA 198 1 ; SPRAGUE, BLAIR and THORNER 1983). Expres- sion of the a and a genes at HMR and HML is blocked by the action of at least five genes: SIRl, SIR2 (MARl ) , SIR3 (CMT), SIR4 and SIR5 (HABER and GEORGE 1979; RINE 1979; RINE et al. 1979; KLAR, FOGEL and MACLEOD 1979; GRANOT, Y. KASSIR and G. SIMCHEN, unpublished results). A mutation in any one of these genes leads to the in situ expression of the information at HMR and HML, independent of mating-type interconversion. Since the mutations in the SIR are recessive and unlinked to HMR and HML, the SIR genes must encode trans-acting regulators. Evidence for cis-acting sites tightly linked to HMR that are necessary for SIR regulation has been provided by duplications involving HMR (KASSIR, HICKS and HERSKOWITZ 1983; HICKS et al. 1984) and by deletions adjacent to HMR (HAWTHORNE 1963; ABRAHAM et al. 1983).

In this paper we present an analysis of both cis- and trans-acting mutations that lead to the partial or full expression of the a information at HMR.

MATERIALS AND METHODS

Media: YEPD (rich medium), SPO (for induction of sporulation) and SD (for scoring nutritional markers) are media that have been described (HICKS and HERSKOWITZ 1976). CAN CYH agar (for scoring resistance to canavanine and cycloheximide) is SD supplemented with all amino acids (except arginine), uracil and adenine, as well as canavanine sulfate (40 rg/ml) and cycloheximide

Strains: Strains are described in Table 1 . Strain 2009 was derived from strain 5584 of J. RINE. The sir mutation in strain 5584 was originally thought to be a mutant allele of SIR4 (RINE 1979) because it did not complement the szr4-9 mutation and complemented mutations in the genes

(1.8 rglml).

484 Y . KASSIR AND G . SIMCHEN

S I R l , SIR2 and SIR3. However, we have recently shown by genetic analysis the mutation in strain 5584 to be nonallelic to sir4-9, and, therefore, we designated it sir5-1 (GRANOT, Y. KASSIR and G. SIMCHEN, unpublished results). Furthermore, the SIR5 gene was cloned and mapped to chromo- some VU (GRANOT, Y. KASSIR and G. SIMCHEN, unpublished results).

Genetic techniques: Mating, sporulation induction, tetrad analysis and mating-type determination have been described (HICKS and HERSKOWITZ 1976).

Isolation of mutants: Strain 540 has the genotype matal /MATa canl-11ICANl cyh2-21/CYH2. Since can l - I1 and cyh2-21 are recessive mutations, this diploid cannot grow on media containing canavanine and cycloheximide. Furthermore, this diploid cannot sporulate due to the matal mu- tation, which results in loss of MATa function that is essential for sporulation (KASSIR and SIMCHEN 1976; STRATHERN, HICKS and HERSKOWITZ 1981). Since CAN1 and CYH2 are unlinked, any mutation that causes strain 540 to regain the ability to sporulate should result in haploid spores containing both canl-11 and cyh2-21 and which would, therefore, be able to grow on CAN CYH media. This double drug resistance phenotype was used as an initial screen for mutations leading to sporulation of strain 540. The ability of mutants identified by this assay to sporulate was confirmed by direct observation of asci in a sporulated culture.

For mutagenesis, cells of strain 540 were grown to logarithmic phase in YEPD and then mu- tagenized by one of two protocols: (1) Cells were mutagenized in water by ultraviolet irradiation at a dose giving 50% viability. The irradiated cells were then grown in liquid YEPD for 3 hr and plated onto YEPD plates. (2) Cells wre mutagenized in water but following the 3 hr of growth, they were irradiated again (in water) with a low dose of ultraviolet light, insufficient to cause further killing. The second irradiation was done in order to induce mitotic recombination and homozygosis of recessive mutations. Following the second irradiation, the cells were plated onto YEPD plates. Colonies arising from either procedure were replica-plated onto SPO plates, incu- bated 4-6 days at 30" and then were replica-plated from the SPO plates onto CAN CYH plates.

