A CIS-Acting Mutation within the MATa Locus of SACCHAROMYCES CEREVISIAE That Prevents Efficient Homothallic Mating-Type Switching

Homothallic strains of Saccharomyces cerevisiae are able to switch from one mating-type to the other as frequently as every cell division. We have identified a cis-dominant mutation of the MATa locus, designated MATa-inc, that can be converted to MATalpha at only about 5% of the normal efficiency. In homothallic MATa-inc/mata* diploids, the MATa-inc locus switched to MATalpha in only one of 30 cases, while the mata* locus switched to MATalpha in all 30 cases. The MATa-inc mutation can be "healed" by a series of switches, first to MATalpha and then to a normal allele of MATa. These data are consistent with the "cassette" model of Hicks, Strathern and Herskowitz (1977), in which mating conversions involve the transposition of wild-type copies of a or alpha information from silent genes elsewhere in the genome. The MATa-inc mutation appears to alter a DNA sequence necessary for the replacement of MATa by MATalpha. The MATa-inc mutation has no other effect on MATa functions. In beterothallic backgrounds, the mutation has no effect on the sensitivity to alpha-factor, synthesis of a-factor, expression of barrier phenotype or ability to mate or sporulate.--The MATa-inc allele does, however, exhibit one pleiotropic effect. About 1% of homothallic MATa-inc cells become completely unable to switch mating type because of mutations at HMa, the locus proposed to carry the silent copy of alpha information.--In addition, we have isolated a less efficient allele of the HO gene.     

A new gene affecting the efficiency of mating-type interconversions in homothallic strains of Saccharomyces cerevisiae

Homothallic strains of Saccharomyes cerevisiae are able to switch efficiently from one mating genotype to another. From a single haploid spore arise both a and mating type cells, which then self-mate to produce a colony consisting almost exclusively of nonmating a/ diploid cells. We have isolated a mutant homothallic strain that gives rise to colonies that show bisexual mating behavior. The mating reaction is always asymmetric, that is, in some colonies a mating is much stronger than mating, while others show greater than a mating.-This mating phenotype arises from the presence of three cell types in a colony: some a/ nonmating diploids and an unequal number of a and haploid cells. The predominant haploid type is that of the original cell that gives rise to the colony. This mixture of cell types arises from a very reduced efficiency of homothallic mating-type interconversions in the mutant strain.-The mutation, designated switch (swi1-1), behaves as a single genetic locus. The mutation is centromere linked, but not linked to the mating type locus or to any of the homothallism genes: HO, HMa and HM. The switch mutation does not affect the efficiency of self-mating, but rather directly affects the frequency of interconversion of mating types.     

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.     

Mating type control in Saccharomyces cerevisiae: a frameshift mutation at the common DNA sequence, X, of the HML alpha locus.

A mutation defective in the homothallic switching of mating type alleles, designated hml alpha-2, has previously been characterized. The mutation occurred in a cell having the HO MATa HML alpha HMRa genotype, and the mutant culture consisted of ca. 10% a mating type cells, 90% nonmater cells of haploid cell size, and 0.1% sporogenous diploid cells. Genetic analyses revealed that nonmater haploid cells have a defect in the alpha 2 cistron at the MAT locus. This defect was probably caused by transposition of a cassette originating from the hml alpha-2 allele by the process of the homothallic mating type switch. That the MAT locus of the nonmater cells is occupied by a DNA fragment indistinguishable from the Y alpha sequence in electrophoretic mobility was demonstrated by Southern hybridization of the EcoRI-HindIII fragment encoding the MAT locus with a cloned HML alpha gene as the probe. The hml alpha-2 mutation was revealed to be a one-base-pair deletion at the ninth base pair in the X region from the X and Y boundary of the HML locus. This mutation gave rise to a shift in the open reading frame of the alpha 2 cistron. A molecular mechanism for the mating type switch associated with the occurrence of sporogenous diploid cells in the mutant culture is discussed.     

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.     

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.     

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.     

Fission yeast switches mating type by a replication-recombination coupled process

Fission yeast exhibits a homothallic life cycle, in which the mating type of the cell mitotically alternates in a highly regulated fashion. Pedigree analysis of dividing cells has shown that only one of the two sister cells switches mating type. It was shown recently that a site- and strand-specific DNA modification at the mat1 locus precedes mating-type switching. By tracking the fate of mat1 DNA throughout the cell cycle with a PCR assay, we identified a novel DNA intermediate of mating-type switching in S-phase. The time and rate of appearance and disappearance of this DNA intermediate are consistent with a model in which mating-type switching occurs through a replication-recombination coupled pathway. Such a process provides experimental evidence in support of a copy choice recombination model in Schizosaccharomyces pombe mating-type switching and is reminiscent of the sister chromatid recombination used to complete replication in the presence of certain types of DNA damage.     

