SUM1, an Apparent Positive Regulator of the Cryptic Mating-Type Loci in SACCHAROMYCES CEREVISIAE

The mating-type information residing at the HML and HMR loci in Saccharomyces cerevisiae is kept unexpressed by the action of at least four MAR (or SIR) loci. To determine possible interactions between the MAR/SIR gene products and to find new regulatory loci, we sought extragenic suppressors of the mar1-1 mutation. A strain with the genotype HMLa MAT alpha HMRa mar1-1 is unable to mate because of the simultaneous expression of a and alpha information. A mutant of this strain was isolated that exhibits an alpha phenotype and, therefore, presumably fails to express the HML and HMR loci. We designate the new locus SUM1 (suppressor of mar). The mutation is recessive, centromere unlinked and does not correspond to the MAT, HML, HMR, SIR1, MAR1, MAR2 (SIR3) or SIR4 loci. The sum1 mutation affects expression of both a and alpha information at the HM loci. Suppression by sum1-1 is neither allele specific nor locus specific as it suppresses a deletion mutation of the MAR1 locus and mutations in SIR3 and SIR4. The sum1-1 mutation has no discernible phenotype in a Mar+ strain. We propose that the MAR/SIR gene products negatively regulate the SUM1 locus, the gene product of which is necessary for expression of the HM loci.     

Efficient production of a ring derivative of chromosome III by the mating-type switching mechanism in Saccharomyces cerevisiae.

The mating-type switches in the yeast Saccharomyces cerevisiae occur by unidirectional transposition of replicas of unexpressed genetic information, residing at HML or HMR, into the mating-type locus (MAT). The source loci, HML and HMR, remain unchanged. Interestingly, when the HM cassettes are expressed, as in marl strains, the HML and HMR cassettes can also efficiently switch, apparently by obtaining genetic information from either of the other two cassettes (Klar et al., Cell 25:517-524, 1981). We have isolated a novel chromosome III rearrangement in heterothallic (marl ho) strains, which is also produced efficiently in marl HO cells, presumably the consequence of a recombination event between HML and HMR. The fusion results in the loss of sequences which are located distal to HML and to HMR and produces a ring derivative of chromosome III. Cells containing such a ring chromosome are viable as haploids; apparently, no essential loci are located distal to the HM loci. The fusion cassette behaves as a standard HM locus with respect to both regulation by the MAR/SIR control and its role in switching MAT.     

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.     

Disruption of a Silencer Domain by a Retrotransposon

A galactose-inducible Ty element carrying the HIS3 gene has been used as an insertional mutagen to generate alpha-factor resistant mutants. This collection of Ty-induced mutations includes insertions into the gene for the alpha-factor receptor (STE2), several nonspecific STE genes, and mutations that lead to the expression of the normally silent HML alpha locus. The hml alpha "on" mutations fall into two classes, those that disrupt trans-acting regulators involved in silencing HML alpha and a novel class of mutations that activate HML alpha by insertion at that locus. The hml alpha::Ty "on" mutations illustrate the unusual ability of these retrotransposons to activate genes by overcoming gene silencing mechanisms. The hml alpha::Ty "on" mutations include examples of multimeric Ty arrays. Single Ty and solo delta insertion derivatives of these Ty multimers restore the ability of the silencing mechanism to repress HML alpha.     

Specific repression of the yeast silent mating locus HMR by an adjacent telomere.

The yeast silent mating loci HML and HMR are located at opposite ends of chromosome III adjacent to the telomeres. Mutations in the N terminus of histone H4 have been previously found to derepress the yeast silent mating locus HML to a much greater extent than HMR. Although differences in the a and alpha mating-type regulatory genes and in the cis-acting silencer elements do not appear to strongly influence the level of derepression at HMR, we have found that the differential between the two silent cassettes is largely due to the position of the HMR cassette relative to the telomere on chromosome III. While HML is derepressed to roughly the same extent by mutations in histone H4 regardless of its chromosomal location, HMR is affected to different extends depending upon its chromosomal positioning. We have found that HMR is more severely derepressed by histone H4 mutations when positioned far from the telomere (cdc14 locus on chromosome VI) but is only minimally affected by the same mutations when integrated immediately adjacent to another telomere (ADH4 locus on chromosome VII). These data indicate that the degree of silencing at HMR is regulated in part by its neighboring telomere over a distance of at least 23 kb and that this form of regulation is unique for HMR and not present at HML. These data also indicate that histone H4 plays an important role in regulating the silenced state at both HML and HMR.     

