Interconversion of Yeast Mating Types III. Action of the Homothallism (HO) Gene in Cells Homozygous for the Mating Type Locus

Mating type interconversion in homothallic Saccharomyces cerevisiae has been studied in diploids homozygous for the mating type locus produced by sporulation of a/a/a/α and a/a/α/α tetraploid strains. Mating type switches have been analyzed by techniques including direct observation of cells for changes in α-factor sensitivity. Another method of following mating type switching exploits the observation that a/α cells exhibit polar budding and a/a and α/α cells exhibit medial budding.—These studies indicate the following: (1) The allele conferring the homothallic life cycle (HO) is dominant to the allele conferring the heterothallic life cycle (ho). (2) The action of the HO gene is controlled by the mating type locus—active in a/a and α/α cells but not in a/α cells. (3) The HO (or HO-controlled) gene product can act independently on two mating type alleles located on separate chromosomes in the same nucleus. (4) A switch in mating type is observed in pairs of cells, each of which has the same change.     

Interconversion of Yeast Mating Types II. Restoration of Mating Ability to Sterile Mutants in Homothallic and Heterothallic Strains

The two mating types of the yeast Saccharomyces cerevisiae can be interconverted in both homothallic and heterothallic strains. Previous work indicates that all yeast cells contain the information to be both a and α and that the HO gene (in homothallic strains) promotes a change in mating type by causing a change at the mating type locus itself. In both heterothallic and homothallic strains, a defective α mating type locus can be converted to a functional a locus and subsequently to a functional α locus. In contrast, action of the HO gene does not restore mating ability to a strain defective in another gene for mating which is not at the mating type locus. These observations indicate that a yeast cell contains an additional copy (or copies) of α information, and lead to the "cassette" model for mating type interconversion. In this model, HMa and hmα loci are blocs of unexpressed α regulatory information, and HMα and hma loci are blocs of unexpressed a regulatory information. These blocs are silent because they lack an essential site for expression, and become active upon insertion of this information (or a copy of the information) into the mating type locus by action of the HO gene.     

Evidence of the Insensitivity of the alpha-inc Allele to the Function of the Homothallic Genes in Saccharomyces Yeasts

The possible function of the α-inc allele (an α mating-type allele that is insensitive to the function of the homothallic gene system) was investigated by means of protoplast fusion. The fusion of protoplasts prepared from haploid strains of α-inc HO HMα HMa and α ho hmα HMa gave rise mainly to nonmating clones (58 of 64 isolates) and a few clones (six of 64 isolates) showing α mating type. Thirty of the 58 nonmating clones showed the diploid cell size and 28 clones had a larger cell size. Tetrad analysis of the nonmating clones with diploid cell size indicated that they were a/α-inc diploid; the normal α allele in α/α-inc cells was preferentially switched to an a allele. This observation further indicated that the HO/ho HMα/hmα HMa/HMa genotype is effective for the conversion of the α to a and that the inconvertibility of the α-inc allele is due to the insensitivity of the mating-type allele to the functional combination of the homothallic genes. It was suspected that fusion products larger than diploid cells might have been caused by multiple fusion of protoplasts.     

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.     

Mating-type locus of Cryptococcus neoformans: a step in the evolution of sex chromosomes

The sexual development and virulence of the fungal pathogen Cryptococcus neoformans is controlled by a bipolar mating system determined by a single locus that exists in two alleles, α and a. The α and a mating-type alleles from two divergent varieties were cloned and sequenced. The C. neoformans mating-type locus is unique, spans >100 kb, and contains more than 20 genes. MAT-encoded products include homologs of regulators of sexual development in other fungi, pheromone and pheromone receptors, divergent components of a MAP kinase cascade, and other proteins with no obvious function in mating. The α and a alleles of the mating-type locus have extensively rearranged during evolution and strain divergence but are stable during genetic crosses and in the population. The C. neoformans mating-type locus is strikingly different from the other known fungal mating-type loci, sharing features with the self-incompatibility systems and sex chromosomes of algae, plants, and animals. Our study establishes a new paradigm for mating-type loci in fungi with implications for the evolution of cell identity and self/nonself recognition.     

