A Suppressor of Mating-Type Locus Mutations in SACCHAROMYCES CEREVISIAE: Evidence for and Identification of Cryptic Mating-Type Loci

A mutation has been identified that suppresses the mating and sporulation defects of all mutations in the mating-type loci of S. cerevisiae. This suppressor, sir1-1, restores mating ability to mat alpha 1 and mat alpha 2 mutants and restores sporulation ability to mat alpha 2 and mata1 mutants. MATa sir1-1 strains exhibit a polar budding pattern and have reduced sensitivity to alpha-factor, both properties of a/alpha diploids. Furthermore, sir1-1 allows MATa/MATa, mat alpha 1/mat alpha/, and MAT alpha/MAT alpha strains to sporulate efficiently. All actions of sir1-1 are recessive to SIR1. The ability of sir1-1 to supply all functions necessary for mating and sporulation and its effects in a cells are explained by proposing that sir1-1 allows expression of mating type loci which are ordinarily not expressed. The ability of sir1-1 to suppress the mat alpha 1-5 mutation is dependent on the HMa gene, previously identified as required for switching of mating types from a to alpha. Thus, as predicted by the cassette model, HMa is functionally equivalent to MAT alpha since it supplies functions of MAT alpha. We propose that sir1-1 is defective in a function. Sir ("Silent-information regulator"), whose role may be to regulate expression of HMa and HM alpha.     

MAR1-a Regulator of the HMa and HMalpha Loci in SACCHAROMYCES CEREVISIAE

A mutation in the MAR1 (mating-type regulator) locus causing sterility in Saccharomyces cerevisiae is reported. The mutation maps on the left arm of linkage group IV between trp1 and cdc2 at a distance of about 27 cM from trp1 and about 31 cM from cdc2. Haploid strains with genotype MATalpha HMalpha HMa mar1-1 and MATa HMalpha HMamar1-1 are sterile. However, MATalpha hmalpha HMa mar1-1 and MATa HMalpha hma mar1-1 strains exhibit alpha and a mating type, respectively. The sterile strains can be "rare mated" with standard strains as a consequence of mutational changes at HMa and HMalpha. It is proposed that the MAR1 locus blocks the expression of MATalpha and MATa information thought to exist at HMa and HMalpha loci, respectively (Hicks, Strathern and Herskowitz, 1977). In a mar1-1 mutant, the expression of both HMalpha and HMa information leads to a nonmating phenotype similar to that of MATa/MATalpha diploids. The genetic evidence reported here is consistent with a central feature of the "cassette model", namely that HMalpha and hma carry MATa information and HMa and hmalpha carry MATalpha information.     

Mutations Leading to Expression of the Cryptic HMR a Locus in the Yeast SACCHAROMYCES CEREVISIAE

Mutations leading to expression of the silent HMRa information in Saccharomyces cerevisiae result in sporulation proficiency in mata1/MAT alpha diploids. An example of such a mutation is sir5-2, a recessive mutation in the gene SIR5. As expected, haploids carrying the sir5-2 mutation are nonmaters due to the simultaneous expression of HMRa and HML alpha, resulting in the nonmating phenotype of an a/alpha diploid. However, sir5-2/sir5-2 mata1/MAT alpha diploids mate as alpha yet are capable of sporulation. The sir5-2 mutation is unlinked to sir1-1, yet the two mutations do not complement each other: mata1/MAT alpha sir5-2/SIR5 SIR1/sir1-1 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 PAS1-1 mutation, Provider of a Sporulation function, is a dominant mutation tightly linked to HMRa. PAS1-1 does not affect the mating ability of a strain, yet it allows diploids lacking a functional MATa locus to sporulate. It is proposed that PAS1-1 leads to partial expression of the otherwise cryptic a1 information at HMRa.     