Monitoring the segregation of matal: matal mutants mate as MATa cells. The phenotype of the matal mutation is evident only in diploids. Therefore, a-mater segregants from crosses involving matal were mated to a wild-type MATa strain and the resulting diploids were tested for the ability to mate and sporulate. mata l /MATa diploids have the a-mating type and cannot sporulate (a mater Spo-), whereas MATa/MATa diploids are nonmater and can sporulate. Some matal segregants in these experiments contain a dominant mutation that suppresses the sporulation defect of the matal mutation. When such segregants were mated to a strains, the resulting diploids were able to sporulate but retained the a-mating type (a mater Spo+). Thus, these diploids were distinguishable from true MATa/MATa diploids.

matal HMLa H M R a segregants containing a sir- mutation (mutants with expressed copies of a genes at both HML and HMR) have the a-mating phenotype. Since the sir- mutations are all recessive, these strains can be distinguished from true MATa strains by mating to a MATa SIR tester strain. matal HMLa HMRa sir- strains, when mated to a MATa strain, form diploids that have the a-mating phenotype and fail to sporulate, since expression of the a genes at HML and HMR is prevented in SIR strains. True MATa cells mated to the same tester form nonmater Spo+ diploids.

Identzjcation of HMR alleles: HMRa and HMRa were distinguished as described (KASSIR, HICKS and HERSKOWITZ 1983). matal and MATa segregants were mated to strain Y138 (HO HMLa MATa HMRa) (prototroph selection). In the presence of an a cassette at HMR, the HO gene promotes homothallic interconversion and the resulting MATaIMATa diploid can sporulate. If the tested segregant has an a cassette at HMR the diploid remains sporulation deficient.

RESULTS

Mutagenized matal /MATa diploid colonies were screened for mutations al- lowing the cells to regain the ability to sporulate. Three classes of mutations predictable upon earlier work are ( 1 ) simple revertants of matal , (2) bypassing of the MAT mutation such as rmel that bypasses mating-type control of spor- ulation and meiosis (KASSIR and SIMCHEN 1976) and (3) mutations leading to

EXPRESSION OF THE HMR LOCUS

TABLE 2

Types of Spa' derivatives obtained from strain 540 (matal/MATa)

485

No. of tetrads Segregation Strain Phenotype checked (in tetrads)" Genotype

540-6 540-7 540-12 540-2 540-14 540-1 7 5406

5402 540 1 5404 5405

540-011

nm Spo+ nm Spo+ nm Spo+ nm Spo+ nm Spo+ nni Spo+ nm Spo+ aspo+ aspo+ aspo+ aspo+ asPo+

5 7 4

17 7

23 32 12 28 35 57

Nonviable

a ,a a,a a,a 2a:2a 2a:2a 2a:2a 2a:2a 2a:2 lethals 2a:2 lethals 4nm spo- 2al-:2a

a l a a l a a l a al. al. a l a al. a lethalla a lethalla a l - la sir5-21sir5-2 al-a PASI/PASI-I

a = a-mater segregants that gave rise to nm Spo' diploid upon mating to MATa (H227) tester strain. a = a-mater segregants that gave rise to nm Spo+ diploid upon mating to MATa (H70) tester strain. al- = a-mater segregant that gave rise to &PO- diploid upon mating to MATa (H70) tester strain.

a When viability was low, e.g., among progeny of 540-6, only the types of progeny are shown.

expression of cryptic a information from HMRa (KASSIR, HICKS and HERSKOW- ITZ 1983). No mutation of the second class has been found.

Screening of 4 1,474 mutagenized colonies gave rise to 47 Spo+ colonies. Of these, 42 were unable to mate, and five retained the a-mating phenotype of the original diploid cells (Table 2). The latter five were derived from the double irradiation procedure (see MATERIALS AND METHODS).

