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.     

Relation between the efficiency of homothallic switching of yeast mating type genes and the distribution of cell types.

Homothallic switching of yeast mating type genes occurs as often as each cell division, so that a colony derived from a single haploid spore soon contains an equal number of MATa and MAT alpha cells. Cells of opposite mating types conjugate, and eventually the colony contains only nonmating MATa/MAT alpha diploids. Mutations that reduce the efficiency of homothallic MAT conversions yield colonies that still contain many haploid cells of the original spore mating type plus a few recently generated cells of the opposite mating type. These (a greater than alpha)- or (alpha greater than a)-mating colonies also contain some nonmating diploid cells. As an alternative to microscopic pedigree analysis to determine the frequency of mating type conversions in a variety of mutant homothallic strains, we analyzed the proportions of MATa, MAT alpha, and MATa/MAT alpha cells in a colony by examining the mating phenotypes of subclones. We developed a mathematical model that described the proportion of cell types in a slow-switching colony. This model predicted that the proportion of nonmating cells would continually increase with the size (age) of a colony derived from a single cell. This prediction was confirmed by determining the proportion of cell types in colonies of an HO swi1 strain that was grown for different numbers of cell divisions. Data from subcloning (a greater than alpha) and (alpha greater than a) colonies from a variety of slow-switching mutations and chromosomal rearrangements were used to calculate the frequency of MAT conversions in these strains.     

The Genetic System Controlling Homothallism in Saccharomyces Yeasts

There are four types of life cycles in Saccharomyces cerevisiae and its related species. A perfect homothallic life cycle (the Ho type) is observed in the classic D strain. Two other types show semi-homothallism; one of them shows a 2-homothallic diploid:2alpha heterothallic haploid segregation (the Hp type) and another, a 2-homothallic:2a segregation (the Hq type). In the segregants from these Ho, Hp, and Hq diploids, each homothallic segregant shows the same segregation pattern as its parental diploid. The fourth type has a heterothallic life cycle showing a 2a:2alpha segregation and the diploids are produced by the fusion of two haploid cells of opposite mating types. The diploids prepared by the crosses of alpha Hp (an alpha haploid segregant from the Hp diploid) to a Hq (an a haploid from the Hq diploid) segregated two types (Type I and II) of the Ho type homothallic clone among their meiotic segregants. Genetic analyses were performed to investigate this phenomenon and the genotypes of the Ho type homothallic clones of Type I and Type II. Results of these genetic analyses have been most adequately explained by postulating three kinds of homothallic genes, each consisting of a single pair of alleles, HO/ho, HMalpha/hmalpha, and HMa/hma, respectively. One of them, the HMalpha locus, was proved to be loosely linked (64 stranes) to the mating-type locus. A spore having the HO hmalpha hma genotype gives rise to an Ho type homothallic diploid (Type I), the same as in the case of the D strain which has the HO HMalpha HMa genotype (Type II). A spore having the a HO hmalpha HMa or alpha HO HMalpha hma genotype will produce an Hp or Hq type homothallic diploid culture, respectively. The other genotypes, a HO HMalpha hma, alpha HO hmalpha HMa, and the genotypes combined with the ho allele give a heterothallic character to the spore culture. A possible molecular hypothesis for the mating-type differentiation with the controlling elements produced by the HMalpha and HMa genes is proposed.     

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.     

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.     

Viability of Physarum polycephalum spores and ploidy of plasmodial nuclei.

Amoebae of Physarum polycephalum carrying the mth mating-type allele may differentiate into plasmodia in the absence of mating. Such plasmodia are haploid and, upon sporulation, produce mainly inviable spores. We have asked whether the viable spores arise from meiotic or mitotic divisions. Using a microfluorometric measurement of the deoxyribonucleic acid content of individual nuclei, we found the fraction of viable spores to be correlated with the proportion of rare, diploid nuclei containing in the generally haploid plasmodium. When homozygous diploid plasmodia were created by heat shocking, spore viability increased dramatically. We suggest that viable spores are produced via meiosis in mth plasmodia, that the mth allele has no effect on sporulation per se, and that the normal source of viable haploid spores is a small fraction of diploid nuclei ubiquitous in haploid plasmodia.     

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.     

