Copyright 0 1983 by the Genetics Society of America

A MUTATION ALLOWING EXPRESSION OF NORMALLY SILENT a MATING-TYPE INFORMATION IN SACCHAROMYCES

CEREVISIAE

HARRY GRUENSPAN' AND NORMAN R. EATON

Biology Department, Brooklyn College of the City University of New York, Brooklyn, New York 11210

Manuscript received June 25, 1980 Revised copy accepted January 26,1983

ABSTRACT

Mating type in haploid cells of the yeast Saccharomyces cerevisiae is deter- mined by a pair of alleles MATa and MATa. Under various conditions haploid mating types can be interconverted. It has been proposed that transpositions of silent cassettes of mating-type information from HML or HMR to MAT are the source of mating type conversions. A mutation described in this work, desig- nated AONl, has the following properties. (1) MATa cells carring AONl are defective in mating. (2) AONl allows MATa/MATa but not MATa/MATa diploids to sporulate: thus, AONl mimics the MATa requirement for sporulation. (3) mata-1 cells that carry AONl are MATa phenocopies, i.e., MATa/mota-1 AONl diploids behave as standard MATa/MATa cells: therefore, AONl sup- presses the defect of mata-1. (4) AONl maps at or near HMRa. (5) Same-site revertants from AONl lose the ability to convert mating type to MATa, indi- cating that reversion is associated with the loss of a functional HMRa locus. In addition, AONl is a dominant mutation. We conclude that AONl is a regulatory mutation, probably cis-acting, that leads to the constitutive expression of silent a mating-type information located at HMRa.

APLOID cells of the yeast Saccharomyces cerevisiae can be divided into H two mating types, a and a (LINDEGREN and LINDEGREN 1943a,b). Cells of one mating type are able to mate efficiently with cells of opposite mating type to produce zygotes that are capable of vegetative growth. Mating type is determined by two codominant alleles of a mating locus that have been denoted MATa and MATa (HERSKOWITZ et al. 1977). Diploid MATa/MATa cells, unlike their MATa/MATa and MATa/MATa counterparts, cannot mate but can be induced to undergo meiosis and sporulation (ROMAN and SANDS 1953). The MAT locus is on chromosome 111, approximately 20 cM from the centromere (HAW- THORNE and MORTIMER 1960).

Cellular mating type remains genetically stable in heterothallic yeast strains. Matings between cells of like mating type occur only at low frequency (HAW- THORNE 1963a; RABIN 1970). In contrast, frequent switching of mating type occurs in homothallic yeast strains (HICKS and HERSKOWITZ 1976). Homothallic

' Present address: Department of Medicine, The New York Hospital, Cornell Medical Center, 525 East 68th Street, New York, New York 10021.

Genetics 104: 219-234 June, 1983.

220 H. GRUENSPAN AND N. R. EATON

yeast spores have been demonstrated to be haploid cells that express either an a or an a mating type. However, clones derived from homothallic spores are capable of sporulation (HAWTHORNE 196313; OSHIMA and TAKANO 1971; HARASH- IMA, NOGI and OSHIMA 1974). Tetrad analysis of cells from such clones results in the observation of two spores of each mating type per tetrad. Outcrossing of tetrads of heterothallic strains shows that the mating-type alleles of all four spores in a tetrad behave as wild-type alleles in terms of mating properties and genetic linkage to other markers (TAKANO and OSHIMA 1970; HICKS and HER-

Homothallic mating-type conversion is under the control of a multigene system. Three different genetic loci are involved, designated HO, HMR, and HMI, (HARASHIMA, NOGI and OSHIMA 1974; HICKS, STRATHERN and KLAR 1979). HO is a dominant allele (HOPPER and HALL 1975; HICKS, STRATHERN and HERSKOWITZ 1977; KLAR and FOGEL 1977). Either HMLa or HMRa is needed to switch to MATa, and either HMLa or HMRa is needed for switches to MAa. HMR maps 40 cM from thr4 on the right arm, and HML maps 30 cM from his4 on the left arm of chromosome III (HARASHIMA and OSHIMA 1976; KLAR et al. 1980).

The “cassette model” of mating-type interconversions has been advanced to explain these findings (HICKS, STRATHERN and HERSKOWITZ 1977). This model proposes that HMR and HML contain copies of mating-locus information. These copies are not expressed and are, therefore, referred to as silent cassettes. Interconversions of mating type are assumed to occur by duplication and subsequent transposition of silent cassettes to MAT. Much data have been presented to support this hypothesis (BLAIR, KUSHNER and HERSHKOWITZ 1979; KLAR, FOGEL and RADIN 1979; KUSHNER, BLAIR and HERSHOWITZ 1979; STRATH- ERN, BLAIR and HERSKOWITZ 1979; KLAR 1980; NASMYTH and TATCHELL 1980).

Several mutations have been isolated that allow the expression of normally silent mating-type information (MCCULLOUGH 1978; HABER and GEORGE 1979; KLAR, FOGEL and MACLEOD 1979; RINE et al. 1979). All of these mutations are recessive and unlinked to either MAT, HMR or HML.

