Hybridization of cells of the same mating type in Saccharomyces yeasts

The problem of mating-type switches in heterothallic yeast cells was investigated. 93% of non-mating hybrids were obtained in a X a crosses. The hybrids obtained in alpha X alpha crosses expressed alpha-mating type predominantly. Hybrids with no major rearrangements or loss of chromosome III were detected among these hybrids. In the selective system for cytoduction in a X a crosses the significant part of all cytoductants were alpha-maters, i.e. those originated through a----alpha switches. In alpha X alpha crosses alpha cytoductants were predominantly obtained either spontaneously or after UV-irradiation, though the frequency of cytoductants after UV-irradiation exceeded the control value several times. So, we developed the method for selection of mating-type "switchers" (a in equilibrium alpha), avoiding the diploid stage, and demonstrated the possibility of hybridization among the alpha-cells without hereditary changes at the MAT locus.     

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.     

Phenotypic expression of primary lesions of genetic material in Saccharomyces yeasts

"Illegitimate" mating of yeasts (alpha x alpha), either spontaneous or induced by uv light or ethyl methanesulfanate, in a selective system for "cytoduction" revealed that about 95% of cytoductants expressed their original (alpha) mating type. Inducing the mating by treating the recipient of cytoplasm with uv light reached two orders of magnitude. An additional copy of MAT alpha in the alpha recipient almost completely eliminated the effect, which means that nonheritable mating type changes observed are formally recessive and are localized within MAT alpha complex. About 1% of cytoductants obtained were nonmating types and some of them were identified as mat alpha l mutants. Radl8 mutant as a recipient showed a considerably elevated spontaneous frequency of illegitimate hybridization and cytoduction. The cytoductants also preserved the original mating type. These facts suggest that nonheritable changes of mating type are due to repairable primary (premutational) lesions in MAT alpha genetic material. The significance of these results for understanding the mechanism of nonheritable variability is discussed.     

Analysis of Y-Linked Mutations to Male Sterility in DROSOPHILA MELANOGASTER

Mating type in haploid cells of the yeast Saccharomyces cerevisiae is determined by a pair of alleles MATa and MAT alpha. Under various conditions haploid mating types can be interconverted. It has been proposed that transpositions of silent cassettes of mating-type information from HML OR HMR to MAT are the source of mating type conversions. A mutation described in this work, designated AON1, has the following properties. (1) MAT alpha cells carring AON1 are defective in mating. (2) AON1 allows MAT alpha/MAT alpha but not MATa/MATa diploids to sporulate; thus, AON1 mimics the MATa requirement for sporulation. (3) mata-1 cells that carry AON1 are MATa phenocopies, i.e., MAT alpha/mata-1 AON1 diploids behave as standard MAT alpha/MATa cells; therefore, AON1 suppresses the defect of mata-1. (4) AON1 maps at or near HMRa. (5) Same-site revertants from AON1 lose the ability to convert mating type to MATa, indicating that reversion is associated with the loss of a functional HMRa locus. In addition, AON1 is a dominant mutation. We conclude that AON1 is a regulatory mutation, probably cis-acting, that leads to the constitutive expression of silent a mating-type information located at HMRa.     

The Saccharomyces cerevisiae recombination enhancer biases recombination during interchromosomal mating-type switching but not in interchromosomal homologous recombination

Haploid Saccharomyces can change mating type through HO-endonuclease cleavage of an expressor locus, MAT, followed by gene conversion using one of two repository loci, HML or HMR, as donor. The mating type of a cell dictates which repository locus is used as donor, with a cells using HML and alpha cells using HMR. This preference is established in part by RE, a locus on the left arm of chromosome III that activates the surrounding region, including HML, for recombination in a cells, an activity suppressed by alpha 2 protein in alpha cells. We have examined the ability of RE to stimulate different forms of interchromosomal recombination. We found that RE exerted an effect on interchromosomal mating-type switching and on intrachromosomal homologous recombination but not on interchromosomal homologous recombination. Also, even in the absence of RE, MAT alpha still influenced donor preference in interchromosomal mating-type switching, supporting a role of alpha 2 in donor preference independent of RE. These results suggest a model in which RE affects competition between productive and nonproductive recombination outcomes. In interchromosome gene conversion, RE enhances both productive and nonproductive pathways, whereas in intrachromosomal gene conversion and mating-type switching, RE enhances only the productive pathway.     