Genetic analyses of a hybrid cross between serotypes A and D strains of the human pathogenic fungus Cryptococcus neoformans

Cryptococcus neoformans has two varieties, var. grubii and var. neoformans, that correspond to serotypes A and D, respectively. Molecular phylogenetic analyses suggest that these two varieties have diverged from each other for approximately 18 million years. The discovery of pathogenic serotype AD hybrid strains in nature indicates that intervariety mating in C. neoformans occurs in the natural environment. However, little is known about the genetic consequences of hybridization in C. neoformans. Here, we analyzed a hybrid population of 163 progeny from a cross between strains of serotypes A (CDC15) and D (JEC20), using 114 codominant nuclear PCR-RFLP markers and 1 direct PCR marker. These markers were distributed on all 14 chromosomes of the sequenced strain JEC21 that was isogenic to one of the parents (JEC20) in our cross. Our analyses identified that of the 163 progeny, 5 were heterozygous at all 115 loci, 1 was completely homozygous and identical to one of the parents (CDC15), and the remaining 157 each contained at least 1 heterozygous locus. Because all 163 progeny inherited mitochondria from the MATa parent JEC20, none of the progeny had a genotype identical to either of the two parents or to a composite of the two parents. All 115 nuclear loci showed three different genotypes in the progeny population, consistent with Mendelian segregation during meiosis. While the linkage analysis showed independent reassortment among loci on different linkage groups, there were significant differences in recombination frequencies among chromosomes and among regions within certain chromosomes. Overall, the linkage-map length from this hybrid cross was much shorter and the recombination frequency much lower than those constructed using serotype D strains, consistent with suppressed recombination in the intervariety cross between strains of serotypes A and D. We discuss the implications of our results in our understanding of the speciation and evolution of the C. neoformans species complex.     

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.     

Diversity of Y region at HML locus in a Saccharomyces cerevisiae strain isolated from a Sardinian wine

Several mutations in genes involved in Saccharomyces mating type switching may affect the homothallic behaviour in wine yeasts. In this study the semi-homothallic (Hq) segregation of a flor wine yeast strain was analysed. We aimed to understand the molecular basis of this behaviour in a flor autochthonous strain, verifying the MAT locus status by a PCR-based HO gene disruption and sequencing of the Y region of the HML, HMR and MAT loci, after nested PCR. Presence of ORFs a1 and a2 in the Y region of the HML locus was found. At the ORF a2 at HML locus, a mutation in the stop codon was found, so the a2 ORF contains 33 more bases.     

Heteroduplex formation and mismatch repair of the "stuck" mutation during mating-type switching in Saccharomyces cerevisiae.

We sequenced two alleles of the MATa locus of Saccharomyces cerevisiae that reduce homothallic switching and confer viability to HO rad52 strains. Both the MATa-stk (J. E. Haber, W. T. Savage, S. M. Raposa, B. Weiffenbach, and L. B. Rowe, Proc. Natl. Acad. Sci. USA 77:2824-2828, 1980) and MATa-survivor (R. E. Malone and D. Hyman, Curr. Genet. 7:439-447, 1983) alleles result from a T----A base change at position Z11 of the MAT locus. These strains also contain identical base substitutions at HMRa, so that the mutation is reintroduced when MAT alpha switches to MATa. Mating-type switching in a MATa-stk strain relative to a MATa Z11T strain is reduced at least 50-fold but can be increased by expression of HO from a galactose-inducible promoter. We confirmed by Southern analysis that the Z11A mutation reduced the efficiency of double-strand break formation compared with the Z11T variant; the reduction was more severe in MAT alpha than in MATa. In MAT alpha, the Z11A mutation also creates a mat alpha 1 (sterile) mutation that distinguishes switches of MATa-stk to either MAT alpha or mat alpha 1-stk. Pedigree analysis of cells induced to switch in G1 showed that MATa-stk switched frequently (23% of the time) to produce one mat alpha 1-stk and one MAT alpha progeny. This postswitching segregation suggests that Z11 was often present in heteroduplex DNA that was not mismatch repaired. When mismatch repair was prevented by deletion of the PMS1 gene, there was an increase in the proportion of mat alpha 1-stk/MAT alpha sectors (59%) and in pairs of switched cells that both retained the stk mutation (27%). We conclude that at least one strand of DNA only 4 bp from the HO cut site is not degraded in most of the gene conversion events that accompany MAT switching.     