In this work we describe a new mutation that alters MAT control over mating and sporulation. The properties of this mutation are consistent with the inter- pretation that the cassette of a mating-type information at HMRa is constitu- tively expressed in affected cells. This mutation differs from others that have been described in that it is dominant and specifically affects information at HMR and not at HML. The mutation has been designated AONI. Evidence is presented to support the hypothesis that AONI strains possess an altered regulatory region at or near HMR.

SKOWITZ 1976).

MATERIALS AND METHODS

Strains: All strains used in this work are listed in Table 1, which includes information about their origin.

Media: The medium used for general purposes, YEPD, contained 2% peptone (Difco), 1% yeast extract (Difco) and 2% glucose. Minimal medium, designated SM, contained 0.7% yeast nitrogen base without amino acids (Difco) and 2% glucose. Often this medium was supplemented in order to select for auxotrophic diploids. Drop-out media, used in the determination of auxotrophic require-

SILENT MATING INFORMATION IN YEAST

TABLE 1

List of strains

221

Strain designation Genotype Source

103-1A 103-8 103-81 EBlO

C63.8D 123-1C

A10701C 119-16 17-15 XMB4-12b A131 6D 131-4B

8785-2C EBX13-15A EBX13-14C EBCB-PA 15 AST-6D

15 AST-9D 15 AST-12B

ASTLUl ASTHll ASTADZ 9DC63-9B D10-2A D35-4B D61-3C CR9C a-ural A277 1300-14B 13R2 a-ural

MATa hisl MAL2 MATa hisl MAL2 MATa his1 MAL2 MATa hisl AONl MALZ MATa ura3 his7 MATa his4 leu2 cry19 thr4 mal- uro3

MATa thr4 MATa his2 MALZ mata-1 leul ura3 ade2 cyh2-21 conl-11 MATa ? supersensitive to a-factor MATa HO HMLa HMRa his4 leu2 lysl-1 MATa HO H M h HMRa his4 leu2 lysl

MATa hisl lysl MATa hisl ura2 con1 AONl MAL2 MATa hisl his4 ura2 AONl MAL2 MATcv hisl adex AONZ mata-1 ura2 ura3 leu2 cyh2 con1 AONl

mate-1 ura3 ade2 canl AONl MAL2 mata-1 u r d ura3 ode2 leul canl AONl

MAL2 mata-1 leul mata-1 his2 mota-1 ade2 MATa cryl-3 thr4 ode2 ura3 ura2 MATa hisl ur02 adex AONl"" MAL2 MATa hisl ura2 AONl"" MAL2 MATa hisl ura2 AON2"" MAL2 MATa leu2 cryl-3 MATa ural MATa lys2 rad18 MATa ura2 his4 can2 MATa ura2 his4 canl rho- MATa ural

Iys2

ura2 ura3 cyh2

MAL2

J. COHEN, J. BLAMIRE Subclone of 103-1A Subclone of 103-1A Subclone of 103-1A J. BLAMIRE L. MELNICK

J. BLAMIRE N. R. EATON D. WYGAL-MASCIOLI, J. HABER J. MCCULLOUGH D. WYGAL-MASCIOLI, J. HABER 15AST-6D X A131

D587-4B X D585-11C EBlO X 1300-14B EBlO X 1300-14B EBlO X CBll EBX13-15A X 17-15

EBS13-15A X 17-15 EBX13-15A X 17-15

EBX13-15A X 17-15 EBX13-15A X 17-15 15 AST-ZD X 17-15 C63.8D X 15AST-9D EBX13-15A X EBCB-ZA EBX13-15A X EBCB-BA EBX13-15A X EBCB-ZA This work J. BLAMIRE J. HABER N. R. EATON Petite from 1300-14B 1. BLAMIRE

ments of spore clones in tetrad analysis, consisted of SM medium supplemented with the appropriate additions of amino acids and nitrogenous bases, as described by SHERMAN, FINK and LUKINS (1970). Sporulation was induced on CSPOE agar plates containing 1% potassium acetate and 0.25% yeast extract. Maltose fermentation was assayed on YEPMAL medium containing 2% maltose, 1% yeast extract and 2% peptone. Bromocresol purple was added as 9 ml of a 4% stock solution per liter of medium; the indicator dye was dissolved in ethanol. Fermentation of all parent strains was tested in Durham tubes. For tetrad analysis, spore clones were assayed on YEPMAL agar plates. These plates were incubated at 30' and read within 18-24 hr after replication. Cryptopleurine medium consisted of YEPD with cryptopleurine added at 3 p ~ ; the drug was added subsequent to autoclaving. Assays of the different phenotypes seen on this medium were performed as described by MELNICK and BLAMIRE (1978a). Cryptopleurine was generously provided by J. Blamire.

When required, the media were solidified by the addition, before autoclaving, of 2% agar.

222 H. GRUENSPAN AND N. R. EATON

Dissection and tetrad analysis were performed by standard techniques, described in detail by MORTIMER and HAWTHORNE (1969).

Liquid matings: Cells of each parent strain were grown for 16-24 hr in YEPD on a roller shaker at 30'. Aliquots of each mating pair were mixed in fresh YEPD, returned to a roller drum for 2 hr and then pelleted by centrifugation. After 5 hr the supernatant was decanted, and minimal medium was added to select for diploids. Cultures were then incubated for 48 hr. After this time cells were plated on solid minimal medium to isolate individual diploid clones. In the experiment in which efficiency of mating was tested, the mating suspension was not resuspended in liquid minimal medium but was plated directly onto solid medium.