Seven of the 42 nonmater Spo+ strains were subjected to tetrad analysis and shown to be MATalMATa diploids (first seven lines, Table 2). In these mutants, the matal mutation either reverted or was lost by a heterothallic switch re- placing the defective matal cassette with a functional a cassette from HMR.

Two of the a-mater Spo+ mutants, 540-a1 and 5402, when sporulated re- sulted in only two viable spores per tetrad, and the viable spores were exclu- sively a's. Although these mutants bear a superficial resemblance to a deletion characterized previously that led to expression of HMRa (HAWTHORNE 1963; STRATHERN et al. 1979), these two mutant diploids clearly had the a-mating type, whereas the previous deletions led to full expression of HMRa and thus a nonmating phenotype. We infer that, in these two a Spo+ mutants, either a deletion (hence the lethality) has resulted in leaky expression of HMRa or an unequal recombination event between HMRa and MAT has resulted in a SAD- like phenotype (KASSIR, HICKS and HERSKOWITZ 1983; HICKS et al. 1984), namely, a fused HMR-MAT locus that is expressed at a low level.

Strain 5405 gave no viable spores in any of the 60 tetrads dissected, and, therefore, no further work has been done with it.

Strain 5401: Strain 5401 (a-mater Spo+ derivative), when sporulated, pro- duced asci in which all four spores were nonmaters and unable to sporulate. All eight segregants of two complete tetrads were mated to MATa (H227) and

486 Y. KASSIR AND G . SIMCHEN

T A B L E 3

Sporulation of diploids constructed by mating progeny of strain 5401 to dqferent tester strains

Sporulation after mating to:

Inferred no. M A T a MATa M A T a H M R a s i r l -1 M A T a H M R a sir5-1 genotype

Segregant H227 H70 XR197-6d 2009

4a 4b 4c 4d 6a 6b 6c 6d

N T + +

N T N T N T + +

N T + +

N T N T N T + +

M A T a sirx matal sirx matal sirx M A T a sirx M A T a sirx M A T a sirx matal sirx matal sirx

N T = not tested.

MATa (H70) tester strains, by low frequency prototroph selection, and the resulting diploids were examined for their ability to sporulate. It is evident (Table 3) that both tetrads have two segregants that contain MATa, since they sporulate when mated to an a strain but not when mated to an a strain. We dissected asci from two such diploids, namely, segregant 4a X MATa and segregant 4d X MATa (12 asci from each) and obtained segregation of two maters to two nonmaters in each tetrad. (“Two maters” indicates either 2a, 2a or 1 a: 1 a.) The other two segregants of strain 540 1 probably contain the matal allele since they fail to sporulate when crossed to either an a or an a strain. Since all four spores in each tetrad failed to mate at normal frequency, we conclude that the mutation present in 5401 is homozygous. As the matal segregants containing this mutation fail to sporulate when crossed to a MATa tester strain, yet sporulate in the original diploid, the mutation responsible for sporulation must be recessive. Since sir mutations are recessive mutations that affect mating and sporulation, we determined by complementation and allelism tests (Table 3) whether the mutation in 5401 is a mutation in a previously defined SIR gene.

Four presumptive matal segregants from strain 540 1, each containing the mutation of interest, were mated to an HMLa MATa HMRa sirl-1 and to HMLa MATa HMRa sir5-1 strains (strains XR 197-6d and 2009, respectively). Diploids homozygous for a recessive sir mutation should express the otherwise silent a1 gene at HMRa and, therefore, be able to sporulate. Thus, one set of diploids were expected to sporulate if the new mutation is in SIRl or SIR5. Surprisingly, both sets of diploids sporulated efficiently (80% sporulation after 48 hr on SPO medium). We conclude that either 5401 has mutations in both SIRl and SIR5 or it has an allele of one that cannot be complemented by one copy of the other.

