Directionality of yeast mating-type interconversion

The mating-type a and α alleles of the yeast Saccharomyces cerevisiae interconvert by a transposition-substitution reaction where replicas of the silent mating loci, at HML and HMR, are transmitted to the expressed mating-type locus (MAT). HML is on the left arm and HMR on the right arm, while MAT is in the middle of chromosome III. Cells with the genotype HMLα HMRa switch mating type efficiently at a frequency of about 86%. Since well over 50% of the cells switch, it is thought that switches do not occur randomly, but are directed to occur to the opposite mating-type allele. In contrast, we report that strains possessing the reverse HMLa HMRα arrangement switch (phenotype) inefficiently at a maximum of about 6%. The basis for this apparent reduced frequency of switching is that these strains preferentially yield futile homologous MAT locus switches—that is, MATa to MATa and MATα to MATα—and consequently, most of these events are undetected. We used genetically marked HM loci to demonstrate that a cells preferentially choose HMR as donor and a cells preferentially choose HML as donor, irrespective of the genetic content of the silent loci. Because of this feature, HMLα HMRa strains generate predominantly heterologous while HMLa HMRα strains produce predominantly homologous MAT switches. The control for directionality of switching therefore is not at the level of transposing heterologous mating-type information, but only at the level of choosing HML versus HMR as the donor. In strains where the preferred donor locus is deleted, the Inefficient donor becomes capable of donating efficiently. Thus the preference seems to be mediated by competition between the HM loci for donating information to MAT.     

Diversity of Mating-Type Chromosome Structures in the Yeast Zygosaccharomyces rouxii Caused by Ectopic Exchanges between MAT-Like Loci

We investigated sex chromosome diversity in Zygosaccharomyces rouxii (Z. rouxii). In the current study, we show that the organization of the mating-type (MAT) locus is highly variable in the Z. rouxii population, indicating the MAT, HML, and HMR loci are translocation hotspots. Although NBRC1130 and CBS732 were originally two stocks of the type strain of the species, only NBRC1130 retains the original karyotype. A reciprocal translocation between the MAT and HMR loci appears to have occurred during the early passage culture of CBS732, which was used for genome sequencing. In NBRC1733, NBRC0686, NBRC0740 and NBRC1053, the terminal region of the chromosome containing the HMR locus was replaced with the chromosomal region to the left of the MAT or HML loci. The translocation events found in NBRC1733, NBRC0686, NBRC0740, and NBRC1053 were reconstructed under our experimental conditions using the DA2 background, and the reconstruction suggests that the frequency of this type of translocation is approximately 10⁻⁷. These results suggest that the MAT and MAT-like loci were the susceptible regions in the genome, and the diversity of mating-type chromosome structures in Z. rouxii was caused by ectopic exchanges between MAT-like loci.     

Control of yeast cell type by the mating type locus: Positive regulation of the α-specific STE3 gene by the MATα 1 product

The mating type locus (MAT) determines the three yeast cell types, a, α, and a/α. It has been proposed that alleles of this locus, MATa and MATα, encode regulators that control expression of unlinked genes necessary for mating and sporulation. Specifically, the α1 product of MATα is proposed to be a positive regulator of α-specific genes. To test this view, we have assayed RNA production from the α-specific STE3 gene in the three cell types and in mutants defective in MATα. The STE3 gene was cloned by screening a yeast genomic clone bank for plasmids that complement the mating defect of ste3 mutants. Using the cloned STE3 gene as a probe, we find that a cells produce STE3 RNA, whereas a and a/a cells do not. Furthermore, matα 1 mutants do not produce STE3 RNA, whereas matα 2 mutants do. These results show that the STE3 gene, required for mating only by α cells, is expressed only in α cells. They show also that production of RNA from the STE3 gene requires the α1 product of MATα. Thus α1 positively regulates at least one α-specific gene by increasing the level of that gene's RNA product.     

Regulation of Nuclear Positioning and Dynamics of the Silent Mating Type Loci by the Yeast Ku70/Ku80 Complex

We have examined the hypothesis that the highly selective recombination of an active mating type locus (MAT) with either HMLα or HMRa is facilitated by the spatial positioning of relevant sequences within the budding yeast (Saccharomyces cerevisiae) nucleus. However, both position relative to the nuclear envelope (NE) and the subnuclear mobility of fluorescently tagged MAT, HML, or HMR loci are largely identical in haploid a and α cells. Irrespective of mating type, the expressed MAT locus is highly mobile within the nuclear lumen, while silent loci move less and are found preferentially near the NE. The perinuclear positions of HMR and HML are strongly compromised in strains lacking the Silent information regulator, Sir4. However, HMLα, unlike HMRa and most telomeres, shows increased NE association in a strain lacking yeast Ku70 (yKu70). Intriguingly, we find that the yKu complex is associated with HML and HMR sequences in a mating-type-specific manner. Its abundance decreases at the HMLα donor locus and increases transiently at MATa following DSB induction. Our data suggest that mating-type-specific binding of yKu to HMLα creates a local chromatin structure competent for recombination, which cooperates with the recombination enhancer to direct donor choice for gene conversion of the MATa locus.     