Rate matings: Strains to be mated were grown to early stationary phase in YEPD liquid. Suspensions were concentrated to 10' cells/ml by centrifugation. YEPD agar plates were spread with 0.2-0.5 ml of each parent. The plates were incubated at 30' for 24 hr. After this time, the plates were replicated to minimal medium. Colonies thought to represent zygote clones appeared after 2- 5 days of incubation at 30'. If mating efficiencies were found to be particularly high, the numbers of cells originally spread onto YEPD plates were reduced. This method is modified from HICKS and HERSKOWITZ (1977) and MELNICK and BLAMIRE (1978a). Colonies formed on minimal medium were picked, and master plates were prepared for further testing.

Cell-to-cell matings: These were performed by placing cells in direct contact with each other on dissection agar. Zygotes were subsequently isolated by micromanipulation. In some cases zygotes formed in liquid medium were isolated by micromanipulation.

Mating-type testing: Mating type was determined by complementation tests. Master plates containing patches taken from 36 spore clones were grown overnight on YEPD medium. These plates were then replicated onto other YEPD plates that were previously spread with tester cells from stationary cultures. After 24 hr of incubation at 30' the mating plates were replicated onto selective minimal medium. MATa AONZ cells generally yielded a variable number of papillae on minimal medium when tested in this way.

The following strains were used as mating testers: a-ural, 123-1C, CRSC, a-ural, A100701C, ASTADZ, ASTHI1, and CB11. Testers a-ural and CR9C were used in all experiments where possible.

Sporulation assay: MATa AONl strains which showed low penetrance of the sterile character could be detected as follows. The minimal-medium mating-test plates for a mata-1 tester were replicated to sporulation medium (CSPOE). After 3-7 days at 30°, cells from each patch were suspended in a small drop of water on a glass slide and observed microscopically. Patches that formed asci containing mature spores were scored as having derived from a MATa AONl parent. Generally, sporulation as judged by this assay was greater than 50%. This assay also differentiates between homothallic and heterothallic maters, providing the mata-1 tester contains HMRa or HMLa. If both AONl and HO segregate in a cross, this assay cannot differentiate between them.

a-Factor assay: The following assay protocol, and tester strain XMB4-12B, were obtained from J. MCCULLOUGH. About 5 X lo6 tester cells were suspended in 1.3 ml of liquid YEPD containing 0.4% agar. This mixture was poured onto the surface of a YEPD plate, and the plate was allowed to dry. Master plates were then replicated onto this lawn, and the plates were incubated at room temperature for about 2 days. Patches were differentiated for a-factor production by halo production around the patch after the incubation period. All of our stock MATa strains tested show a positive result by this assay, whereas MATa AONI strains do not.

Assay for mata-1 strains: MATa-I strains could be assayed for AONZ by a sporulation assay. Prototrophs formed with the tester a-ural were replicated to sporulation medium. Patches were then observed microscopically to detect sporulation as described. MATa-1 strains carrying AONl generally show greater than 50% sporulation with tester a-ural by this assay.

Determination of the switched parent in rare matings: Rare matings between two MATa cells can occur when one of the cells converts mating type prior to mating. The converted parent can be identified by marking one of the parental strains with the marker cryl-3. This marker is known to be closely linked to MAT (SKOGERSON, McLAucHLrN and WAKAMATA 1973). If the coupling of MAT and CRYI-3 is altered by the rare mating, then the drug-resistant parent has switched mating type. If coupling is maintained, the sensitive parent must have switched mating type. Alternatively, patches of sterile auxotrophic diploids can be replicated to cryptopleurine medium. It is known that some of the papillae arising on this medium are the result of reciprocal recombinations between the centromere and the cryl-3 locus (KLAR and FOGEL 1977). The mating specificity of these patches

SILENT MATING INFORMATION IN YEAST 223

determines the coupling between cryl-3 and MAT directly. This assay has been verified by standard analysis of the sterile diploids (L. MELNICK, personal communication).

Assay for transposable a-information: MATO and mota-1 strains were mated to a strain of genotype HO HMLa MATa HMRa. Prototrophic mating products were then transferred to sporu- lation medium. A positive sporulation assay was assumed to indicate that the test strain contained either HMLa or HMRa or both and a negative assay to indicate that a strain carried alleles that are inactive in either transposition or a-mating function. The validity of this assay was demonstrated by its ability to detect a mutation at HMRa which could alternatively be identified by linkage to MAL2. This assay is also consistent with the observations of HICKS (1978).

Construction of MATa/MATa ond MATa/MATa diploids: In these experiments individual MATa/MATa cells were plated on YEPD and exposed to 380-500 ergs/"' of UV light according to ROMAN and JACOB (1958). The UV light source was a 15-watt General Electric germicidal lamp which delivered 60-63 ergs/mm*/sec at the distance used. Irradiated clones were tested for mating ability against MATa and MATa testers, and mating clones were purified by subcloning before use in additional experiments.