Coconversion of flanking sequences with homothallic switching

Homothallic switching in S. cerevisiae involves replacing the DNA of the expressed allele at the mating type locus (MAT) with a duplicate of sequences from the unexpressed loci HML or HMR. The MATa and MAT alpha alleles differ by a DNA substitution that is flanked by sequences in common to MAT, and the donor loci HML and HMR. Using restriction site polymorphisms between MAT and the donor loci, we demonstrate that the extent of MAT DNA that is replaced during switching is variable and that there is a gradient of coconversion across the X region. Coconversion events occur on both sides of the double-strand cleavage by the HO gene product. The two cells produced after a switch often differ at the flanking site, indicating a DNA heteroduplex intermediate.     

The Action of Homothallism Genes in Saccharomyces Diploids during Vegetative Growth and the Equivalence of hma and HMalpha Loci Functions

The action of homothallism genes in vegetatively growing diploid cells was examined. The results demonstrate that homothallism genes function during regular vegetative growth cycles as well as during the first few divisions after spore germination. A procedure based on ultraviolet-induced reciprocal mitotic recombination monitored by homozygosity for cryptopleurine resistance (a recessive marker closely linked to the mating-type locus) allowed us to identify and recover Saccharomyces cerevisiae colonies sectored for the mating-type locus i.e., a/a and alpha/alpha. Homothallism genes can switch a/a or alpha/alpha vegetative diploid cells, generated from a strain with genotype a/alpha HO/ho HMalpha/HMalpha HMa/HMa, to a/alpha diploids or a/a/alpha/alpha tetraploids during a given mitotic division cycle. We found that both a/a and alpha/alpha sectors generated from a strain with genotype a/alpha HO/HO hmalpha/hmalpha hma/HMa switch to a/alpha diploids or a/a/alpha/alpha tetraploids. This finding supports Naumov and Tolstorukov's suggestion (1973) that the hm a allele provides for the same functions as the HMalpha allele, namely, a switch at the mating-type locus from alpha to a. The HO allele is dominant to ho but hma and HMa alleles are codominant. A loose linkage between the mating-type and the HMalpha loci ( approximately 55cM), confirming Harashima, Nogi and Oshima (1974) data, was observed.     

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 MATalpha and MATa strains. At 37 degrees 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 MATalpha and MATa loci. At 24 degrees 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.     

MAR1-a Regulator of the HMa and HMalpha Loci in SACCHAROMYCES CEREVISIAE

A mutation in the MAR1 (mating-type regulator) locus causing sterility in Saccharomyces cerevisiae is reported. The mutation maps on the left arm of linkage group IV between trp1 and cdc2 at a distance of about 27 cM from trp1 and about 31 cM from cdc2. Haploid strains with genotype MATalpha HMalpha HMa mar1-1 and MATa HMalpha HMamar1-1 are sterile. However, MATalpha hmalpha HMa mar1-1 and MATa HMalpha hma mar1-1 strains exhibit alpha and a mating type, respectively. The sterile strains can be "rare mated" with standard strains as a consequence of mutational changes at HMa and HMalpha. It is proposed that the MAR1 locus blocks the expression of MATalpha and MATa information thought to exist at HMa and HMalpha loci, respectively (Hicks, Strathern and Herskowitz, 1977). In a mar1-1 mutant, the expression of both HMalpha and HMa information leads to a nonmating phenotype similar to that of MATa/MATalpha diploids. The genetic evidence reported here is consistent with a central feature of the "cassette model", namely that HMalpha and hma carry MATa information and HMa and hmalpha carry MATalpha information.     

``alternative Self-Diploidization'''' or ``asd'''' Homothallism in Saccharomyces Cerevisiae: Isolation of a Mutant, Nuclear-Cytoplasmic Interaction and Endomitotic Diploidization

A mutant of Saccharomyces cerevisiae representing a novel life cycle, named "alternative self-diploidization" or "ASD" homothallism, was obtained fortuitously. In this life cycle, MAT alpha (or MATa) haplophase and MAT alpha/MAT alpha (or MATa/MATa) diplophase alternate. Germinated cells are haploid and mating. They soon become nonmating and sporogenous as they vegetatively grow. They sooner or later diploidize presumably via endomitosis. The diploid cells haploidize via normal meiosis. A single recessive nuclear mutation, named asd 1-1, is responsible for "ASD" homothallism. In the rho 0 cytoplasm, asd 1-1 cells mate even if at a low efficiency and fail to diploidize. Since pet mutations do not have such effects, we conclude that a certain mitochondrial function other than respiration is required for manifestation of "ASD" homothallism. That is, "ASD" homothallism is the result of some sort of nuclear-cytoplasmic interaction.     