Evidence for Co-Dominance of the Homothallic Genes, HMalpha/hmalpha and HMa/hma, IN SACCHAROMYCES YEASTS

To investigate the dominance and recessiveness of the homothallism genes, HMα/hmα and HMa/hma, for mating-type conversion, we constructed hybrids with various configurations of the homothallic genes by fusion of protoplasts prepared from haploid strains having identical mating types. Eight different combinations of the homothallic genes were tested for their function by observing the mating and sporulation abilities of the fusion products. With few exceptions, nonmating and sporogenous fusion products were obtained from the following combinations: α HO hmα HMa + α ho hmα hma, α HO hmα HMa + α ho HMα hma, α HO hmα HMa + α ho HMα HMa, a HO HMα hma + a ho hmα hma, a HO HMα hma + a ho hmα HMa and a HO HMα hma + a ho HMα HMa. All the fusion products from the α HO hmα HMa + α ho hmα HMa and a HO HMα hma + a ho HMα hma combinations showed mating types identical to those of the respective haploid strains. These results clearly support the co-dominance of the HMα/hmα and HMa/hma alleles and indicate that the hmα allele has the same function as the HMa allele and that the hma allele has the same function as the HMα allele.     

Global chromatin structure of 45,000 base pairs of chromosome III in a- and alpha-cell yeast and during mating-type switching

Directionality of yeast mating-type switching has been attributed to differences in chromatin structure for the left arm of chromosome III. We have mapped the structure of ~45 kbp of the left arm of chromosome III in a and α cells in logarithmically growing cultures and in a cells during switching. Distinctive features of chromatin structure were the occurrence of DNase I-hypersensitive sites in the promoter region of nearly every gene and some replication origins and the presence of extended regions of positioned nucleosomes in ~25% of the open reading frames. Other than the recombination enhancer, chromatin structures were identical in the two cell types. Changes in chromatin structure during switching were confined to the recombination enhancer. This unbiased analysis of an extended region of chromatin reveals that significant features of organized chromatin exist for the entire region, and these features are largely static with respect to mating type and mating-type switching. Our analysis also shows that primary chromatin structure does not cause the documented differences in recombinational frequency of the left arm of chromosome III in yeast a and α cells.     

Mating Type and Sporulation in Yeast I. Mutations Which Alter Mating-Type Control over Sporulation

In Saccharomyces cerevisiae, meiosis and spore formation as well as mating are controlled by mating-type genes. Diploids heterozygous for mating type (aα) can sporulate but cannot mate; homozygous aa and αα diploids can mate, but cannot sporulate. From an αα diploid parental strain, we have isolated mutants which have gained the ability to sporulate. Those mutants which continue to mate as αα cells have been designated CSP (control of sporulation). Upon sporulation, CSP mutants yield asci containing 4α spores. The mutant gene which allows αα cells to sporulate is unlinked to the mating-type locus and also acts to permit sporulation in aa diploid cells. Segregation data from crosses between mutant αα and wild-type aa diploids and vice versa indicate (for all but one mutant) that the mutation which allows constitutive sporulation (CSP) is dominant over the wild-type allele. Some of the CSP mutants are temperature-sensitive, sporulating at 32°, but not at 23°. In addition to CSP mutants, our mutagenesis and screening procedure led to the isolation of mutants which sporulate by virtue of a change in the mating-type locus itself, resulting in loss of ability to mate.     

Pleiotropic Mutations at the TUP1 Locus That Affect the Expression of Mating-Type-Dependent Functions in SACCHAROMYCES CEREVISIAE

The umr7–1 mutation, previously identified in a set of mutants that had been selected for defective UV-induced mutagenesis at CAN1, affects other cellular functions, including many of those regulated by the mating-type locus (MAT) in heterothallic Saccharomyces cerevisiae. The recessive umr7–1 allele, mapping approximately 20 cM distal to thr4 on chromosome III, causes clumpy growth in both a and α cells and has no apparent effect on a mating functions. However, α umr7 meiotic segregants fail to express several α-specific functions (e.g., high-frequency conjugation with a strains, secretion of the hormone α-factor and response to the hormone a-factor). In addition, α umr7 cells exhibit some a-specific characteristics, such as the barrier phenotype (Bar⁺) that prevents diffusion of α-factor and an increased mating frequency with α strains. The most striking property of α umr7 strains is their altered morphology, in which mitotic cells develop an asymmetric pear shape, like that of normal a cells induced to form "shmoos" by interaction with α-factor. Some a/α-specific diploid functions are also affected by umr7; instead of polar budding patterns, a/α umr7/umr7 diploids have medial budding like a/a, α/α and haploid strains. Moreover, a/α umr7/umr7 diploids have lost the ability to sporulate and are Bar⁺ like a or a/a strains. Revertant studies indicate that umr7–1 is a single point mutation. The umr7 mutant fails to complement mutants of both tup1 (selected for deoxythymidine monophosphate utilization) and cyc9 (selected for high iso-2-cytochrome c levels), and all three isolates have similar genetic and phenotypic properties. It is suggested that the product of this gene plays some common central role in the complex regulation of the expression of both MAT-dependent and MAT-independent functions.     