Mating-Type Differentiation by Transposition of Controlling Elements in SACCHAROMYCES CEREVISIAE

The nonfunctional mutation of the homothallic gene HML alpha, designated hml alpha, produced two mutant alleles, hml alpha-1 and hml alpha-2. Both mutant clones were mixed cultures consisting of a mating-type cells and nonmating haploid cells. The frequencies of the two cell types were different, and a few diploid cells able to sporulate were found in the hml alpha-2 mutant. Conversions of an a mating-type cell to nonmater, and vice versa, were observed in both mutants. The conversion of an a mating phenotype to nonmating is postulated to occur by alteration of the a mating type to the sterile mating-type allele in the hml alpha-1 mutant. In tetrad dissection of prototrophic diploids that were obtained by rare mating of hml alpha-1 mutants with a heterothallic strain having the MATa ho HMRa HMLa genotype, many mating-deficient haploid segregants were found, while alpha mating-type segregants were observed in a similar diploid using an hml alpha-2 mutant. The mating-type-deficient haploid segregants were supposed to have the sterile alpha mating-type allele because the nonmating genetic trait always segregated with the mating-type locus. Sporogenous diploid cells obtained in the hml alpha-2 mutant clone had the MATa/MAT alpha HO/HO HMRa/HMRa hml alpha-2/hml alpha-2 genotype. These observations suggested that the hml alpha-1 allele produces a transposable element that gives rise to the sterile alpha mating type by transposition into the mating-type locus, and that the hml alpha-2 allele produces an element that provides alpha mating-type information, but is defective in the structure for transposition.     

The Action of Homothallism Genes in Saccharomyces Diploids during Vegetative Growth and the Equivalence of hma and HMalpha Loci Functions

The action of homothallism genes in vegetatively growing diploid cells was examined. The results demonstrate that homothallism genes function during regular vegetative growth cycles as well as during the first few divisions after spore germination. A procedure based on ultraviolet-induced reciprocal mitotic recombination monitored by homozygosity for cryptopleurine resistance (a recessive marker closely linked to the mating-type locus) allowed us to identify and recover Saccharomyces cerevisiae colonies sectored for the mating-type locus i.e., a/a and alpha/alpha. Homothallism genes can switch a/a or alpha/alpha vegetative diploid cells, generated from a strain with genotype a/alpha HO/ho HMalpha/HMalpha HMa/HMa, to a/alpha diploids or a/a/alpha/alpha tetraploids during a given mitotic division cycle. We found that both a/a and alpha/alpha sectors generated from a strain with genotype a/alpha HO/HO hmalpha/hmalpha hma/HMa switch to a/alpha diploids or a/a/alpha/alpha tetraploids. This finding supports Naumov and Tolstorukov's suggestion (1973) that the hm a allele provides for the same functions as the HMalpha allele, namely, a switch at the mating-type locus from alpha to a. The HO allele is dominant to ho but hma and HMa alleles are codominant. A loose linkage between the mating-type and the HMalpha loci ( approximately 55cM), confirming Harashima, Nogi and Oshima (1974) data, was observed.     

Analysis of Y-Linked Mutations to Male Sterility in DROSOPHILA MELANOGASTER

Mating type in haploid cells of the yeast Saccharomyces cerevisiae is determined by a pair of alleles MATa and MAT alpha. 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, designated AON1, has the following properties. (1) MAT alpha cells carring AON1 are defective in mating. (2) AON1 allows MAT alpha/MAT alpha but not MATa/MATa diploids to sporulate; thus, AON1 mimics the MATa requirement for sporulation. (3) mata-1 cells that carry AON1 are MATa phenocopies, i.e., MAT alpha/mata-1 AON1 diploids behave as standard MAT alpha/MATa cells; therefore, AON1 suppresses the defect of mata-1. (4) AON1 maps at or near HMRa. (5) Same-site revertants from AON1 lose the ability to convert mating type to MATa, indicating that reversion is associated with the loss of a functional HMRa locus. In addition, AON1 is a dominant mutation. We conclude that AON1 is a regulatory mutation, probably cis-acting, that leads to the constitutive expression of silent a mating-type information located at HMRa.     

