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.     

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     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Chromosomal loci of genes controlling site-specific restriction endonucleases of Bacillus subtilis

Summary In Saccharomyces cerevisiae, diploid strains which are respiratory deficient (e.g., rho^–) or are homozygous for the mating-type locus (i.e., either a/a or /) are unable to sporulate. In order to induce sporulation in these nonsporulating strains, the technique of protoplast fusion mediated by polyethylene glycol was adopted. In this study, the products of protoplast fusion were induced to sporulate without reversion to normal cells.Protoplasts from a respiratory-deficient diploid strain were mixed with those from a respiratory-competent haploid one carrying mitochondrial drug resistance markers, treated with 30% polyethylene glycol-4000 and 25 mM CaCl₂, and incubated in 0.1 M potassium acetate containing 0.8 M sorbitol as an osmotic stabilizer. After two days' incubation, asci with three to eight spores were formed at a frequency of 1×10^–3 to 2×10^–4. Sporulation was also observed in products of fusion between an a/a diploid and haploid strains and between an / diploid and a haploid strains. The analysis of the genotypes of spores revealed that when fusion products were cultured under conditions for sporulation, karyogamy did not take place, diploid nuclei underwent meiosis, and both diploid and haploid nuclei were able to develop into spores.     

Stringent mating-type-regulated auxotrophy increases the accuracy of systematic genetic interaction screens with Saccharomyces cerevisiae mutant arrays

A genomic collection of haploid Saccharomyces cerevisiae deletion strains provides a unique resource for systematic analysis of gene interactions. Double-mutant haploid strains can be constructed by the synthetic genetic array (SGA) method, wherein a query mutation is introduced by mating to mutant arrays, selection of diploid double mutants, induction of meiosis, and selection of recombinant haploid double-mutant progeny. The mechanism of haploid selection is mating-type-regulated auxotrophy (MRA), by which prototrophy is restricted to a particular haploid genotype generated only as a result of meiosis. MRA escape leads to false-negative genetic interaction results because postmeiotic haploids that are supposed to be under negative selection instead proliferate and mate, forming diploids that are heterozygous at interacting loci, masking phenotypes that would be observed in a pure haploid double-mutant culture. This work identified factors that reduce MRA escape, including insertion of terminator and repressor sequences upstream of the MRA cassette, deletion of silent mating-type loci, and utilization of α-type instead of a-type MRA. Modifications engineered to reduce haploid MRA escape reduced false negative results in SGA-type analysis, resulting in >95% sensitivity for detecting gene–gene interactions.     

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.     

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.     

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.     

Interconversion of Yeast Mating Types I. Direct Observations of the Action of the Homothallism (HO) Gene

The HO gene promotes interconversion between a and α mating types. As a consequence, homothallic diploid cells are formed by mating between siblings descended from a single α HO or a HO spore. In order to determine the frequency and pattern of the mating-type switch, we have used a simple technique by which the mating phenotype can be assayed without losing the cell to the mating process itself. Specifically, we have performed pedigree analysis on descendants of single homothallic spores, testing these cells for sensitivity to α-factor.

The switch from α to a and vice versa is detectable after a minimum of two cell divisions. 50% of the clones tested showed switching by the four-cell stage. Of the four cells descended from a single cell, only the oldest cell and its immediate daughter are observed to change mating type. This pattern suggests that one event in the switching process has occurred in the first cell division cycle. Restriction of the switched mating-type to two particular cells may reflect the action of the homothallism system followed by nonrandom segregation of DNA strands in mitosis.

The mating behavior of cells which have sustained a change in mating type due to the HO gene is indistinguishable from that of heterothallic strains.

     

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.     

Initiation of Yeast Sporulation by Partial Carbon, Nitrogen, or Phosphate Deprivation

In this paper we show that partial deprivation of a carbon source, a nitrogen source, or phosphate in the presence of all other nutrients needed for growth initiates meiosis and sporulation of Saccharomyces cerevisiae homothallic strain Y55. For carbon deprivation experiments, cells were grown in synthetic medium (pH 5.5) containing an excess of one carbon source and then transferred to the same medium containing different concentrations of the same carbon source. In the case of transfer to different acetate concentrations, the log optical density at 600 nm increased at the previous rate until the cells had used up all of the acetate, whereupon the cells entered a stationary phase and did not sporulate. The same was observed with ethanol. In contrast, at different concentrations of dihydroxy-acetone or pyruvate, cells grew at different rates and sporulated optimally at intermediate concentrations (50 to 75 mM). The response to galactose was similar but reflected the presence of a low-affinity galactose transport system and the induction of a high-affinity galactose transport system. Cells could also sporulate when a glucose medium ran out of glucose, apparently because they initiated sporulation during the subsequent lag period and then used the produced ethanol as a carbon source. For phosphate deprivation experiments, cells growing with excess ethanol or pyruvate and phosphate were transferred to the same medium containing limiting amounts of phosphate. First, they used up the intracellular phosphate reserves for rapid growth, and then they sporulated optimally when an intermediate concentration (30 μM) of phosphate had been added to the medium. For nitrogen deprivation experiments, cells grown with excess acetate, ethanol, or pyruvate and NH₄⁺ were transferred to the same medium from which all nitrogen had been removed. These cells sporulated well in acetate medium but poorly in ethanol and pyruvate media. However, the sporulation frequency in the latter media could be increased greatly by adding intermediate concentrations (1 mM) of the slowly metabolizable amino acids glycine, histidine, or phenylalanine. If one assumes that the sporulation response to partial deprivation of carbon-, nitrogen-, or phosphorus-containing compounds reflects control by a single metabolite, the intracellular concentration of this metabolite may decide at the START position (G1 phase) of the cell cycle whether a/α cells enter mitosis or meiosis.     

Diploid Strains of the Pathogenic Basidiomycete Cryptococcus neoformans Are Thermally Dimorphic

Sia, R. A., Lengeler, K. B., and Heitman, J. 2000. Diploid strains of the pathogenic basidiomycete Cryptococcus neoformans are thermally dimorphic. Cryptococcus neoformans is an opportunistic human pathogenic fungus with a defined sexual cycle. Clinical and environmental isolates of C. neoformans are haploid, and the diploid stage of the lifecycle is thought to be transient and unstable. In contrast, we find that diploid strains are readily obtained following genetic crosses of congenic MATα and MATa strains. At 37°C, the diploid strains grow as yeast cells with a single nucleus that is larger than a haploid nucleus, contains a 2n content of DNA by FACS analysis, and is heterozygous for the MATα and MATa loci. At 24°C, these diploid self-fertile strains filament and sporulate, producing recombinant haploid progeny in which meiotic segregation has occurred. In contrast to dikaryotic filament cells that are typically linked by fused clamp connections during mating, self-fertile diploid strains produce monokaryotic filament cells with unfused clamp connections. We also show that these diploid strains can be transformed and sporulated and that an integrated selectable marker segregates in a mendelian fashion. The diploid state could play novel roles in the lifecycle and virulence of the organism and can be exploited for the analysis of essential genes. Finally, the observation that dimorphism is thermally regulated suggests similarities between the lifecycle of C. neoformans and other thermally dimorphic human pathogenic fungi, including Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, Paracoccidioides brasiliensis, and Sporothrix schenkii.