Mutants of SACCHAROMYCES CEREVISIAE Resistant to the α Mating-Type Factor

Mutants that are resistant to α-factor have been isolated from a mating-type haploid strains of yeast by direct selection on agar medium containing partially purified α-factor. All resistant mutants isolated were found to be sterile. They were characterized and compared with mutants previously isolated as nonmating. Among 93 able to mate at low frequency and to sporulate, none showed linkage to the mating-type locus. The results support the hypothesis that the response to α-factor by cells of mating-type a is essential for mating.     

Mating Type and Sporulation in Yeast. II. Meiosis, Recombination, and Radiation Sensitivity in an α_entitystart_#945_entit Diploid with Altered Sporulation Control

In wild-type S. cerevisiae, diploid cells must be heterozygous at the mating-type locus in order to sporulate. In the preceding paper, we described a number of mutants (CSP mutants), isolated from nonsporulating aa and αα parent strains, in which sporulation appeared to be uncoupled from control by mating type. The characterization of one of these mutants (CSP1) is now extended to other processes controlled by mating type. This mutant is indistinguishable from αα cells and unlike aα cells for mating factor production and response, zygote formation, intragenic mitotic recombination, and for X-ray sensitivity. The mutant apparently undergoes a full round of DNA synthesis in sporulation medium, but with delayed kinetics. Only 20% of the cells complete sporulation. Among spores in completed asci, the frequency of both intra- and intergenic recombination is the same as it is for spores produced by aα cells. However, experiments in which cells were shifted from sporulation medium back to minimal growth medium gave a frequency of meiotic recombination between ade2 or leu2 heteroalleles only 25% to 29% as high for CSP1 αα diploid or CSP1 aa disomic cells as for aα diploid or disomic cells. Because the latter result, indicating recombination defectiveness, measured recombinant production in the entire cell population, whereas the result indicating normal recombination sampled only completed spores, we infer that all meiotic recombination events occurring in the population of CSP1 αα cells are concentrated in those few cells which complete sporulation. This high degree of correlation between meiotic recombination and the completion of meiosis and sporulation suggests that recombination may be required for proper meiotic chromosome segregation in yeast just as it appears to be in maize and in Drosophila     

Alpha-pheromone-induced "shmooing" and gene regulation require white-opaque switching during Candida albicans mating

A 14-mer α-pheromone peptide of Candida albicans was chemically synthesized and used to analyze the role of white-opaque switching in the mating process. The α-pheromone peptide blocked cell multiplication and induced “shmooing” in a/a cells expressing the opaque-phase phenotype but not in a/a cells expressing the white-phase phenotype. The α-pheromone peptide induced these effects at 25°C but not at 37°C. An analysis of mating-associated gene expression revealed several categories of gene regulation, including (i) MTL-homozygous-specific, pheromone stimulated, switching-independent (CAG1 and STE4); (ii) mating type-specific, pheromone-induced, switching-independent (STE2); and (iii) pheromone-induced, switching-dependent (FIG1, KAR4, and HWP1). An analysis of switching-regulated genes revealed an additional category of opaque-phase-specific genes that are downregulated by α-pheromone only in a/a cells (OP4, SAP1, and SAP3). These results demonstrate that α-pheromone causes shmooing, the initial step in the mating process, only in a/a cells expressing the opaque phenotype and only at temperatures below that in the human host. These results further demonstrate that although some mating-associated genes are stimulated by the α-pheromone peptide in both white- and opaque-phase cells, others are stimulated only in opaque-phase cells, revealing a category of gene regulation unique to C. albicans in which α-pheromone induction requires the white-opaque transition. These results demonstrate that in C. albicans, the mating process and associated gene regulation must be examined within the context of white-opaque switching.     