Mating type regulates the radiation-associated stimulation of reciprocal translocation events in Saccharomyces cerevisiae

Both ultraviolet (UV) and ionizing radiation were observed to stimulate mitotic, ectopic recombination between his3 recombinational substrates, generating reciprocal translocations in Saccharomyces cervisiae (yeast). The stimulation was greatest in diploid strains competent for sporulation and depends upon both the ploidy of the strain and heterozygosity at the MAT locus. The difference in levels of stimulation between MATa/MATα diploid and MATα haploid strains increases when cells are exposed to higher levels of UV radiation (sevenfold at 150 J/m²), whereas when cells are exposed to higher levels of ionizing radiation (23.4 krad), only a twofold difference is observed. When the MATα gene was introduced by DNA transformation into a MATa/matα::LEU2 ⁺ diploid, the levels of radiation-induced ectopic recombination approach those obtained in a strain that is heterozygous at MAT. Conversely, when the MATA gene was introduced by DNA transformation into a MATα haploid, no enhanced stimulation of ectopic recombination was observed when cells were irradiated with ionizing radiation but a threefold enhancement was observed when cells were irradiated with UV The increase in radiation-stimulated ectopic recombination resulting from heterozygosity at MAT correlated with greater spontaneous ectopic recombination and higher levels of viability after irradiation. We suggest that MAT functions that have been previously shown to control the level of mitotic, allelic recombination (homolog recombination) also control the level of mitotic, radiation-stimulated ectopic recombination between short dispersed repetitive sequences on non-homologous chromosomes.     

Precise Mapping of the Homothallism Genes HML and HMR in SACCHAROMYCES CEREVISIAE

The HML and HMR loci carry unexpressed copies of MATa and MATα information, and a replica of that information is transposed to MAT during mating-type interchange in Saccharomyces yeasts. A negative control mechanism keeps silent the information located at the HML and HMR loci. We mapped these loci by constructing strains in which these loci are expressed. In these strains, the mating type of the segregants is dependent upon the allele at HML and HMR. This novel approach is independent of their switching function. HML is located on the left arm of chromosome III distal to his4 by about 26.8 centimorgans (cM). HMR maps on the right arm of the same chromosome distal to thr4 by about 39.8 cM and proximal to MAL2 by about 1.0 cM. The results allow the exact placement of these loci and are in accord with the observations made by Harashima and Oshima (1976).     

Interconversion of Yeast Cell Types by Transposable Genes

The a and α cell types of budding yeast Saccharomyces cerevisiae are controlled by alternate alleles of the mating-type locus (MAT), MATa and MATα. The cell types can be interconverted by switching alleles of MAT. The loci HMRa and HMLα, which are loosely linked to MAT, are involved in mating-type switching. Experimental evidence for their role in MAT interconversion is presented. As a result of switching, the homothallic and heterothallic strains containing the amber and ochre mutations within the HMRa locus yield corresponding amber and ochre mutant mata loci. Similarly, the hmlα mutant strain generates matα mutant alleles. That is, specific mutations from HMRa and HMLα are transmitted to MAT. A replica of the mating-type coding information originating from these loci is transposed to MAT, where it replaces the existing information. Furthermore, "Hawthorne deletions" in strains containing hmra-amber/ochre result in production of mata-amber/ochre alleles. Therefore, genetic information for MATa resides at HMRa. The switches occur in a defined set of clonally related cells. Thus, the efficient interconversion of yeast cell types is mediated by an unidirectional transfer of genetic information between nonallelic sites in a nonrandom and programmed fashion. The results are inconsistent with the "flip-flop" models, but satisfy a key prediction of the general controlling element and the specific cassette models proposed for mating-type interchange.     

