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.     

Intermediates of recombination during mating type switching in Saccharomyces cerevisiae.

We have identified two novel intermediates of homothallic switching of the yeast mating type gene, from MATa to MAT alpha. Following HO endonuclease cleavage, 5' to 3' exonucleolytic digestion is observed distal to the HO cut, creating a 3'-ended single-stranded tail. This recision is more extensive in a rad52 strain unable to switch. Surprisingly, the proximal side of the HO cut is protected from degradation; this stabilization depends on the presence of the silent copy donor sequences. A second intermediate was identified by a quantitative application of the polymerase chain reaction (PCR). The Y alpha-MAT distal covalent fragment of the switched product appears 30 min prior to the appearance of the MAT proximal Y alpha junction. No covalent joining of MAT distal to HML distal sequences is detected. We suggested that the MAT DNA distal to the HO cut invades the intact donor and is extended by DNA synthesis. This step is prevented in a rad52 strain. These intermediates are consistent with a model for MAT switching in which only the distal side of the HO cut is initially active in strand invasion and transfer of information from the donor.     

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.     

A mutation that permits the expression of normally silent copies of mating-type information in Saccharomyces cerevisiae

Studies of heterothallic and homothallic strains of Saccharomyces cerevisiae have led to the suggestion that mating-type information is located at three distinct sites on chromosome 3, although only information at the mating-type (MAT) locus is expressed (Hicks, Strathern and Herskowitz, 1977). We have found that the recessive mutation cmt permits expression of the normally silent copies of mating-type information at the HMa and HM alpha loci. In haploid strains carrying HMa and HM alpha, the cmt mutation allows the simultaneous expression of both a and alpha information, leading to a nonmating ("MATa/MAT alpha") phenotype. The effects of cmt can be masked by changing the mating-type information at HMa or HM alpha. For example, a cell of genotype MATa hma HM alpha cmt has an a mating type, while a MAT alpha hma HM alpha cmt strain is nonmating. Expression of mating-type information at the HM loci can correct the mating and sporulation defects of the mata* and mat alpha 10 alleles. Meiotic segregants recovered from cmt/cmt diploids carrying the mat mutations demonstrate that these mutants are not "healed" to normal MAT alleles, as is the case in parallel studies using the homothallism gene HO.--All of the results are consistent with the notion that the HMa and hm alpha alleles both code for alpha information, while HM alpha and hma both code for a information. The cmt mutation demonstrates that these normally silent copies of mating-type and sporulation information can be expressed and that the information at these loci is functionally equivalent to that found at MAT. The cmt mutation does not cause interconversions of mating-type alleles at MAT, and it is not genetically linked to MAT, HMa, HM alpha or HO. In cmt heterozygotes, cmt becomes homozygous at a frequency greater than 1% when the genotype at the MAT locus is mata*/MAT alpha or mat alpha 10/MATa.     

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.     

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.     

Physical monitoring of mating type switching in Saccharomyces cerevisiae.

The kinetics of mating type switching in Saccharomyces cerevisiae can be followed at the DNA level by using a galactose-inducible HO (GAL-HO) gene to initiate the event in synchronously growing cells. From the time that HO endonuclease cleaves MAT a until the detection of MAT alpha DNA took 60 min. When unbudded G1-phase cells were induced, switched to the opposite mating type in "pairs." In the presence of the DNA synthesis inhibitor hydroxyurea, HO-induced cleavage occurred but cells failed to complete switching. In these blocked cells, the HO-cut ends of MATa remained stable for at least 3 h. Upon removal of hydroxyurea, the cells completed the switch in approximately 1 h. The same kinetics of MAT switching were also seen in asynchronous cultures and when synchronously growing cells were induced at different times of the cell cycle. Thus, the only restriction that confined normal homothallic switching to the G1 phase of the cell cycle was the expression of HO endonuclease. Further evidence that galactose-induced cells can switch in the G2 phase of the cell cycle was the observation that these cells did not always switch in pairs. This suggests that two chromatids, both cleaved with HO endonuclease, can interact independently with the donors HML alpha and HMRa.     

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.     

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.     

Mating type control in Saccharomyces cerevisiae: a frameshift mutation at the common DNA sequence, X, of the HML alpha locus.