Assay of radl8: The mutation rod18 was assayed by replicating test patches onto YEPD plates and then irradiating these plates for varying lengths of time. A 1-min exposure was found to best differentiate rad18 from wild-type clones.

RESULTS

The mutation AONZ was found as a spontaneous sterile isolate of the MATa strain 103-1A. MATa strains carrying AONZ are not affected in mating ability. Mating efficiency varied among different MATa AONZ strains and among various subclones of a single MATa AONZ clone. The most severely affected clones mate at frequencies of the same order of magnitude as the genetic reversion frequency. MATa AONZ clones are deficient in a-factor pheremone production. These findings are detailed elsewhere (GRUENSPAN 1979).

Effects of AONl in MATa/MATa and MATa/MATa diploids: During the original purification of AONZ from 103-1A, some self-sporulating subclones were found. Asci produced by these subclones yielded up to four viable spores, each of which displayed a sterile phenotype. This finding was the first indication that AONZ affected the control of sporulation in addition to the ability to mate. To study the effect of AONZ on sporulation, MATa/MATa and MATa/MATa diploids carrying AONl were constructed by UV-induced mitotic recombination of MATa/MATa diploids (described in MATERIALS AND METHODS). MATa/MATa diploids containing AONZ could be differentiated from background MATa/ MATa diploids in which a mitotic recombination had not been induced, because MATa/MATa diploid are unable to mate (even at low frequency) with the MATa mating-type tester. The recombinant diploids were then tested for their mating and sporulation abilities.

The results of these experiments are summarized in Table 2. Both MATa/ MATa and MATa/MATa diploids derived from DO in the control cross showed confluent patch mating and did not sporulate. This was also true of the MATa/ MATa diploids derived from D1. However, all MATa/MATa diploids derived from D1 were defective in mating and also sporulated. Two were analyzed further: all spores were either sterile or a-maters. No tetrads yielded a-mating spores. The segregation pattern of AONZ was not determined because of difficulties in the accurate assay of AONZ at this stage of the work.

The results with DO and D1 indicated that AONZ specifically promoted the sporulation of MATa/MATa diploids but not MATa/MATa diploids. However,

224 H. GRUENSPAN AND N. R. EATON

TABLE 2

Sporulation of diploids in which homozygosity at MAT had been induced by UV irradiation

a-Mating diploids a-Mating diploids Dip- loids"

Capable Capable Capable Dip 1 o i d Re- of spor- Re- of spor- Re- of spor- designa-

covered ulation covered ulation covered ulation Parents tion

Control 4 0 5 0 0 0

7 0 0 0 13 13

3 0 1 0 3 3

Experimental 5 0 0 0 5 5

3 0 2 0 3 3

6 0 5 0 0 0

Total experi- 24 0 8 0 24 24 mental

103-8 X 8785- DO 2 c

EBIOb X 8785- D1 2 c

EBCB-XA* EBX13-14Cb x D2'

EBX13-15Ab X D5

EBX13-15Ab x D10"

EBX13-15Ab X D35'

EBCB-2Ab

E B C B - ~ A ~

EBCB-ZA~

Nonmating diploids are not MATa/MATa diploids (see text). Denotes strain that carries AONZ. Denotes diploid strain whose MATa parent had undergone reversion at AONZ before mating.

if AONl were located on the right arm of chromosome 111, distal to MAT (as was later determined), then it would be quite possible that all MATa/MATa diploids constructed might not carry AONl.

Additional experiments are performed using diploids D2, D5, D10 and D35 that inherited AONl from the MATa parent. In each of these cases, all MATa/ MATa diploids constructed in these experiments are expected to carry at least one copy of AONZ. Yet, none of these sporulated. AONZ, therefore, does not promote sporulation of MATa/MATa strains.

Not all the constructed MATa/MATa diploids sporulated. This is explained by the fact that a reversion event at AONl in the MATa parent of the diploid prior to mating would allow construction of MATa/MATa diploids not carrying AONZ.

Four MATa/MATa AONl diploids from these experiments were analyzed further. Again, no a-mating spores were recovered.

Suppression of mata-1 b y AON1: The genetic characterization of AONl was made possible by the use of mata-l strain 17-15. mata-l is a defective MAT allele (KASSIR and SIMCHEN 1976). mata-2 haploids mate as typical MATa strains. However, the resulting MATa/mata-I diploid cells mate with MATa cells and do not sporulate. AONl suppresses both of these defects. Three MATa AONl strains, EB10, EBX13-15A and EBX13-14C, were tested and formed mating products with 17-15 that were capable of sporulation. Strains 103-81 and EB10,

SILENT MATING INFORMATION IN YEAST 225

which are isogenic to the previous strains except for AONZ, did not form sporulation-competent products with 17-15. This shows that AONZ is respon- sible for overcoming the sporulation defect of mata-2, and that AONZ acts in a dominant fashion.