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.     

The alpha-mating type locus of Cryptococcus neoformans contains a peptide pheromone gene.

The opportunistic fungal pathogen Cryptococcus neoformans has two mating types, MATa and MAT alpha. The MAT alpha strains are more virulent. Mating of opposite mating type haploid yeast cells results in the production of a filamentous hyphal phase. The MAT alpha locus has been isolated in this study in order to identify the genetic differences between mating types and their contribution to virulence. A 138-bp fragment of MAT alpha-specific DNA which cosegregates with alpha-mating type was isolated by using a difference cloning method. Overlapping phage and cosmid clones spanning the entire MAT alpha locus were isolated by using this MAT alpha-specific fragment as a probe. Mapping of these clones physically defined the MAT alpha locus to a 35- to 45-kb region which is present only in MAT alpha strains. Transformation studies with fragments of the MAT alpha locus identified a 2.1-kb XbaI-HindIII fragment that directs starvation-induced filament formation in MATa cells but not in MAT alpha cells. This 2.1-kb fragment contains a gene, MF alpha, with a small open reading frame encoding a pheromone precursor similar to the lipoprotein mating factors found in Saccharomyces cerevisiae, Ustilago maydis, and Schizosaccharomyces pombe. The ability of the MATa cells to express, process, and secrete the MAT alpha pheromone in response to starvation suggests similar mechanisms for these processes in both cell types. These results also suggest that the production of pheromone is under a type of nutritional control shared by the two cell types.     

Altered Mating-Type Identity in the Fungus Podospora Anserina Leads to Selfish Nuclei, Uniparental Progeny, and Haploid Meiosis

In wild-type crosses of the filamentous ascomycete Podospora anserina, after fertilization, only nuclei of opposite mating type can form dikaryons that undergo karyogamy and meiosis, producing biparental progeny. To determine the role played by the mating type in these steps, the four mat genes were mutagenized in vitro and introduced into a strain deleted for its mat locus. Genetic and cytological analyses of these mutant strains, crossed to each other and to wild type, showed that mating-type information is required for recognition of nuclear identity during the early steps of sexual reproduction. In crosses with strains carrying a mating-type mutation, two unusual developmental patterns were observed: monokaryotic cells, resulting in haploid meiosis, and uniparental dikaryotic cells providing, after karyogamy and meiosis, a uniparental progeny. Altered mating-type identity leads to selfish behavior of the mutant nucleus: it migrates alone or paired, ignoring its wild-type partner in all mutant X wild-type crosses. This behavior is nucleus-autonomous because, in the same cytoplasm, the wild-type nuclei form only biparental dikaryons. In P. anserina, mat genes are thus required to ensure a biparental dikaryotic state but appear dispensable for later stages, such as meiosis and sporulation.     

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.     

Homothallic mating type switching generates lethal chromosome breaks in rad52 strains of Saccharomyces cerevisiae.

In homothallic cells of Saccharomyces cerevisiae, a or alpha mating type information at the mating type locus (MAT) is replaced by the transposition of the opposite mating type allele from HML alpha or HMRa. The rad52-1 mutation, which reduces mitotic and abolishes meiotic recombination, also affects homothallic switching (Malone and Esposito, Proc. Natl. Acad. Sci. U.S.A. 77:503-507, 1980). We have found that both HO rad52 MATa and HO rad52 MAT alpha cells die. This lethality is suppressed by mutations that substantially reduce but do not eliminate homothallic conversions. These mutations map at or near the MAT locus (MAT alpha inc, MATa-inc, MATa stk1) or are unlinked to MAT (HO-1 and swi1). These results suggest that the switching event itself is involved in the lethality. With the exception of swi1, HO rad52 strains carrying one of the above mutations cannot convert mating type at all. MAT alpha rad52 HO swi1 strains apparently can switch MAT alpha to MATa. However, when we analyzed these a maters, we found that few, if any, of them were bona fide MATa cells. These a-like cells were instead either deleted for part of chromosome III distal to and including MAT or had lost the entire third chromosome. Approximately 30% of the time, an a-like cell could be repaired to a normal MATa genotype if the cell was mated to a RAD52 MAT alpha-inc strain. The effects of rad52 were also studied in mata/MAT alpha-inc rad52/rad52 ho/HO diploids. When this diploid attempted to switch mata to MATa, an unstable broken chromosome was generated in nearly every cell. These studies suggest that homothallic switching involves the formation of a double-stranded deoxyribonucleic acid break or a structure which is labile in rad52 cells and results in a broken chromosome. We propose that the production of a double-stranded deoxyribonucleic acid break is the lethal event in rad52 HO cells.     