To map the new sir mutation relative to SIRl and SZR5, one spore clone (4b, Table 3) was mated to the sirl-1 and the sir5-1 tester strains and the resulting diploids, strains 540 1-7 and 5401-8, respectively, were examined by

EXPRESSION OF THE HMR LOCUS

TABLE 4

Phenotypes of the various genotypes among the meiotic segregants expected from strain 5401-7 and 5401-8

487

Genotype sir SIR

matal HMRa nm a matal HMRa a a MATa HMRa nm a MATa HMRa a or

a = a-mater; or = a-mater; nm = nonmater; a- indicates that mating of the or-mater segregant to MATa strain results in m a t a l l MATa Spo- diploid (see MATERIALS AND METHODS). All segregants are assumed to be HMLa.

-

tetrad analysis. Because of the complex genotype of the diploid, the segregation of expected mating types is somewhat baroque and is explained in Table 4. If sirx, the new mutation, is allelic to the sir mutation in the tester strain, then no SIR+ recombinants should be found. Thus, the most easily verifiable con- sequence of allelism of sir mutations is the complete absence of segregants with the a-mating type. This prediction was fulfilled in the cross with sir5-1 (Table 5), whereas in the cross to the strain containing sirl-1, a mater segregants were observed, and the types of tetrads expected from two unlinked mutations were evident. Thus, sirx is a mutant allele of SIR4 that we designate sir5-2. We conclude that the genotype of strain 540 1 was matal/MATa sir5-2/sir5-2. We have also shown that a matal/MATa sirl-l/SIRl sir5-2lSIR5 diploid sporulate, i.e., recessive mutations in two different SIR genes do not comple- ment each other.

Strain 5404: Strain 5404 (a-mater Spo' derivative) gave four viable spores per tetrad with Pa-mater and 2a-mater segregants in each of 57 asci that were examined. All a-mater segregants were mated to a MATa tester strain (H70), and the resulting diploids were checked for their ability to mate and to sporulate. Fifty-three segregants gave rise to a-mater Spo+ diploids, whereas the rest (61) gave rise to a-mater Spo- diploids. Therefore, we conclude that strain 5404 contained the matal allele and that it is heterozygous for a mu- tation that can provide a sporulation functions to diploids. Furthermore, this mutation must be dominant. We have designated this mutation PASl-1 for Provider of a Sporulation functions. In some cases sporulation provided by PASl-1 is very efficient, allowing 80% of the diploid cells to sporulate within 2 days in sporulation medium (see later for exceptions.)

If the PASl-1 mutation is able to provide all a functions necessary for spor- ulation, then any diploid containing both MATa and PASl-1 should sporulate. T o test this hypothesis, we examined all 114 a-mater segregants from 5404 for the ability to sporulate, when mated to strains of three different genotypes: MATa, matal and matala2 (a double mutant inactivating both genes of MATa). The data presented in Table 6 demonstrate that all 114 a segregants, when mated to MATa strains, formed diploids capable of sporulation, as expected. Fifty-one segregants were unable to sporulate when mated to a variety of matal

488 Y. KASSIR AND G. SIMCHEN

TABLE 5

Type of tetrads obtained from strains 5401-7 and 5401-8

No. of tetrads obtained from:

Type of tetrad 5401-7 540 1-8

2nm:a:a- 2nm:2a 2nm:2a- nm:a:a:a- nm a:2a- 2nm:a:a nm:2a:a

Strains 5401-7 and 5401-8 resulted from mating 5401-4b (matal HMRa sirx) to XR197-6b ( M A T d M R a sirl-1) and to 2009 (MATaHMRa sir5-1), respectively.