STE16, a New Gene Required for Pheromone Production by a Cells of Saccharomyces cerevisiae

Genes required for mating by a and alpha cells of Saccharomyces cerevisiae (STE, "sterile," genes) encode products such as peptide pheromones, pheromone receptors, and proteins responsible for pheromone processing. a-specific STE genes are those required for mating by a cells but not by alpha cells. To identify new a-specific STE genes, we have employed a novel strategy that enabled us to determine if a ste mutant defective in mating as a is also defective in mating as alpha without the need to do crosses. This technique involved a strain (K12-14b) of genotype mata1 HML alpha HMR alpha sir3ts, which mates as a at 25 degrees and as alpha at 34 degrees. We screened over 40,000 mutagenized colonies derived from K12-14b and obtained 28 a-specific ste mutants. These strains contained mutations in three known a-specific genes--STE2, STE6 and STE14--and in a new gene, STE16. ste16 mutants are defective in the production of the pheromone, a-factor, and exhibit slow growth. Based on the distribution of a-specific ste mutants described here, we infer that we have identified most if not all nonessential genes that can give rise to a-specific mating defects.     

Homothallic switching of yeast mating type cassettes is initiated by a double-stranded cut in the MAT locus

A double-stranded DNA cut has been observed in the mating type (MAT) locus of the yeast Saccharomyces cerevisiae in cultures undergoing homothallic cassette switching. Cutting is observed in exponentially growing cells of genotype HO HML alpha MAT alpha HMR alpha or HO HMLa MATa HMRa, which switch continuously, but not in a/alpha HO/HO diploid strains, in which homothallic switching is known to be shut off. Stationary phase cultures do not exhibit the cut. Although this site-specific cut occurs in a sequence (Z1) common to the silent HML and HMR cassettes and to MAT, only the Z1 sequence at the MAT locus is cut. The cut at MAT occurs in the absence of the HML and HMR donor cassettes, suggesting that cutting initiates the switching process. An assay for switching on hybrid plasmids containing mata- cassettes has been devised, and deletion mapping has shown that the cut site is required for efficient switching. Thus a double-stranded cut at the MAT locus appears to initiate cassette transposition-substitution and defines MAT as the recipient in this process.     

Duplicated Genes Producing Transposable Controlling Elements for the Mating-Type Differentiation in SACCHAROMYCES CEREVISIAE

Mutation of the two homothallic genes, HML alpha/HMLa and HMRa/HMR alpha, in homothallic strains of Saccharomyces cerevisiae was studied. Of 11 mutants of the HML alpha gene, eight were due to a phenotypic mutation from HML alpha to HMLa, i.e., a mutation causing a change in function of the original HML allele to that of the other HML allele (functional mutation), and three were due to a defective mutation at the HML alpha gene, i.e., a mutation causing a nonfunctional allele (nonfunctional mutation). All 14 mutants of the HMRa gene, on the other hand, were due to a phenotypic mutation from HMRa to HMR alpha i.e., a functional mutation. Phenotypic reverse mutations, i.e., HMLa to HML alpha and HMR alpha to HMRa, were also observed in the cultivation of EMS (ethyl methanesulfonate) treated spores having the HO HMR alpha HMLa genotype. Mutation from heterothallic cells to homothallism was observed in a nonfunctional mutant of the HML alpha gene, by mutagenesis with EMS, but not in the functional mutants of the HML alpha and HMRa genes or in the authentic strains having the alpha HO HMR alpha HML alpha (alpha Hp) and a HO HMRa HMLa (a Hq) genotypes. These observations suggest that the functional mutation is not caused by the direct mutation from a homothallic allele to the opposite, but by replacement of a transposable genic element produced from a homothallic locus with a region of a different homothallic locus. These observations also support the controlling-element model and the cassette model, which have been proposed to explain the mating-type differentiation by the homothallic genes.     

Mating-type control in Saccharomyces cerevisiae: isolation and characterization of mutants defective in repression by a1-alpha 2.

The alpha 2 protein, the product of the MAT alpha 2 cistron, represses various genes specific to the a mating type (alpha 2 repression), and when combined with the MATa1 gene product, it represses MAT alpha 1 and various haploid-specific genes (a1-alpha 2 repression). One target of a1-alpha 2 repression is RME1, which is a negative regulator of a/alpha-specific genes. We have isolated 13 recessive mutants whose a1-alpha 2 repression is defective but which retain alpha 2 repression in a genetic background of ho MATa HML alpha HMRa sir3 or ho MAT alpha HMRa HMRa sir3. These mutations can be divided into three different classes. One class contains a missense mutation, designated hml alpha 2-102, in the alpha 2 cistron of HML, and another class contains two mat alpha 2-202, in the MAT alpha locus. These three mutants each have an amino acid substitution of tyrosine or acid substitution of tyrosine or phenylalanine for cysteine at the 33rd codon from the translation initiation codon in the alpha 2 cistron of HML alpha or MAT alpha. The remaining 10 mutants make up the third class and form a single complementation group, having mutations designated aar1 (a1-alpha 2 repression), at a gene other than MAT, HML, HMR, RME1, or the four SIR genes. Although a diploid cell homozygous for the aarl and sir3 mutations and for the MATa, HML alpha, and HMRa alleles showed alpha mating type, it could sporulate and gave rise to asci containing four alpha mating-type spores. These facts indicate that the domain for alpha2 repression is separable from that for a1-alpha2 protein interaction or complex formation in the alpha2 protein and that an additional regulation gene, AAR1, is associated with the a1-alpha2 repression of the alpha1 cistron and haploid-specific genes.