Segregation and genetic mapping of AON1: Strain EBX13-15A (MATa AONZ) was crossed with strain 17-15 (mata-2) to show that both mata-1 and AONZ segrate as single Mendelian units. The AONZ allele was scored in MATa spores by the low frequency (papillated patch) mating of these segregants with the MATa tester. Mata-2 segregants were scored by their ability to form mating products with the MATa RME+ mating tester and by the inability of the resulting prototrophs to sporulate. The results of this experiment for 13 complete tetrads are shown in Table 3. Both mata-Z and MATa AONZ segregants were recovered in all of these tetrads. However, a-mating clones capable of forming sporulation proficient diploids were also recovered. These clones were likely to be of the genotype mata-Z AONZ. To verify this genotype, crosses of three such spore clones to MATa strains were analyzed. The mata-2 strains used, desig- nated 15AST-6D, 15AST-9D, and 15AST-l2B, were crossed to MATa strain 123- 1C. Strains 15AST-6D and 15AST-9D were also crossed to strain C63.8D. Both mata-Z and AONZ were recovered in all crosses. This analysis showed that AONZ segregated in the 2 2 manner expected for a single gene and also that the sporulation assay we describe provides a method to identify mata-2 AONl segregants.

Strain 17-15 carries a recessive marker, designated rme (KASSIR and SIMCHEN 1976), which, when present in homozygous form, allows MATa/mata-Z diploids to sporulate. The sporulation assay of AONZ in mata-1 strains, however, is not influenced by rme since the standard a-mating testers used, a-ural, 123-1C and C63.8D, do not sporulate with 17-15 and, thus, are RME+. In the assay of MATa AONZ strains with the mata-2 testers mentioned before the sporulation-positive strains are invariably affected in mating ability.

TABLE 3

Tetrad analysis for the cross EBX13-15A (MATa AON1) X 17-15 (mata-1)

Low frequency a-Mater non- a-Mater sporu- Tetrad-type a-maters a-mater (MATa sporulator lator (mata-1

Tetrad AONl/MAT (MATol) AONI) (mota-1) AONI)

1 2 3 4 5 6 7 0 9

10 11 12 13

T PD T T T T T PD T T PD T T

1 0 1 1 1 1 1 0 1 1 0 1 1

1 2 1 1 1 1 1 2 1 1 2 1 1

1 2 1 1 1 1 1 2 1 1 2 1 1

1 0 1 1 1 1 1 0 1 1 0 1 1

226 H. GRUENSPAN AND N. R. EATON

Table 4 shows tetrad data for the genetic analysis of AONZ. The crosses in sections 4a through 4d were originally performed to confirm the mata-2 AONZ genotype that was suggested for various progeny of the cross EBX13-15A X 17- 15 (Table 3). During analysis of the results, the excess of parental vs. nonparental ditype tetrads in section 4a hinted at loose linkage between MAT and AONZ. Linkage of AONZ to thr4, shown in Table 4b, further suggested that AONl was situated on the right arm of chromosome III. To locate AONZ more precisely, linkage to MAL2 was determined. Table 4c shows these loci to be separated by 6.8 cM.

To determine the position of AONl more precisely, rad28 was included in a mapping cross as a third linked marker. The gene RAD28 is known to lie on the right arm of chromosome 111 proximal to the centromere from MAL2 Strain A277 (MATa lys2 rad28 mal-) was crossed to strain 15AST-6D (mata-2 ura2 ura3 leu1 cyh2 MAL2 AON1). All markers segregated as expected from a diploid. The tetrad-type data for the three possible marker pairings are sum- marized in Table 4. A tetrad in which a gene conversion had taken place was not classified according to type. AONZ was found to map between the RAD28 and MAL2 loci and to be situated 5.4 cM proximal to the centromere from MAL2. This is the previously determined map position of HMRa. (HARASHIMA and OSHIMA 1976) which is the proposed silent copy of a-mating-type informa- tion.

The gene order was also verified by analyzing the 11 tetratype asci between

TABLE 4

Genetic mopping of AONl

Distance Section Reference genes PD NPD T Total (cm) Crosses involved

~ _____ ____ ~~

a MAT/AONl 17 9 74 100 64.0 123-1C X 15AST-6D 123-1C X 15AST-9D 123-1C X 15AST-12B C63-ED X 15AST-6D C63-ED X 15AST-9D C63.8D X 15AST-90

b THR4/AON1 12 2 19 33 47.0 C63.8D X 15AST-9D C63.8D X 15AST-6D

c MALZ/AONl 95 0 15 110 6.8 123-1C X 15AST-6D 123-1C X 15AST-9D 123-1C X 15AST-12B C63.8D X 15AST-6D C63.8D X 15AST-9D

d CRY/MAT 22 0 5 27 9.3 C63.8D X 15AST-6D C63.8D X 15AST-9D

e RADlE/AONI 40 1 56 97 32.0 A277 X 15AST-6D f RADIS/MALZ 36 2 63 102 36.8 A277 X 15AST-6D g MALZ/AONl 91 0 11 101 5.4 A277 X 15AST-6D

SILENT MATING INFORMATION IN YEAST 227

MAL2 and AONZ (Table 4g). Six of these asci were also tetratype for the RADZ8/MAL2 and the RADZ8/AONI gene pairs. In four of the remaining five asci the RAD18/AONZ gene pair retained parental linkage, whereas the asci were tetratype for the RADZ8/MAL2 linkage. In the remaining tetrad the RAD18/AONZ pair was tetratype, whereas RAD28 and MAL2 segregated in nonparental linkage. These data imply the gene order RAD28 AONl MAL2, because this order requires the fewest number of crossovers and is consistant with the map distances for these three markers.