Mutations Affecting Donor Preference during Mating Type Interconversion in Saccharomyces Cerevisiae

Homothallic strains of Saccharomyces cerevisiae can convert mating type from a to α or α to a as often as every generation, by replacing genetic information specifying one mating type at the expressor locus, MAT, with information specifying the opposite mating type. The cryptic mating type information that is copied and inserted at MAT is contained in either of two loci, HML or HMR. The particular locus selected as donor during mating type interconversion is regulated by the allele expressed at MAT. MATa cells usually select HML, and MATα cells usually select HMR, a process referred to as donor preference. To identify factors required for donor preference, we isolated and characterized a number of mutants that frequently selected the nonpreferred donor locus during mating type interconversion. Many of these mutants were found to harbor chromosome rearrangements or mutations at MAT or HML that interfered with the switching process. However, one mutant carried a recessive allele of CHL1, a gene previously shown to be required for efficient chromosome segregation during mitosis. Homothallic strains of yeast containing a null allele of CHL1 exhibited almost random selection of the donor locus in a MATa background but were normal in their ability to select HMR in a MATα background. Our results indicate that Chl1p participates in the process of donor selection and are consistent with a model in which Chl1p helps establish an intrinsic bias in donor preference.     

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.     

Directionality of Fission Yeast Mating-Type Interconversion Is Controlled by the Location of the Donor Loci

Cells of homothallic strains of Schizosaccharomyces pombe efficiently switch between two mating types called P and M. The phenotypic switches are due to conversion of the expressed mating-type locus (mat1) by two closely linked silent loci, mat2-P and mat3-M, that contain unexpressed information for the P and M mating types, respectively. In this process, switching-competent cells switch to the opposite mating type in 72-90% of the cell divisions. Hence, mat2-P is a preferred donor of information to mat1 in M cells, whereas mat3-M is a preferred donor in P cells. We investigated the reason for the donor preference by constructing a strain in which the genetic contents of the donor loci were swapped. We found that switching to the opposite mating type was very inefficient in that strain. This shows that the location of the silent cassettes in the chromosome, rather than their content, is the deciding factor for recognition of the donor for each cell type. We propose a model in which switching is achieved by regulating accessibility of the donor loci, perhaps by changing the chromatin structure in the mating-type region, thus promoting an intrachromosomal folding of mat2 or mat3 onto mat1 in a cell type-specific fashion. We also present evidence for the involvement of the Swi6 and Swi6-mod trans-acting factors in the donor-choice mechanism. We suggest that these factors participate in forming the proposed folded structure.     

Evidence of natural hybridization among homothallic members of the basidiomycete Armillaria mellea sensu stricto

Populations of Armillaria mellea (Basidiomycota, Agaricales) across much of its range are heterothallic; homothallic populations occur only in Africa (A. mellea ssp. africana), China (China Biological Species CBS G), and Japan (A. mellea ssp. nipponica). Monosporous isolates of heterothallic A. mellea are haploid and their mating behaviour is consistent with the requirement of two different alleles at two mating-type loci (tetrapolar mating system) to create a diploid individual. In contrast, monosporous isolates of homothallic A. mellea are putatively diploid; they bypass the haploid phase by undergoing karyogamy in the basidium (a unique type of secondary homothallism/pseudohomothallism). In order to determine the genetic origin of this homothallism, we analyzed genetic variation of 47 heterothallic isolates from China, Europe, and North America, and 14 homothallic isolates from Africa, China, and Japan. Gene trees and mutational networks were constructed for partial mitochondrial gene ATP synthase subunit 6 (ATP6) and for the following nuclear genes: actin (ACTIN), elongation factor subunit 1-alpha (EFA), glyceraldehyde 3-phosphate dehydrogenase (GPD), and the RNA polymerase subunit II (RPB2). Homothallic isolates from Africa and Japan shared a common mitochondrial ATP6 haplotype with homothallic isolates from China, and are likely introductions. Homothallic isolates from China that shared a common mitochondrial haplotype with all European isolates did not share European nuclear haplotypes, as revealed by median-joining networks, but instead clustered with haplotypes from China or were intermediate between those of China and Europe. Such mitochondrial-nuclear discordance in homothallic isolates from China is indicative of hybridization between lineages originating from China and Europe.     