Mating-Type Regulation of Methyl Methanesulfonate Sensitivity in SACCHAROMYCES CEREVISIAE

Heterozygosity at the mating-type locus (MAT) in Saccharomyces cerevisiae has been shown previously to enhance X-ray survival in diploid cells. We now show that a/α diploids are also more resistant to the radiomimetic agent methyl methanesulfonate (MMS) than are diploids that are homozygous at MAT (i.e., either a/a or α/α). Log-phase a/α cultures exhibit biphasic MMS survival curves, in which the more resistant fraction consists of budded cells (those cells in the S and G2 phases of the cell cycle). Survival curves for log-phase cultures of a/a or α/α diploids have little if any biphasic nature, suggesting that the enhanced S- and G2-phase repair capacity of a/α cells may be associated with heterozygosity at MAT. The survival of cells arrested at the beginning of the S phase with hydroxyurea indicates that MAT-dependent MMS repair is limited to S and G2, whereas MAT-independent repair can occur in G1.     

Mating-Type Functions for Meiosis and Sporulation in Yeast Act through Cytoplasm

Given a nutritional regime marked by a low nitrogen level and the absence of fermentable carbon sources, conventional a/α diploid cells of Saccharomyces cerevisiae exhibit a complex developmental sequence that includes a round of premeiotic DNA replication, commitment to meiosis and the elaboration of mature tetrads containing viable ascospores. Ordinarily, haploid cells and diploid cells of genotype a/a and α/α fail to display these reactions under comparable conditions. Here, we describe a simple technique for sporulation of α/α and a/a cells. Cells of genotype α/α are mated to haploid a cells carrying the kar1 (karyogamy defective) mutation to yield heterokaryons containing the corresponding diploid and haploid nuclei. The kar1 strains mate normally, but nuclei in the resultant zygotes do not fuse. When heterokaryotic cells are inoculated into sporulation media, they produce asci with six spores. Four spores carry genotypes derived from the diploid nucleus and the other two possess the markers originating from the haploid nucleus, i.e., the diploid nucleus divides meiotically while the haploid nucleus apparently divides mitotically. Similarly, the a/a genome is "helped" to sporulate as a consequence of mating with α kar1 strains. The results allow us to conclude that the mating-type functions essential for meiosis and sporulation are communicated and act through the cytoplasm and that sporulation can be dissociated from typical meiosis. This procedure will facilitate the genetic analysis of strains that are otherwise unable to sporulate.     

Regulation of Mating and Meiosis in Yeast by the Mating-Type Region

A supposed sporulation-deficient mutation of Saccharomyces cerevisiae is found to affect mating in haploids and in diploids, and to be inseparable from the mating-type locus by recombination. The mutation is regarded as a defective a allele and is designated a*. This is confirmed by its dominance relations in diploids, triploids, and tetraploids. Tetrad analysis of tetraploids and of their sporulating diploid progeny suggests the existence of an additional locus, RME, which regulates sporulation in yeast strains that can mate. Thus the recessive homozygous constitution rme/rme enables the diploids a*/α, a/a*, and α/α to go through meiosis. Haploids carrying rme show apparent premeiotic DNA replication in sporulation conditions. This new regulatory locus is linked to the centromere of the mating-type chromosome, and its two alleles, rme and RME, are found among standard laboratory strains.     

Dependence on Mating Type for the Overproduction of Iso-2-Cytochrome c in the Yeast Mutant CYC7–H2

The CYC7–H2 mutation causes an approximately 20-fold overproduction of iso–2–cytochromo c in a and α haploid strains of the yeast Saccharomyces cerevisiae due to an alteration in the nontranslated regulatory region that is presumably contiguous with the structural region. In this investigation, we demonstrated that heterozygosity at the mating type locus, a/α or a/a/α/α, prevents expression of the overproduction, while homozygosity, a/a and α/α, and hemizygosity, a/0 and α/0, allow full expression of the CYC7–H2 mutation, equivalent to the expression observed in a and α haploid strains. There is no decrease in the overproduction of iso-2-cytochrome c in a/α diploid strains containing either of the other two similar mutations, CYC7–H1 and CYC7–H3. It appears as if active expression of one or another of the mating-type alleles is required for the overproduction of iso-2-cytochrome c in CYC7–H2 mutants.     

The Saccharomyces cerevisiae RAD54 gene is important but not essential for natural homothallic mating-type switching

Homothallic Saccharomyces cerevisiae strains switch their mating-type in a specific gene conversion event induced by a DNA double strand break made by the HO endonuclease. The RAD52 group genes control recombinational repair of DNA double strand breaks, and we examined their role in native homothallic mating-type switching. Surprisingly, we found that the Rad54 protein was important but not essential for mating-type switching under natural conditions. As an upper limit, we estimate that 29% of the rad54 spore clones can successfully switch their mating-type. The RAD55 and RAD57 gene products were even less important, but their presence increased the efficiency of the process. In contrast, the RAD51 and RAD52 genes are essential for homothallic mating-type switching. We propose that mating-type switching in RAD54 mutants occurs stochastically with a low probability, possibly reflecting different states of chromosomal structure.     