Revertants: The mutation AONZ causes MATa cells to be sterile. Often MATa AONZ haploids mate at such low frequency that a significant percentage of the zygotes formed in rare matings arises from reversions of AONZ. Therefore, an assay was devised to screen diploids for their genetic constitution at AONZ. Diploid clones were tested for mating efficiency by the patch-replica plate assay described in MATERIALS AND METHODS. Normally, MATa/MATa diploids show papillae or low-frequency mating with both mating-type testers when assayed in this way. Mating results from mitotic recombination at MAT which yields MATa/MATa and MATa/MATa diploids at a detectable frequency (JAMES and LEE-WHITING 1955). If a diploid cell inherits AONZ from it MATa parent, the MATa/MATa recombinants will be sterile, as previously demonstrated. MATa AONI/MATa diploid strains should, therefore, be capable of mating at low frequency only with the MATa mating-type tester, but no papillae should be seen with the MATa tester. In contrast, such diploids carrying a reversion of AON inherited from the MATa parent would show low-frequency mating with testers of both mating types. This would be true even if the diploid contained an AONZ allele inherited from the MATa parent, as AONZ and MAT are both located on the right arm of chromosome III and could be linked in mitotic recombination. This assay should detect reversions that take place at or near AONZ and dominant suppressor mutations that are unlinked to AONI. Unlinked recessive suppressors would probably be missed. Haploid revertants could be recovered by sporulation of the MATa/MATa diploids tested.

Revertants of AONZ were isolated from diploid clones of a cross of EBX13- 15A (MATa AONZ) to EBCB-BA (MATa AONI) to EBCB-BA (MATa AONI). One hundred individual diploid clones were tested for mating ability with both mating-type testers. Of these, 87 showed no papillae with the MATa mating- tester, nine showed a significant number of papillae (3-lo), and four showed one papillation. Asci derived from five diploids that were capable of low- frequency mating with the a-tester were analyzed. Haploid revertant spore clones were recovered in numbers expected for a dominant suppressor in all cases (Table 5). As a control, three diploid clones that did not show low- frequency mating with the MATa tester were also analyzed. No fertile MATa clones were obtained from the latter three diploids.

Three independent MATa revertant spore clones were chosen for further analysis. These were designated D10-BA, D35-4B and D61-3C. Each of these strains were derived from a different diploid clone. Each revertant was crossed to strain 17-15 (mata-I). None of the mating products was capable of sporulation. The revertants were also tested for a-factor production by a “halo” assay. All

228 H. GRUENSPAN AND N. R. EATON

TABLE 5

Confirmation of the detection of AONl in diploid clones obtained from the cross EBX13-15A X EBCB-BA

MATa more clones

lfa (MATa Diploid no. lfa" mating Total AONI) Fertile (MATa) Spore viability

5 9 25 10 35 61 66 85

17 30 10 14 13 16 18 17

17 30 10 6 4 7 8 8

0 0 0 8 9 9 10 9

70 69 63 80 70 70 88 88

a 1fa denotes low-frequency mating with the MATa tester.

produced halos. These results indicated that the mating and sporulation effects of AONZ revert together, i.e., that both phenotypes are controlled by a single gene.

The revertants were each crossed to MATa strain 13R2. Tetrad analysis resulted in the recovery of 110 a-mating spore clones. None of the recovered spore clones was sterile. The absence of nonmating spore clones eliminates the possibility that these revertants are due to unlinked suppressor mutations. Therefore, all of these revertants are assumed to be altered at a site near or at AONZ.

Switching function in revertants: Revertants of AONl might be expected to restore the wild-type allele, to be switched to HMRa or to retain AONl and carry secondary suppressor mutations that alter the proposed silent MATa cassette. The latter types of revertants would be unable to convert mating type to MATa. Experiments were performed in order to assess whether revertants could switch to functional a-mating strains.

First, MATa AONl strains were tested for their ability to convert their mating type to MATa. Rare diploids were selected from crosses involving MATa AONl strains EBlO and EBX13-15A and MATa cryl-3 strain C63.8D. a-mating and sterile diploids were recovered. Ten complete tetrads were analyzed from one sterile diploid derived from the mating of EBlO and C63.8D. In each tetrad the cryl-3 MATa linkage of C63.8D was maintained. Since MATa segregated with the wild-type CRY allele, EBlO must have switched mating type prior to mating. Two diploids were analyzed in the case of EBX13-15A. Again, cryl-3 MATa and CRY+ MATa linkages were seen in the progeny. Therefore, these AONZ strains can provide information for switching MAT to a functional MATa allele. AONZ segregated normally in the tetrads that were analyzed. This indicates that AONl is not lost in heterothallic mating-type interconversions.

Rare diploids were isolated from crosses of each of the revertants DlO-ZA, D35-4B and D61-3C to strain C63.8D. A summary of the types of diploids obtained in these matings is shown in Table 6. Twenty-one independent sporulating diploids were isolated from all three matings. Twenty of these were

SILENT MATING INFORMATION IN YEAST 229

analyzed. The diploids were found to fall into two classes. One class produced asci with 60-90% viable spore clones. The other class of diploids showed poor spore clone viability, with no ascus containing more than two viable spores.