Control of yeast cell type by the mating type locus: I. Identification and control of expression of the a-specific gene BAR1

The MATα allele of the yeast mating type locus confers the α mating phenotype and contains two complementation groups, MATα1 and MATα2. The α1–α2 hypothesis proposes that MATα1 is a positive regulator of α-specific genes and that MATα2 is a negative regulator of a-specific genes. According to this hypothesis, matα2 mutants, which are defective in mating and in production of extracellular α-factor, express both a-specific functions (because they lack MATα2 product) and α-specific functions (because they contain MATα1 product). Failure to produce extracellular α-factor results from antagonism between these functions; in particular, because α-factor (an α-specific function) is degraded by an a-specific function. If this view is correct, matα2 mutants should acquire the ability to produce α-factor if they also carry a defect in the gene(s) responsible for α-factor degradation. We have isolated a derivative of a matα2 mutant that produces α-factor and have characterized the suppressor mutation in this strain. (1) This strain carries a mutation (bar1-1) tightly linked to HIS6 (on chromosome IX) that allows matα2 mutants to produce α-factor. (2) It does not allow matα1 mutants to produce α-factor. (3) Haploids of the a mating type bearing the bar1-1 mutation still mate, but are unable to act as a barrier to the diffusion of α-factor. MATa bar1-1 cells display increased sensitivity to α-factor. (4) A mutation (sst1?2) that causes increased sensitivity to α-factor is allelic to bar1-1 and also allows α-factor synthesis by matα2 mutants. The ability of matα2 bar1 double mutants to produce extracellular α-factor indicates that matα2 mutants do produce α-factor but that it is degraded by the Barrier function. These results suggest that BAR1 is normally expressed only in a cells, and is negatively regulated in α cells by the MATα2 product.     

Development of growth selection systems to isolate a-type or α-type of yeast cells spontaneously emerging from <Emphasis Type="Italic">MAT</Emphasis> a/α diploids

Background

Manufacture of MAT a and MAT α yeast cells is required for crossbreeding, a procedure that permits hybridization and the generation of new heterozygous strains. Crossbreeding also can be performed with a- and α-type of cells, which have the same mating abilities as MAT a and MAT α haploid cells, respectively.

Results

In this work, we describe a method to generate a- and α-type of cells via the naturally-occurring chromosomal aberration in parental MAT a/α diploids. We successfully designed suitable genetic circuits for expression of the URA3 selection marker gene to permit isolation of a- and α-type of cells, respectively, on solid medium lacking uracil. Furthermore we succeeded in generation of zygotes by mating of both the manufactured a- and α-type of yeast cells.

Conclusions

This process does not require exposure to mutagens such as UV irradiation, thereby avoiding the accumulation of undesirable mutations that would detract from the valuable traits that are under study. All the genetic modifications in the current study were introduced into yeast cells using plasmids, meaning that these traits can be removed without altering the genome sequence. This approach provides a reliable and versatile tool for scientific research and industrial yeast crossbreeding.

     

Healing of Broken Linear Dicentric Chromosomes in Yeast

In yeast, meiotic recombination between a linear chromosome III and a haploid-viable circular chromosome will yield a dicentric, tandemly duplicated chromosome. Spores containing apparently intact dicentric chromosomes were recovered from tetrads with three viable spores. The spore containing the dicentric inherited URA3 (part of the recombinant DNA used to join regions near the ends of the chromosome into a circle) as well as HML, HMR and MAL2 (located near the two ends of a linear but deleted from the circle). The Ura⁺ Mal⁺ colonies were highly variegated, giving rise to as many as seven distinctly different stable ("healed") derivatives, some of which were Ura⁺ Mal ⁺, others Ura⁺ Mal^- and others Ura ^- Mal⁺. The colonies were also sectored for five markers (HIS4, LEU2, CRY1, MAT and THR4) initially heterozygous in the tandemly duplicated dicentric chromosome.—Southern blot and genetic analyses have demonstrated that these stable derivatives arose from mitotic break-age of the dicentric chromosome, followed by one of several different healing events. The majority of the stable derivatives contained circular or linear chromosomes apparently resulting from homologous recombination between a broken chromosome end and a homologous region on the other end of the original dicentric duplicated chromosome. A smaller proportion of events resulted in apparently uniquely healed linear chromosomes in which the broken chromosome acquired a new telomere. In two instances we recovered chromosome III partially duplicated with a novel right end. We have also found one derivative that had also experienced rearrangement of repeated DNA sequences found adjacent to yeast telomeres.     