Many globally isolated AD hybrid strains of Cryptococcus neoformans originated in Africa

Interspecific and intervarietal hybridization may contribute to the biological diversity of fungal populations. Cryptococcus neoformans is a pathogenic yeast and the most common fungal cause of meningitis in patients with AIDS. Most patients are infected with either of the two varieties of C. neoformans, designated as serotype A (C. neoformans var. grubii) or serotype D (C. neoformans var. neoformans). In addition, serotype AD strains, which are hybrids of these two varieties, are commonly isolated from clinical and environmental samples. While most isolates of serotype A and serotype D are haploid, AD strains are diploid or aneuploid, and contain two sets of chromosomes and two mating type alleles, MATa and MATalpha, one from each of the serotypes. The global population of serotype A is dominated by isolates with the MATalpha mating type (Aalpha); however, about half of the globally analyzed AD strains possess the extremely rare serotype A MATa allele (Aa). We previously described an unusual population of serotype A in Botswana, in which 25% of the strains contain the rare MATa allele. Here we utilized two methods, phylogenetic analysis of three genes and genotyping by scoring amplified fragment length polymorphisms, and discovered that AD hybrid strains possessing the rare serotype A MATa allele (genotype AaDalpha) cluster with isolates of serotype A from Botswana, whereas AD hybrids that possess the MATalpha serotype A allele (AalphaDa and AalphaDalpha) cluster with cosmopolitan isolates of serotype A. We also determined that AD hybrid strains are more resistant to UV irradiation than haploid serotype A strains from Botswana. These findings support two hypotheses: (i) AaDalpha strains originated in sub-Saharan Africa from a cross between strains of serotypes A and D; and (ii) this fusion produced hybrid strains with increased fitness, enabling the Botswanan serotype A MATa genome, which is otherwise geographically restricted, to survive, emigrate, and propagate throughout the world.     

Haploid fruiting in Cryptococcus neoformans is not mating type alpha-specific

Under appropriate conditions, haploid Cryptococcus neoformans cells can undergo a morphological switch from a budding yeast form to develop hyphae and viable basidiospores, which resemble those produced by mating. This process, known as haploid fruiting, was previously thought to occur only in MATalpha strains. We identified two new strains of C. neoformans var. neoformans serotype D that are MATa type and are able to haploid fruit. Further, a MATa reference strain, B-3502, also produced hyphae and fruited after prolonged incubation on filament agar. Over-expression of STE12a dramatically enhanced the ability of all MATa strains tested to filament. Segregation analysis of haploid fruiting ability confirmed that haploid fruiting is not MATalpha-specific. Our results indicate that MATa cells are intrinsically able to haploid fruit and previous observations that they do not were probably biased by the examination of a small number of genetically related isolates that have been maintained in the laboratory for many years.     

Recessive Mutations from Natural Populations of NEUROSPORA CRASSA That Are Expressed in the Sexual Diplophase

Wild-collected isolates of Neurospora crassa Shear and Dodge were systematically examined for recessive mutations affecting the sexual phase of the life cycle, which is essentially diploid. Seventy-four of 99 wild-collected isolates from 26 populations in the United States, India and Pakistan carried one or more recessive mutations that reduced fertility significantly when homozygous; mutations affecting spore morphology were also detected. Limited complementation tests indicate that most of the 106 recovered mutations are unique.--The recessive diplophase (= sexual phase) mutations were uncovered by crossing each wild-collected isolate to a marked two-chromosome double-reciprocal translocation strain as "balancer." Surviving progeny receive approximately 60% of their genome from the wild parent, but receive the mating-type allele from the "balancer" parent. These progeny were backcrossed to the wild parent and were also crossed with a standard laboratory strain (fl). Reduced fertility in the backcross vs. normal fertility in the cross with the laboratory standard signals the presence of a recessive mutation in the wild-collected isolate.--Most of the mutants (95 of 106) fall into two major classes: those producing barren perithecia with no or few viable ascospores (51) and those with spore maturation defects (44). Most of the recessive barrens result either from an early block in meiosis of ascus development (25) or from a late disturbance in postmeiotic ascus behavior (18).--These recessive mutations are formally equivalent to recessive lethals in higher eukaryotes and may be important in determining the breeding structure of natural Neurospora populations.