The linkage relationship between cryl-3 and MAT were examined for each of the 13 diploids that showed typical spore viability; cryl-3 was found to be coupled to MATa in each case. This is shown in Table 7. Therefore, strain C63.8D must have switched mating type prior to mating in all cases.

In the course of the preceding experiment, 221 MATa spore clones were tested for mating ability. None was found to be sterile. This further supports the conclusion that the reversion events are tightly linked to AONl.

In another experiment, rare mating products were isolated from D10-2A, D35- 4B and D61-3C and strain 9DC63-9B (MATa cryl-3). Sterile, sporulating diploid patches were replicated onto cryptopleurine-containing medium. Since diploids were heterozygous for the cryl-3 allele, resistant papillations arose because of

TABLE 6

Types of diploids obtained in rare matings between three revertants of AONl and tester strain C63.8D

Prototrophs ca- 60-90% Prototrophs iso- pable of sporula- spore viabil- Less than 50%

Mating pair lated tion ity spore viability

D10-2A X C63.8D 36 6 4 2 DB5-4B X C63.8D 33 8" 6 1 D61-3C X C63.8D 28 7 3 4

" One of this group of diploids was not analyzed.

TABLE 7

Coupling of CRY1-3 and MAT in a/a diploids obtained in rare matings between three revertants of AONl and tester strain C63.8D

MATa PD:NPD:T in Diploid designa- spores ana- Spore clone complete tet-

tion lyzed MATa cry' MAT8 cry' Asci viability rads

D10-7 19 0 15 10 85% 4:0:0 DlO-18 16 0 12 8 88% 4:0:0 D10-19 20 0 17 11 86% 5:0:0 D10-27 18 0 18 11 82% 5:0:0

D35-10 18 0 15 11 75% 3:00 D35-13 14 0 12 9 72% 1:0:0 D35-16 16 0 13 10 73% 3:0:0 D35-21 16 0 11 10 70% 3:0:0 D35-24 17 0 11 10 70% 1:0:0 D35-28 16 0 13 10 75% 3:00

D61-16 18 0 17 10 90% 8:0:0 D61-17 14 0 10 9 60% 3:0:0 D61-23 18 1 12 10 80% 3:0:0

"The designation to the left of a hyphen indicates the revertant parent of the specific diploid clone. These parents are DlO-ZA, D35-4B and D61-3C.

230 H. GRUENSPAN AND N. R. EATON

mitotic recombination. Any single reciprocal mitotic recombination between the centromere and cryl-3 should render both the drug marker and the closely coupled MAT locus homozygous (KLAR and FOGEL 1977). Therefore, several papillae from each diploid were assayed for mating type. This test established the MAT allele to which cryl-3 was linked. It should be noted that, although a number of cryptopleurine-resistant colonies arise independently of any recom- bination involving MAT, these will not affect the assay.

The results of the rare-mating experiments to strain 9DC63-9B are shown in Table 8. In the cases of D10-2A and D61-3C, no a-mating cryptopleurine- resistant papillae were recovered from the diploids generated. This observation indicated that only strain 9DC63-9B switched mating type prior to conjugation and suggests that the two former revertants are incapable of switching to "normal" a-mating haploid cells. However, the three sterile diploid clones capable of forming a-mating, cryptopleurine-resistant papillae recovered from strain D35-4B show that this revertant is capable of switching. Possibly the frequency of mating-type switch is significantly lower in strain D35-4B.

Twenty-six of the sterile diploids recovered from all three selected rare matings were found to produce only sterile, cryptopleurine-resistant papilla- tions. These diploids could result from Hawthorne-type deletions that occurred prior to mating in the strain carrying cryl-3. If this were the case, all reciprocal recombinants for cryl-3 would be homozygous for a lethal deletion.

A class of diploids was also seen that did not form resistant papillae on cryptopleurine medium. Two were found in the case of D10-2A, three in the case of D35-4B and 31 in the case of D61-3C. We can produce no straightforward explanation of these results.

Rare matings were also selected in crosses with MATa strain 6D131-4B. This strain is incapable of mating-type switch to MATa, so that any switches detected should have taken place in the partner strain. The results of these crosses are shown in Table 9. In the case of rare matings with D10-ZA, none of the 134

TABLE 8

The results of rare mating experiment with CRY' strain 9DC63-9B

Revertant CRYS strain

D10-2A D35-4B D61-3C

Mating frequency" Diploids screened a-Mater diploids Sterile diploids Diploids forming cry' papillae Diploids not forming cry' papillae a Mating cry' papillae Sterile cry' papillae a-Mating cry' papillae

4.5 x 10-~ 332 279

53 51 2

44 7 0

4.1 X 2.4 X lo-' 259 353 217 275 42 78 39 47 3 31

25 41 11 6 3 0

Generally more than 25 papillae were tested for mating phenotype. Therefore, viable reciprocal

a This frequency is not specific to any one mating partner, as forced matings were performed recombinants homozygous for mating type should be detected if they can be formed.

with equal numbers of both parents.