A CIS-Acting Mutation within the MATa Locus of SACCHAROMYCES CEREVISIAE That Prevents Efficient Homothallic Mating-Type Switching

Homothallic strains of Saccharomyces cerevisiae are able to switch from one mating-type to the other as frequently as every cell division. We have identified a cis-dominant mutation of the MATa locus, designated MATa-inc, that can be converted to MATalpha at only about 5% of the normal efficiency. In homothallic MATa-inc/mata* diploids, the MATa-inc locus switched to MATalpha in only one of 30 cases, while the mata* locus switched to MATalpha in all 30 cases. The MATa-inc mutation can be "healed" by a series of switches, first to MATalpha and then to a normal allele of MATa. These data are consistent with the "cassette" model of Hicks, Strathern and Herskowitz (1977), in which mating conversions involve the transposition of wild-type copies of a or alpha information from silent genes elsewhere in the genome. The MATa-inc mutation appears to alter a DNA sequence necessary for the replacement of MATa by MATalpha. The MATa-inc mutation has no other effect on MATa functions. In beterothallic backgrounds, the mutation has no effect on the sensitivity to alpha-factor, synthesis of a-factor, expression of barrier phenotype or ability to mate or sporulate.--The MATa-inc allele does, however, exhibit one pleiotropic effect. About 1% of homothallic MATa-inc cells become completely unable to switch mating type because of mutations at HMa, the locus proposed to carry the silent copy of alpha information.--In addition, we have isolated a less efficient allele of the HO gene.     

Heteroduplex formation and mismatch repair of the "stuck" mutation during mating-type switching in Saccharomyces cerevisiae.

We sequenced two alleles of the MATa locus of Saccharomyces cerevisiae that reduce homothallic switching and confer viability to HO rad52 strains. Both the MATa-stk (J. E. Haber, W. T. Savage, S. M. Raposa, B. Weiffenbach, and L. B. Rowe, Proc. Natl. Acad. Sci. USA 77:2824-2828, 1980) and MATa-survivor (R. E. Malone and D. Hyman, Curr. Genet. 7:439-447, 1983) alleles result from a T----A base change at position Z11 of the MAT locus. These strains also contain identical base substitutions at HMRa, so that the mutation is reintroduced when MAT alpha switches to MATa. Mating-type switching in a MATa-stk strain relative to a MATa Z11T strain is reduced at least 50-fold but can be increased by expression of HO from a galactose-inducible promoter. We confirmed by Southern analysis that the Z11A mutation reduced the efficiency of double-strand break formation compared with the Z11T variant; the reduction was more severe in MAT alpha than in MATa. In MAT alpha, the Z11A mutation also creates a mat alpha 1 (sterile) mutation that distinguishes switches of MATa-stk to either MAT alpha or mat alpha 1-stk. Pedigree analysis of cells induced to switch in G1 showed that MATa-stk switched frequently (23% of the time) to produce one mat alpha 1-stk and one MAT alpha progeny. This postswitching segregation suggests that Z11 was often present in heteroduplex DNA that was not mismatch repaired. When mismatch repair was prevented by deletion of the PMS1 gene, there was an increase in the proportion of mat alpha 1-stk/MAT alpha sectors (59%) and in pairs of switched cells that both retained the stk mutation (27%). We conclude that at least one strand of DNA only 4 bp from the HO cut site is not degraded in most of the gene conversion events that accompany MAT switching.     

Mapping of the Homothallic Genes, HMα and HMa, in Saccharomyces Yeasts

Two of the three homothallic genes, HM alpha and HMa, showed direct linkage to the mating-type locus at approximately 73 and 98 strans (57 and 65 centimorgans [cM], respectively, whereas, the other, HO, showed no linkage to 25 standard markers distributed over 17 chromosomes including the mating-type locus. To determine whether the HM alpha and HMa loci located on the left or right side of the mating-type locus, equations for three factor analysis of three linked genes were derived. Tetrad data were collected and were compared with expected values by chi 2 statistics. Calculations indicated that the HM alpha gene is probably located on the right arm at 95 strans (65 cM) from the centromere and the HMa locus at approximately 90 strans (64 cM) on the left arm of chromosome III.     

Homothallic mating type switching generates lethal chromosome breaks in rad52 strains of Saccharomyces cerevisiae.