TABLE 6

Segregation of PASl-1 among the MATa progeny of strain 5404

Sporulation following mating to:

Presumed H227 17-16 17-15 alfl-128 No. of genotype a al-Rh4E al-rme a l - a l r m e segregants

- - - MA TaPAS 1 + MA TapAS 1 - 1 + + N T +

+ MATaPAS 1 - 1 + MATaPASl-1 + + N T

- - -

51 31 30

2 ~~ ~

N T = not tested.

or matala2 strains. These segregants were inferred to contain the wild-type PAS1 gene. The remaining 63 segregants were capable of forming sporulation- competent diploids when mated to the matal strain, the matala2 strain or to both. Therefore, these segregants contain the PASI-I mutation. Not all of the PASI-I segregants were able to sporulate with every matal or matala2. Since there are many irrelevant explanations for lack of sporulation of any single diploid, we interpret sporulation with any one or more of the tester strains as indicating the presence of PASI-I . By these criteria, PASI-IIPASI heterozy- gous strains display in tetrads 2:2 segregation for the ability to provide a sporulation function. PASI-I/PASI-I homozygous strains display 4:O segrega- tion of this phenotype (Table 7). In these tests the phenotype of the PASI-I mutation was quantitatively different from what it was in the original PASI-I diploid in which the sporulation levels were high. In the tests of MATa PASI- 1 segregants, sporulation was poor, 5-15% after 6-10 days. The basis of this difference in PASI-1 penetrance appears to be due to the mating type of the strain that contributes the PASI-I mutation. When PASI-I is inherited through the MATa parent the phenotype is weak. When a diploid cell inherits PASI-I through the a parent (matal or matala2) then its phenotype is strong. This dependence of PASl-I phenotype in a diploid on the genotype of the PASI-I

EXPRESSION OF THE HMR LOCUS 489

TABLE 7

Segregation of PASI-I in dgerent strains

No. of progeny containing PASZ-lltetrad

Strain Genotype 4PASI-I JPASI-1 PPASI-I IPASI-I OPASI-I

5404 PASI-I/PASI 0 4 52 1 0 5404-2 PASl - 1 /PAS1 0 0 5 0 0 5404-3 PASI-I/PASI 0 0 8 0 0 5404- 1 PASl - 1 /PAS1 -I 8 0 0 0 0

TABLE 8

Mapping of PAS 1 - 1

Gene PD NPD T Total cM Strain

PAS 1 -I :MAT 87 38 31 1 436 61.8 5404-10 PAS 1 -I :HMR 53 0 0 53 0

a Accummulated data from strains: 5404, 5404-2, 5404-3, 5404-10, 5404-47a X Y130, 5404- 47a X 919, 5404-8a X K394, 5404-47a X 720 and 5404-47a X 349-1.

parent was observed in crosses using many strains from different backgrounds (data not shown).

PASI-I was able to suppress the sporulation defects of the following diploids: mataI /MATa(5404) , mataIa2/MATa(5404-5) and MATaIMATa (5404-7) . How- ever, PASI-l did not enable the following diploids to sporulate: mata2/MATa, mata2/matal, and MATaIMATa. In short, PASI-I provided sporulation to any diploid that expressed the MATa2 function but did not provide sporulation to any diploid lacking this function. Therefore, PASl-I provides a and only a functions necessary for sporulation.

A reasonable hypothesis for the PASI-I mutation is that it allows partial expression of the otherwise cryptic a1 gene at HMR. To test this hypothesis, the PASI-I mutation was mapped relative to HMR. A HMLa matal PASl-I HMRa strain (5404-53b) was mated to an HMLorMATorHMRa strain (Y 131) to form diploid 5404-10 . Since matal /MATa PASI- l /PASI and MATaIMATa PASI-I/PASI diploids sporulate due to PASI-I , the HMR allele could be de- termined only among the PASl segregants. The matal PAS1 and MATa PASl segregants were mated to an HO HMLa MATa H M R a strain ( Y 1 3 8 ) and dip- loids were isolated by prototroph selection (see MATERIALS AND METHODS). The data in Table 8 indicate that PASl-I is tightly linked to HMR and is approxi- mately 6 2 cM distal to MAT.