SILENT MATING INFORMATION IN YEAST 231

TABLE 9

Rare mating of revertants from AONl with strain 6D131-4B (HO HMLol MATa HMRa)

Revertant

D10-2A D35-4B D61-3C

Mating frequency" 2.2 x 10-7 1.9 x 10-~ 2.7 x 1 0 - ~ Diploids assayed 134 162 124 Sterile diploids 0 0 38

a Rare matings were obtained from mixtures of equal numbers (2 X la8 cells) of cells spread onto a YEPD plate. Therefore, the mating frequency is underestimated, and is not specific for a particular mating partner.

diploid clones assayed was sterile. All were a-maters. This observation is again consistent with the assertion that the reversion event in D10-2A is caused by an alternation of HMR function. It was also the case that none of the 162 mating products formed with D35-4B was sterile, again suggesting that the frequency of switch in D35-4B may be low.

An unexpected result was seen in the case of D61-3C: 38 of 162 diploids obtained were sterile and capable of sporulation. These diploids have not been analyzed further. This result implies that D61-3C may well contain a silent copy of a-mating-type information. However, results from other analyses (Tables 8 and next paragraph) indicate that this copy is not active in transposition to MAT.

It is known that strains of genotype HO HMLa MATa HMRa are capable of low-frequency mating with MATa strains (HICKS 1978). The diploids that are formed in this way are capable of sporulation if they contain at least one functional HMRa or HMLa allele. Therefore, strain A131 (HO HMLa MATa HMRa) was used to test the revertants for functionally silent MATa information. Diploids formed by mating D10-2A and D61-3C with A131 were incapable of sporulation, as was the case with 6D131-4B (HO HMLa MATa HMRa) which contains no silent MATa information. This indicates that these two revertants are not capable of homothallic conversion to MATa because they either do not contain silent a-mating-type information or such information cannot function in transposition. In contrast, all other MATa strains tested (including D35-4B) have functionally silent MATa information by this assay.

DISCUSSION

Many lines of evidence indicate that the mutation AONl allows the consti- tutive expression of normally silent a-mating-type information. AONl causes MATa cells to be low-efficiency maters. MATa cells carrying AONZ are defec- tive in a-factor production. These cells were also observed to be polar budders (experiments not presented). This budding pattern is usually diagnostic for MATa/MATa diploid cells (HICKS, STRATHERN and HERSKOWITZ 1977; CRAN- DALL, EGEL and MACKAY 1977).

AONZ has been found to allow MATa/MATa diploid cells to sporulate. However, MATa/MATa diploids that carry AONZ are not capable of sporula-

232 H. GRUENSPAN AND N. R. EATON

tion. Thus, AONZ provides the MATa information required for sporulation but not the MATa information.

MATa AONZ/mata-1 diploids behave as true MATa/MATa cells. Therefore, AONZ maps 5-6 cM proximal to the centromere from MAL2 on the right arm of chromosome Ill. This is the map position from HMR (HARASHIMA and OSHIMA 1976). The site also corresponds to the end point of Hawthorne’s deletion (HAWTHORNE 1963a) which presumably fuses genetic information at HMRa to mating-type regulatory signals at or near MAT.

Evidence suggests that the AONZ gene can replace the ability of MATa to suppress the action of the HO gene in diploid strains containing the MATa locus. For example, strains of the genotype mata-Z/MATa, HO/+ are able to switch to MATa/MATa, HO/+ diploids. In contrast, strains of the genotype mata-Z/MATa, HO/+, AONZ/+ appear to be unable to undergo homothallic switching of mating type (GRUENSPAN 1979).

The genetic properties of AONZ are consistent with the hypothesis that the mutational site in AONZ resides within a DNA sequence that exerts control over the expression of HMRa. This mutation could be similar to an operator- constitutive or an up-promoter mutation. This is suggested because AONZ is a dominant mutation that is closely linked to the gene that it controls.

Others have reported mutations leading to the apparent constitutive expres- sion of HMR and HML. These mutations have been called pb (MCCULLOUGH 1978), cmt (HABER AND GEORGE 1979), mar &LAR, FOGEL and MACLEOD 1979) and sirl-1 (RINE et al. 1979). These mutations represent at least two different loci. All such mutations are recessive and unlinked to MAT. A particular pb mutation at one locus is thought to be amber-suppressible. These properties are consistent with the hypothesis that these mutations reside at structural genes that code for proteins involved in the negative regulation of HMR and HML.

The cassette model posits that the HMR and HML loci contain transposable cassettes of normally silent mating-type information. However, observations have been made that suggest that other cassettes of mating-type information may be present. The mutation Ar (MELNICK and BLAMIRE 1978b) and SAD (KASSIR, HOPPER and MACKAY 1978) appear to allow expression of such infor- mation. These genes allow MATa/MATa but not MATa/MATa diploids to sporulate. This phenotype is similar to that of AONZ. However, neither Ar or SAD strains have been found to be mating deficient. In addition, the action of SAD1 does not depend on which allele is present at HMR, and SAD1 does not map at HMR.

We have shown that AONZ exerts control over the expression of HMRa. We hope that further work with AONZ strains will provide information about the control of cassette transcription and the mechanism of cassette transposition.

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Corresponding editor: F. SHERMAN