In homothallic cells of Saccharomyces cerevisiae, a or alpha mating type information at the mating type locus (MAT) is replaced by the transposition of the opposite mating type allele from HML alpha or HMRa. The rad52-1 mutation, which reduces mitotic and abolishes meiotic recombination, also affects homothallic switching (Malone and Esposito, Proc. Natl. Acad. Sci. U.S.A. 77:503-507, 1980). We have found that both HO rad52 MATa and HO rad52 MAT alpha cells die. This lethality is suppressed by mutations that substantially reduce but do not eliminate homothallic conversions. These mutations map at or near the MAT locus (MAT alpha inc, MATa-inc, MATa stk1) or are unlinked to MAT (HO-1 and swi1). These results suggest that the switching event itself is involved in the lethality. With the exception of swi1, HO rad52 strains carrying one of the above mutations cannot convert mating type at all. MAT alpha rad52 HO swi1 strains apparently can switch MAT alpha to MATa. However, when we analyzed these a maters, we found that few, if any, of them were bona fide MATa cells. These a-like cells were instead either deleted for part of chromosome III distal to and including MAT or had lost the entire third chromosome. Approximately 30% of the time, an a-like cell could be repaired to a normal MATa genotype if the cell was mated to a RAD52 MAT alpha-inc strain. The effects of rad52 were also studied in mata/MAT alpha-inc rad52/rad52 ho/HO diploids. When this diploid attempted to switch mata to MATa, an unstable broken chromosome was generated in nearly every cell. These studies suggest that homothallic switching involves the formation of a double-stranded deoxyribonucleic acid break or a structure which is labile in rad52 cells and results in a broken chromosome. We propose that the production of a double-stranded deoxyribonucleic acid break is the lethal event in rad52 HO cells.     

Switching of a Mating-Type a Mutant Allele in Budding Yeast SACCHAROMYCES CEREVISIAE

Aimed at investigating the recovery of a specific mutant allele of the mating type locus (MAT) by switching a defective MAT allele, these experiments provide information bearing on several models proposed for MAT interconversion in bakers yeast, Saccharomyces cerevisiae. Hybrids between heterothallic (ho) cells carrying a mutant MAT a allele, designated mata-2, and MAT alpha ho strains show a high capacity for mating with MATa strains. The MAT alpha/mata-2 diploids do not sporulate. However, zygotic clones obtained by mating MAT alpha homothallic (HO) cells with mata-2 ho cells are unable to mate and can sporulate. Tetrad analysis of such clones revealed two diploid (MAT alpha/MATa):two haploid segregants. Therefore, MAT switches occur in MAT alpha/mata-2 HO/ho cells to produce MAT alpha/Mata cells capable of sporulation. In heterothallic strains, the mata-2 allele can be switched to a functional MAT alpha and subsequently to a functional MATa. Among 32 MAT alpha to MATa switches tested, where the MAT alpha was previously derived from the mata-2 mutant, only one mata-2 like isolate was observed. However, the recovered allele, unlike the parental allele, complements the matalpha ste1-5 mutant, suggesting that these alleles are not identical and that the recovered allele presumably arose as a mutation of the Mat alpha locus. No mata-2 was recovered by HO-mediated switching of MAT alpha (previously obtained from mata-2 by HO) in 217 switches analyzed. We conclude that in homothallic and heterothallic strains, the mata-2 allele can be readily switched to a functional MAT alpha and subsequently to a functional MATa locus. Overall, the results are in accord with the cassette model (HICKS, STRATHERN and HERSKOWITZ )977b) proposed to explain MAT interconversions.     

Transcriptional control of the sporulation-specific glucoamylase gene in the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, glucoamylase activity appears specifically in sporulating cells heterozygous for the mating-type locus (MAT). We identified a sporulation-specific glucoamylase gene (SGA) and show that expression of SGA is positively regulated by the mating-type genes, both MATa1 and MAT alpha 2. Northern blot analysis revealed that control of SGA is exerted at the level of RNA production. Expression of SGA or the consequent degradation of glycogen to glucose in cells is not required for meiosis or sporulation, since MATa/MAT alpha diploid cells homozygous for an insertion mutation at SGA still formed four viable ascospores.     