A mutation defective in the homothallic switching of mating type alleles, designated hml alpha-2, has previously been characterized. The mutation occurred in a cell having the HO MATa HML alpha HMRa genotype, and the mutant culture consisted of ca. 10% a mating type cells, 90% nonmater cells of haploid cell size, and 0.1% sporogenous diploid cells. Genetic analyses revealed that nonmater haploid cells have a defect in the alpha 2 cistron at the MAT locus. This defect was probably caused by transposition of a cassette originating from the hml alpha-2 allele by the process of the homothallic mating type switch. That the MAT locus of the nonmater cells is occupied by a DNA fragment indistinguishable from the Y alpha sequence in electrophoretic mobility was demonstrated by Southern hybridization of the EcoRI-HindIII fragment encoding the MAT locus with a cloned HML alpha gene as the probe. The hml alpha-2 mutation was revealed to be a one-base-pair deletion at the ninth base pair in the X region from the X and Y boundary of the HML locus. This mutation gave rise to a shift in the open reading frame of the alpha 2 cistron. A molecular mechanism for the mating type switch associated with the occurrence of sporogenous diploid cells in the mutant culture is discussed.     

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.     

rme1 Mutation of Saccharomyces cerevisiae: map position and bypass of mating type locus control of sporulation.

Sporulation in Saccharomyces cerevisiae normally occurs only in MATa/MAT alpha diploids. We show that mutations in RME1 bypassed the requirements for both a and alpha mating type information in sporulation and therefore allowed MATa/MATa and MAT alpha/MAT alpha diploids to sporulate. RME1 was located on chromosome VII, between LEU1 and ADE6.     

Isolation and genetic analysis of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones.

Eight independently isolated mutants which are supersensitive (Sst-) to the G1 arrest induced by the tridecapeptide pheromone alpha factor were identified by screening mutagenized Saccharomyces cerevisiae MATa cells on solid medium for increased growth inhibition by alpha factor. These mutants carried lesions in two complementation groups, sst1 and sst2. Mutations at the sst1 locus were mating type specific: MATa sst1 cells were supersensitive to alpha factor, but MAT alpha sst1 cells were not supersensitive to a factor. In contrast, mutations at the sst2 locus conferred supersensitivity to the pheromones of the opposite mating type on both MATa and MAT alpha cells. Even in the absence of added alpha pheromone, about 10% of the cells in exponentially growing cultures of MATa strains carrying any of three different alleles of sst2 (including the ochre mutation sst2-4) had the aberrant morphology ("shmoo" shape) that normally develops only after MATa cells are exposed to alpha factor. This "self-shmooing" phenotype was genetically linked to the sst2 mutations, although the leakiest allele isolated (sst2-3) did not display this characteristic. Normal MATa/MAT alpha diploids do not respond to pheromones; diploids homozygous for an sst2 mutation (MATa/MAT alpha sst2-1/sst2-1) were still insensitive to alpha factor. The sst1 gene was mapped to within 6.9 centimorgans of his6 on chromosome IX. The sst2 gene was unlinked to sst1, was not centromere linked, and was shown to be neither linked to nor centromere distal to MAT on the right arm of chromosome III.     

a/alpha-specific effect on the mms3 mutation on ultraviolet mutagenesis in Saccharomyces cerevisiae.

A new gene involved in error-prone repair of ultraviolet (UV) damage has been identified in Saccharomyces cerevisiae by the mms3-1 mutation. UV-induced reversion is reduced in diploids that are homozygous for mms3-1, only if they are also heterozygous (MATa/MAT alpha) at the mating type locus. The mms3-1 mutation has no effect on UV-induced reversion either in haploids or MATa/MATa or MAT alpha/MAT alpha diploids. The mutation confers sensitivity to UV and methyl methane sulfonate in both haploids and diploids. Even though mutation induction by UV is restored to wild-type levels in MATa/MATa mms3-1/mms3-1 or MAT alpha/MAT alpha mms3-1/mms3-1 diploids, such strains still retain sensitivity to the lethal effects of UV. Survival after UV irradiation in mms3-1 rad double mutant combinations indicates that mms3-1 is epistatic to rad6-1 whereas non-epistatic interactions are observed with rad3 and rad52 mutants. When present in the homozygous state in MATa/MAT alpha his1-1/his1-315 heteroallelic diploids, mms3-1 was found to lower UV-induced mitotic recombination.     