DISCUSSION

The mutations described here that allow mata l /MATa diploids to sporulate have as a common theme the activation of the cryptic a information at HMR. Mutations analogous to rmel (KASSIR and SIMCHEN 1976) were not found. Since only one mutant allele of RMEl exists, a mutation such as rmel may be

490 Y. KASSIR AND G. SIMCHEN

rare and may not be a simple loss of function. Since the seven mutations from the nonmater Spo+ class that were analyzed all proved to be MATalMATa diploids, we assume that the majority or even all members of this class are identical. We suggest that these mutants arose by heterothallic interconversion events, possibly stimulated by the UV-irradiation.

The sir5-2 and PASl-1 mutations are relevant to the nature of repression of the silent mating-type loci. These loci are regulated by the products of the SIRl , SIR2, SIR3, SIR4 and SIR5 genes. An analysis of a large number of mutations in these genes identified recessive alleles of one gene that appeared to belong to more than one complementation group. That is, some sir mutants were inefficiently complemented in diploids containing one defective allele of a different SIR gene (RINE 1979). Our sir5-2 mutation appears to be of the same type. This mutation maps to SIR5 and is recessive, yet it fails to comple- ment other recessive mutations in both SIRl and SIR5. The nonrecessive na- ture of sir5-2 in diploids containing a sir1 mutation is consistent with the two mutant products interacting and thus interfering with the products from the wild-type gene. Alternatively, regulation of HMR and HML may be insensitive to a 50% decrease in the relative level of any one SIR gene product but may be unable to tolerate a 50% decrease in the level of two of the products. It will be interesting to examine the complementation of sir5-2 in diploids het- erozygous for SIR2, SIR3 and SIR4 mutations. The one phenotype of sir5-2 that still eludes explanation is the difference between its phenotype in haploids and diploids. Namely, sir4-2 haploids are nonmaters, whereas matal /MATa sir4-2/sir4-2 is an a-mater. We offer no explanation, although we would like to mention a somewhat similar phenomenon, namely, matal sirl-Z is a bimater, whereas MATa sirl-1 is an a-mater (RINE et al. 1979) .

PASl-1 is a dominant mutation linked to HMR that results in the leaky expression of the a1 gene at HMRa. Could PASI-1 affect the site(s) of SIR action at HMRa? ABRAHAM et al. ( 1 9 8 3 ) have shown that two regions-to the left and to the right of HMR (and HML), designated E and I, respectively- are required for the normal repression of the silent copy. The E and I sites lie outside the homologous information (X Y and ZI) , about 900 and 1000 bp, respectively, away from the central promotor of HMR; E and I extend to a maximum of 260 and 85 bp, respectively. Deletions extending into the E site result in full expression of HMR and consequently affect both mating and sporulation behavior. Deletions extending to the I site result in partial expres- sion and affect only sporulation. These results agree with those of KASSIR, HICKS and HERSKOWITZ ( 1 9 8 3 ) and HICKS et al. ( 1 9 8 4 ) that the site of SIR action lies to the left of HMR. It was, therefore, suggested that this site cor- responds to the E site (ABRAHAM et al. 1983) .

The nature of the PASl-Z mutation is not known. It might be a small deletion of the I site but not a large one, as Southern blot analysis of PASl-1 strains showed the proper length of HMR (data not shown; a 200-bp deletion could easily be visualized). A deletion of the E site is less probable as PASl-Z does not affect mating and MATa PASl-1 HMRa strains are bonafide a-maters. PASl-1 could also be a point mutation at E, at I or at another site. If so, its

EXPRESSION OF THE HMR LOCUS 491

molecular characterization would be of importance to the understanding of the repression of HMR.

An interesting feature of the PASl-1 mutation is the cis effect from MAT on its expression. matal PASl-1 HMRa strains give rise to mata l /MATa diploids that sporulate well, whereas MATa PASl-1 HMRa give rise to mata l /MATa diploids that sporulate poorly. We offer no explanation for this, as more work is required to establish this phenomenon.

We thank JASPER RINE for his considerable help in writing the manuscript, ANNE SHALOM for her assistance and advice and JEANNE MARGOLSKEE for reading the manuscript. This research was supported by a grant from the United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel.

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Communicating editor: 1. HERSKOWITZ