The Genetic System Controlling Homothallism in Saccharomyces Yeasts

There are four types of life cycles in Saccharomyces cerevisiae and its related species. A perfect homothallic life cycle (the Ho type) is observed in the classic D strain. Two other types show semi-homothallism; one of them shows a 2-homothallic diploid:2alpha heterothallic haploid segregation (the Hp type) and another, a 2-homothallic:2a segregation (the Hq type). In the segregants from these Ho, Hp, and Hq diploids, each homothallic segregant shows the same segregation pattern as its parental diploid. The fourth type has a heterothallic life cycle showing a 2a:2alpha segregation and the diploids are produced by the fusion of two haploid cells of opposite mating types. The diploids prepared by the crosses of alpha Hp (an alpha haploid segregant from the Hp diploid) to a Hq (an a haploid from the Hq diploid) segregated two types (Type I and II) of the Ho type homothallic clone among their meiotic segregants. Genetic analyses were performed to investigate this phenomenon and the genotypes of the Ho type homothallic clones of Type I and Type II. Results of these genetic analyses have been most adequately explained by postulating three kinds of homothallic genes, each consisting of a single pair of alleles, HO/ho, HMalpha/hmalpha, and HMa/hma, respectively. One of them, the HMalpha locus, was proved to be loosely linked (64 stranes) to the mating-type locus. A spore having the HO hmalpha hma genotype gives rise to an Ho type homothallic diploid (Type I), the same as in the case of the D strain which has the HO HMalpha HMa genotype (Type II). A spore having the a HO hmalpha HMa or alpha HO HMalpha hma genotype will produce an Hp or Hq type homothallic diploid culture, respectively. The other genotypes, a HO HMalpha hma, alpha HO hmalpha HMa, and the genotypes combined with the ho allele give a heterothallic character to the spore culture. A possible molecular hypothesis for the mating-type differentiation with the controlling elements produced by the HMalpha and HMa genes is proposed.     

SAD mutation of Saccharomyces cerevisiae is an extra a cassette.

Sporulation of Saccharomyces cerevisiae ordinarily requires the a1 function of the a mating type locus. SAD is a dominant mutation that allows strains lacking a1 (MAT alpha/MAT alpha and mata1/MAT alpha diploids) to sporulate. We provide functional and physical evidence that SAD is an extra cassette in the yeast genome, distinct from those at HML, MAT, and HMR. The properties of SAD strains indicate that the a cassette at SAD produces a limited amount of a1 product, sufficient for promoting sporulation but not for inhibiting mating and other processes. These conclusions come from the following observations. (i) SAD did not act by allowing expression of HMRa: mata1/MAT alpha diploids carrying SAD and only alpha cassettes at HML and HMR sporulated efficiently. (ii) SAD acted as an a cassette donor in HML alpha HMR alpha strains and could heal a mata1 mutation to MATa as a result of mating type interconversion. (iii) The genome of SAD strains contained a single new cassette locus, as determined by Southern hybridization. (iv) Expression of a functions from the SAD a cassette was limited by Sir: sir- SAD strains exhibited more extreme phenotypes than SIR SAD strains. This observation indicates that SAD contains not only cassette information coding for a1 (presumably from HMRa) but also sites for Sir action.     

Gene Conversion of the Mating-Type Locus in SACCHAROMYCES CEREVISIAE

Tetrad analysis of MATa/MAT alpha diploids of Saccharomyces cerevisiae generally yields 2 MATa:2MAT alpha meiotic products. About 1 to 1.8% of the tetrads yield aberrant segregations for this marker. Described here are experiments that determine whether the aberrant meiotic segregations at the mating-type locus are ascribable to gene conversions or to MAT switches, that is, to mating-type interconversions. Diploid strains incapable of switching MATa to MAT alpha, or the converse, nevertheless display changes of MATa to MAT alpha, or the reverse. These events must be attributed to gene conversion. Further, we suggest that MATa and MAT alpha alleles may represent nonhomologous sequences of DNA since they fail to display postmeiotic segregations.     

Relation between the efficiency of homothallic switching of yeast mating type genes and the distribution of cell types.