Regulation of transcription and promoter mapping of the structural genes for nitrogenase (nifHDK) of Azospirillum brasilense Sp7

A galactose-inducible HO gene was used to induce mating type switching in heterothallic Saccharomyces cerevisiae cells arrested in G1, in rad52 mutants defective in DNA damage repair, and in cells lacking the donor cassettes. The HO-cleaved MAT intermediate is stable over significant lengths of time, i.e. HO cleavage is not coupled to the subsequent gene conversion event. The in vivo cleavage site was mapped to single base resolution by primer extension experiments on total genomic DNA. Cells arrested in G1 with alpha-factor switched mating type thus demonstrating that switches can occur in the absence of replication of the genome. rad52 mutants did not produce MAT DNA of the opposite mating type indicating that the block is prior to the gene duplication stage of the switch. In strains in which the HM donor cassettes are deleted the cut MAT DNA was degraded after induction of the HO gene.     

Homothallic switching of Saccharomyces cerevisiae mating type genes by using a donor containing a large internal deletion.

Homothallic switching of the mating type genes of Saccharomyces cerevisiae occurs by a gene conversion event, replacing sequences at the expressed MAT locus with a DNA segment copied from one of two unexpressed loci, HML or HMR. The transposed Ya or Y alpha sequences are flanked by homologous regions that are believed to be essential for switching. We examined the transposition of a mating type gene (hmr alpha 1-delta 6) which contains a 150-base-pair deletion spanning the site where the HO endonuclease generates a double-stranded break in MAT that initiates the gene conversion event. Despite the fact that the ends of the cut MAT region no longer share homology with the donor hmr alpha 1-delta 6, switching of MATa or MAT alpha to mat alpha 1-delta 6 was efficient. However, there was a marked increase in the number of aberrant events, especially the formation of haploid-inviable fusions between MAT and the hmr alpha 1-delta 6 donor locus.     

Rules of Donor Preference in Saccharomyces Mating-Type Gene Switching Revealed by a Competition Assay Involving Two Types of Recombination

Mating type (MAT) switching in Saccharomyces cerevisiae is initiated by a double-strand break (DSB) created at MAT by HO endonuclease. MATa cells activate the entire left arm of chromosome III; thus MATa preferentially recombines with the silent donor HML. In contrast, MATα cells inactivate the left arm, including HML, and thus preferentially recombine with HMR, 100 kb to the right of MAT. We present a novel competition assay, in which the DSB at MAT can be repaired either by MAT switching or by single-strand annealing (SSA) between two URA3 genes flanking MAT. With preferred donors, MATa or MATα switching occurs 65-70% of the time in competition with SSA. When HML is deleted, 40% of MATa cells recombine with the ``wrong' donor HMR; however, when HMR is deleted, only 18% of MATα cells recombine with HML. In interchromosomal switching, with donors on chromosome III and MAT on chromosome V, MATa retains its strong preference for HML and switching is efficient, when the chromosome III recombination enhancer is present. However, MATα donor preference is lost and interchromosomal switching is very inefficient. These experiments demonstrate the utility of using competition between two outcomes to measure the relative efficiency of recombination.     

Donor Locus Selection during Saccharomyces Cerevisiae Mating Type Interconversion Responds to Distant Regulatory Signals

Mating type interconversion in homothallic strains of the yeast Saccharomyces cerevisiae results from directed transposition of a mating type allele from one of the two silent donor loci, HML and HMR, to the expressing locus, MAT. Cell type regulates the selection of the particular donor locus to be utilized during mating type interconversion: MATa cells preferentially select HML alpha and MAT alpha cells preferentially select HMRa. Such preferential selection indicates that the cell is able to distinguish between HML and HMR during mating type interconversion. Accordingly, we designed experiments to identify those features perceived by the cell to discriminate HML and HMR. We demonstrate that discrimination does not derive from the different structures of the HML and HMR loci, from the unique sequences flanking each donor locus nor from any of the DNA distal to the HM loci on chromosome III. Moreover, we find that the sequences flanking the MAT locus do not function in the preferential selection of one donor locus over the other. We propose that the positions of the donor loci on the left and right arms of chromosome III is the characteristic utilized by the cell to distinguish HML and HMR. This positional information is not generated by either CEN3 or the MAT locus, but probably derives from differences in the chromatin structure, chromosome folding or intranuclear localization of the two ends of chromosome III.     

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.