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.     

Mitochondria are inherited from the MATa parent in crosses of the basidiomycete fungus Cryptococcus neoformans

Previous studies demonstrated that mitochondrial DNA (mtDNA) was uniparentally transmitted in laboratory crosses of the pathogenic yeast Cryptococcus neoformans. To begin understanding the mechanisms, this study examined the potential role of the mating-type locus on mtDNA inheritance in C. neoformans. Using existing isogenic strains (JEC20 and JEC21) that differed only at the mating-type locus and a clinical strain (CDC46) that possessed a mitochondrial genotype different from JEC20 and JEC21, we constructed strains that differed only in mating type and mitochondrial genotype. These strains were then crossed to produce hyphae and sexual spores. Among the 206 single spores analyzed from six crosses, all but one inherited mtDNA from the MATa parents. Analyses of mating-type alleles and mtDNA genotypes of natural hybrids from clinical and natural samples were consistent with the hypothesis that mtDNA is inherited from the MATa parent in C. neoformans. To distinguish two potential mechanisms, we obtained a pair of isogenic strains with different mating-type alleles, mtDNA types, and auxotrophic markers. Diploid cells from mating between these two strains were selected and 29 independent colonies were genotyped. These cells did not go through the hyphal stage or the meiotic process. All 29 colonies contained mtDNA from the MATa parent. Because no filamentation, meiosis, or spore formation was involved in generating these diploid cells, our results suggest a selective elimination of mtDNA from the MATalpha parent soon after mating. To our knowledge, this is the first demonstration that mating type controls mtDNA inheritance in fungi.     

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.     

Alpha mating type-specific expression of mutations leading to constitutive agglutinability in Saccharomyces cerevisiae.

Two mutants of Saccharomyces cerevisiae have been isolated and characterized. The mutants were constitutively agglutinable at 36 degrees C, the temperature at which wild-type cells agglutinate only after induction by mating pheromone. The mutant cells had other properties specific for the normal alpha cell type, i.e., conjugation with a cells, response to a mating pheromone, and production of alpha mating pheromone. The two mutations, cag1 and cag2, were recessive and expressed only in alpha cells. cag1 is linked very closely to the MAT locus, but cag2 is unlinked to the MAT locus. These cag mutations complemented ste3-1. These results indicate that CAG genes are novel alpha-specific genes involved in the regulation of sex agglutinin synthesis.     

Interaction between genetic background and the mating-type locus in Cryptococcus neoformans virulence potential

The study of quantitative traits provides a window on the interactions between multiple unlinked genetic loci. The interaction between hosts and pathogenic microbes, such as fungi, involves aspects of quantitative genetics for both partners in this dynamic equilibrium. One important pathogenic fungus is Cryptococcus neoformans, a basidiomycete yeast that can infect the human brain and whose mating system has two mating type alleles, a and alpha. The alpha mating-type allele has previously been linked to increased virulence potential. Here congenic C. neoformans strains were generated in the two well-characterized genetic backgrounds B3501alpha and NIH433a to examine the potential influence of genes outside of the mating-type locus on the virulence potential of mating type. The congenic nature of these new strain pairs was established by karyotyping, amplified fragment length polymorphism genotyping, and whole-genome molecular allele mapping (congenicity mapping). Virulence studies revealed that virulence was equivalent between the B3501 a and alpha congenic strains but the alpha strain was more virulent than its a counterpart in the NIH433 genetic background. These results demonstrate that genomic regions outside the mating type locus contribute to differences in virulence between a and alpha cells. The congenic strains described here provide a foundation upon which to elucidate at genetic and molecular levels how mating-type and other unlinked loci interact to enable microbial pathogenesis.