Homothallic switching of yeast mating type genes occurs as often as each cell division, so that a colony derived from a single haploid spore soon contains an equal number of MATa and MAT alpha cells. Cells of opposite mating types conjugate, and eventually the colony contains only nonmating MATa/MAT alpha diploids. Mutations that reduce the efficiency of homothallic MAT conversions yield colonies that still contain many haploid cells of the original spore mating type plus a few recently generated cells of the opposite mating type. These (a greater than alpha)- or (alpha greater than a)-mating colonies also contain some nonmating diploid cells. As an alternative to microscopic pedigree analysis to determine the frequency of mating type conversions in a variety of mutant homothallic strains, we analyzed the proportions of MATa, MAT alpha, and MATa/MAT alpha cells in a colony by examining the mating phenotypes of subclones. We developed a mathematical model that described the proportion of cell types in a slow-switching colony. This model predicted that the proportion of nonmating cells would continually increase with the size (age) of a colony derived from a single cell. This prediction was confirmed by determining the proportion of cell types in colonies of an HO swi1 strain that was grown for different numbers of cell divisions. Data from subcloning (a greater than alpha) and (alpha greater than a) colonies from a variety of slow-switching mutations and chromosomal rearrangements were used to calculate the frequency of MAT conversions in these strains.     

Mechanism of MAT alpha donor preference during mating-type switching of Saccharomyces cerevisiae.

During homothallic switching of the mating-type (MAT) gene in Saccharomyces cerevisiae, a- or alpha-specific sequences are replaced by opposite mating-type sequences copied from one of two silent donor loci, HML alpha or HMRa. The two donors lie at opposite ends of chromosome III, approximately 190 and 90 kb, respectively, from MAT. MAT alpha cells preferentially recombine with HMR, while MATa cells select HML. The mechanisms of donor selection are different for the two mating types. MATa cells, deleted for the preferred HML gene, efficiently use HMR as a donor. However, in MAT alpha cells, HML is not an efficient donor when HMR is deleted; consequently, approximately one-third of HO HML alpha MAT alpha hmr delta cells die because they fail to repair the HO endonuclease-induced double-strand break at MAT. MAT alpha donor preference depends not on the sequence differences between HML and HMR or their surrounding regions but on their chromosomal locations. Cloned HMR donors placed at three other locations to the left of MAT, on either side of the centromere, all fail to act as efficient donors. When the donor is placed 37 kb to the left of MAT, its proximity overcomes normal donor preference, but this position is again inefficiently used when additional DNA is inserted in between the donor and MAT to increase the distance to 62 kb. Donors placed to the right of MAT are efficiently recruited, and in fact a donor situated 16 kb proximal to HMR is used in preference to HMR. The cis-acting chromosomal determinants of MAT alpha preference are not influenced by the chromosomal orientation of MAT or by sequences as far as 6 kb from HMR. These data argue that there is an alpha-specific mechanism to inhibit the use of donors to the left of MAT alpha, causing the cell to recombine most often with donors to the right of MAT alpha.     

Expression of the BAR1 gene in Saccharomyces cerevisiae: induction by the alpha mating pheromone of an activity associated with a secreted protein.

We have demonstrated and partially characterized the genetic control and pheromonal regulation of a soluble activity, produced only by mating-type a cells, that inhibits the action of the alpha mating pheromone, alpha-factor, on mating-type a cells. This activity was found to be associated with a heat-stable protein and to be secreted by MATa BAR1, mat alpha 2 BAR1, and mat alpha 1 mat alpha 2 BAR1 strains, but not by MAT alpha BAR1, MATa/MAT alpha BAR1, mat alpha 1 BAR1, or MATa barl strains, demonstrating that it is under the control of both the MAT alpha 2 and the BAR1 genes. Secretion of this activity was also found to be stimulated to as much as five times the basal level by exposure of the cells to alpha-factor. This stimulation was maximal after 6 h at a pheromone concentration of approximately 2 U/ml. An assay for this activity was developed by using a refined, quantitative assay for alpha-factor. The pheromone activity of samples added to wells in an agar plate was related to the size of the halo of growth inhibition produced in a lawn of mutant cells that are abnormally sensitive. The alpha-factor-inhibiting activity was related to a reduction of the halo size when active samples were added to the lawn. Although the assay for alpha-factor was found to be relatively insensitive to pH over a range of several units, the alpha-factor-inhibiting activity displayed a sharp pH optimum at approximately 6.5. The properties of this activity have important implications concerning the role of the BAR1 gene product in recovery of mating-type a cells from cell division arrest by alpha-factor.