Construction and Characterization of Isogenic Series of Saccharomyces cerevisiae Polyploid Strains

Tetraploid cells of Saccharomyces cerevisiae are generated spontaneously in a homothallic MATa/MATα diploid population at low frequency (approximately 10⁻⁶ per cell) through the homozygosity of mating-type alleles by mitotic recombination followed by homothallic switching of the mating-type alleles. To isolate tetraploid clones more effectively, a selection method was developed that used a dye plate containing 40 mg each of eosin Y and amaranth in synthetic nutrient agar per liter. It was possible to isolate tetraploid clones on the dye plate at a frequency of 1 to 3% among the colonies colored dark red in contrast to the light red of the original diploid colonies. Isogenic series of haploid to tetraploid clones with homozygous or heterozygous genomic configurations were easily constructed with the tetraploid strains. No significant differences in specific growth rate or fermentative rate were observed corresponding to differences in ploidy, although the haploid clones showed a higher frequency of spontaneous respiratory-deficient cells than did the others. However, a significant increment in the fermentative rate in glucose nutrient medium was observed in the hybrid strains constructed with two independent homozygous cell lines. These observations strongly suggest that the polyploid strains favored by the brewing and baking industries perform well not because of the physical increment of the cellular volume by polyploidy but because of the genetic complexity or heterosis by heterozygosity of the genome in the hybrid polyploid cells.     

αADα Hybrids of Cryptococcus neoformans: Evidence of Same-Sex Mating in Nature and Hybrid Fitness

Cryptococcus neoformans is a ubiquitous human fungal pathogen that causes meningoencephalitis in predominantly immunocompromised hosts. The fungus is typically haploid, and sexual reproduction involves two individuals with opposite mating types/sexes, α and a. However, the overwhelming predominance of mating type (MAT) α over a in C. neoformans populations limits α–a mating in nature. Recently it was discovered that C. neoformans can undergo same-sex mating under laboratory conditions, especially between α isolates. Whether same-sex mating occurs in nature and contributes to the current population structure was unknown. In this study, natural αADα hybrids that arose by fusion between two α cells of different serotypes (A and D) were identified and characterized, providing definitive evidence that same-sex mating occurs naturally. A novel truncated allele of the mating-type-specific cell identity determinant SXI1α was also identified as a genetic factor likely involved in this process. In addition, laboratory-constructed αADα strains exhibited hybrid vigor both in vitro and in vivo, providing a plausible explanation for their relative abundance in nature despite the fact that AD hybrids are inefficient in meiosis/sporulation and are trapped in the diploid state. These findings provide insights on the origins, genetic mechanisms, and fitness impact of unisexual hybridization in the Cryptococcus population.     

Protein Degradation, Meiosis and Sporulation in Proteinase-Deficient Mutants of SACCHAROMYCES CEREVISIAE

During the process of sporulation, a/α diploids degrade about 50% of their vegetative proteins. This degradation is not sporulation specific, for asporogenous diploids of a/a mating type degrade their vegetative proteins in a fashion similar to that of their a/α counterparts. Diploids lacking carboxypeptidase Y activity, prc1/prc1, show about 80% of wild-type levels of protein degradation, but are unimpaired in the production of normal asci. Diploids lacking proteinase B activity, prb1/prb1, show about 50% of wild-type levels of protein degradation. The effect on degradation of the proteinase B deficiency is epistatic to the degradation deficit attributable to the carboxypeptidase Y deficiency. The prb1 homozygotes undergo meiosis and produce spores, but the asci and, possibly, the spores are abnormal. Diploids homozygous for the pleiotropic pep4–3 mutation show only 30% of the wild-type levels of degradation when exposed to a sporulation regimen, and do not undergo meiosis or sporulation. Neither proteinase B nor carboxypeptidase Y is necessary for germination of spores.——Approximately half of the colonies arising from a/a or α/α diploids exposed to the sporulation regiment that express an initially heterozygous drug-resistance marker (can1) appear to arise from mating-type switches followed by meiosis and sporulation.