Cell cycle inhibition of yeast spheroplasts

Summary Osmotically stabilized yeast spheroplasts are capable of extensive DNA synthesis. Although the rate of DNA synthesis in spheroplasts is approximately one-third that of intact cells, the relative amounts of nuclear and mitochondrial DNA synthesized by spheroplasts is very similar to the relative amounts synthesized by intact cells. Furthermore, nuclear but not mitochondrial DNA synthesis is inhibited in MATa spheroplasts by the application of the yeast mating pheromone, -factor. Similarly, DNA synthesis is reversibly temperature-sensitive in spheroplasts created from cdc7 and cdc8 mutant cells.     

Control of yeast cell type by the mating type locus: II. Genetic interactions between MATα and unlinked α-specific STE genes

The alleles of the yeast mating type locus, MATα and MATa, determine the yeast cell types, a,α, and a/α. It has been proposed that the MATα2 product negatively regulates expression of unlinked a-specific genes, and that the MATα1 product positively regulates expression of unlinked α-specific genes. The behavior of mutants defective in MATα2, which are deficient in mating and in production of α-factor, can thus be attributed to antagonism between a-specific and α-specific functions expressed simultaneously in matα2^? strains. If this view is correct, then elimination by mutation of the specific functions required to mate as α may allow matα2 mutants to mate as a. In order to test this possibility, we examined the interactions between matα2 mutations and various unlinked mutations that cause α cells but not a cells to be mating defective (α-specific STE mutations). Three α-specific mutations (ste3, ste13 and kex2) were found to be non-allelic. Furthermore, although matα2 mutants mate weakly as a, matα2, ste3 double mutants, but not matα2 ste13 or matα2 kex2 double mutants, mate efficiently as a. The ability of matα2 ste3 strains to mate as a supports the view that matα2 mutants express a-specific mating functions, and suggests that a mating functions are expressed constitutively in MATa cells. The mating behaviour of the matα2 ste3 double mutant is consistent with the proposal that STE3 is positively regulated by the MATα1 product.     

A novel role for the mating type (MAT) locus in the maintenance of cell wall integrity in Saccharomyces cerevisiae

The cell wall and stress response component (Wsc) protein family in the yeast Saccharomyces cerevisiae is encoded by at least three genes, WSC1, WSC2, and WSC3. The Wsc proteins are putative upstream activators of the RHO1-regulated PKC1-MAP kinase cascade, and are required for maintenance of cell wall integrity and the stress response. Deletion of WSC1 causes a cell lysis defect that is exacerbated by deleting WSC2 or WSC3. This cell lysis defect can be rescued by adding osmotic stabilizers, such as 1?M sorbitol, to the medium, and by overexpressing PKC1 or RHO1. To advance our understanding of the function of the WSC genes, we performed a genetic screen to identify other components of the pathways they regulate. Here we report our findings. MAT a 1 and MATα2 were identified as dosage-dependent suppressors of the lysis defect of a wscΔ mutant. Overexpression of MAT a 1 or MATα2 was found to suppress the heat shock sensitivity, in addition to the lysis defect, of the wscΔ mutant. Phenotypic suppression by these two genes, MAT a 1 and MATα2, is significantly stronger when they are overexpressed in cells of the opposite mating type. Deletion of MAT a 1 exacerbates the lysis defect of haploid and diploid wscΔ strains. Our results suggest that the MAT locus plays a role in responses similar to those regulated by WSC and provide evidence for a regulatory effect of the MAT locus outside the realm of cell type determination.     

Dpb11, the budding yeast homolog of TopBP1, functions with the checkpoint clamp in recombination repair

Dpb11 is required for the loading of DNA polymerases α and on to DNA in chromosomal DNA replication and interacts with the DNA damage checkpoint protein Ddc1 in Saccharomyces cerevisiae. The interaction between the homologs of Dpb11 and Ddc1 in human cells and fission yeast is thought to reflect their involvement in the checkpoint response. Here we show that dpb11-1 cells, carrying a mutated Dpb11 that cannot interact with Ddc1, are defective in the repair of methyl methanesulfonate (MMS)-induced DNA damage but not in the DNA damage checkpoint at the permissive temperature. Epistatic analyses suggested that Dpb11 is involved in the Rad51/Rad52-dependent recombination pathway. Ddc1 as well as Dpb11 were required for homologous recombination induced by MMS. Moreover, we found the in vivo association of Dpb11 and Ddc1 with not only the HO-induced double-strand break (DSB) site at MAT locus but also the donor sequence HML during homologous recombination between MAT and HML. Rad51 was required for their association with the HML donor locus, but not with DSB site at the MAT locus. In addition, the association of Dpb11 with the MAT and HML locus after induction of HO-induced DSB was dependent on Ddc1. These results indicate that, besides the involvement in the replication and checkpoint, Dpb11 functions with Ddc1 in the recombination repair process itself.