Directional bias during mating type switching in Saccharomyces is independent of chromosomal architecture

Haploid Saccharomyces cells have the remarkable potential to change mating type as often as every generation, a process accomplished by an intrachromosomal gene conversion between an expressor locus MAT and one of two repositories of mating type information, HML or HMR. The particular locus selected as donor is dictated by the mating type of the cell, a bias that ensures productive mating type interconversion. Here we use green fluorescent protein tagging of the expressor and donor loci on chromosome III to show that this preference for donor locus does not result from a predetermined organization of chromosome III: HML and MAT as well as HMR and MAT remain separated in cells of both mating types. In fact, cells in which the inappropriate donor locus is artificially tethered to MAT still predominantly select the correct donor. We find, though, that initiation of switching leads to a rapid association of the correct donor locus with MAT. Thus, in mating type switching in Saccharomyces, donor preference is imposed at commitment to recombination rather than at physical contact of interacting DNA strands.     

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.     

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.     

Physical monitoring of mating type switching in Saccharomyces cerevisiae.

The kinetics of mating type switching in Saccharomyces cerevisiae can be followed at the DNA level by using a galactose-inducible HO (GAL-HO) gene to initiate the event in synchronously growing cells. From the time that HO endonuclease cleaves MAT a until the detection of MAT alpha DNA took 60 min. When unbudded G1-phase cells were induced, switched to the opposite mating type in "pairs." In the presence of the DNA synthesis inhibitor hydroxyurea, HO-induced cleavage occurred but cells failed to complete switching. In these blocked cells, the HO-cut ends of MATa remained stable for at least 3 h. Upon removal of hydroxyurea, the cells completed the switch in approximately 1 h. The same kinetics of MAT switching were also seen in asynchronous cultures and when synchronously growing cells were induced at different times of the cell cycle. Thus, the only restriction that confined normal homothallic switching to the G1 phase of the cell cycle was the expression of HO endonuclease. Further evidence that galactose-induced cells can switch in the G2 phase of the cell cycle was the observation that these cells did not always switch in pairs. This suggests that two chromatids, both cleaved with HO endonuclease, can interact independently with the donors HML alpha and HMRa.     

A novel switch-activating site (SAS1) and its cognate binding factor (SAP1) required for efficient mat1 switching in Schizosaccharomyces pombe.

The pattern of parental DNA strand inheritance at the mating type locus (mat1) determines the pattern of mat1 switching in a cell lineage by regulating the formation of the site-specific double-stranded break (DSB) required for mating type interconversion in Schizosaccharomyces pombe. To study the molecular basis of this programmable cell type change, we conducted structural and functional analyses of the DNA sequence flanking the DSB at mat1. We have identified and characterized a DNA-binding activity that interacts with a specific sequence located 140 bp from the DSB site. Deletion analysis of DNA sequences located distal to mat1 cassette revealed the presence of at least two switch-activating sites (SAS1 and SAS2), both of which are required for generating an efficient level of DSBs and consequently, for efficient switching. We found that SAS1 overlaps with the target site of the DNA-binding activity called SAP1 (for switch-activating protein). Point mutations generated in the SAS1 element that adversely affect binding of SAP1 protein in vitro were found to reduce the efficiency of switching in vivo, suggesting the requirement of SAP1 for switching. Pedigree analysis revealed that SAS1 is equally required for initial switching (one switch in four grand-daughters of a cell) and for consecutive switching (where the sister of a recently switched cell switches again), indicating that the two developmentally asymmetric cell divisions required to generate a particular pattern of switching share the same molecular control mechanism.     

Homothallic switching of Saccharomyces cerevisiae mating type genes by using a donor containing a large internal deletion.

Homothallic switching of the mating type genes of Saccharomyces cerevisiae occurs by a gene conversion event, replacing sequences at the expressed MAT locus with a DNA segment copied from one of two unexpressed loci, HML or HMR. The transposed Ya or Y alpha sequences are flanked by homologous regions that are believed to be essential for switching. We examined the transposition of a mating type gene (hmr alpha 1-delta 6) which contains a 150-base-pair deletion spanning the site where the HO endonuclease generates a double-stranded break in MAT that initiates the gene conversion event. Despite the fact that the ends of the cut MAT region no longer share homology with the donor hmr alpha 1-delta 6, switching of MATa or MAT alpha to mat alpha 1-delta 6 was efficient. However, there was a marked increase in the number of aberrant events, especially the formation of haploid-inviable fusions between MAT and the hmr alpha 1-delta 6 donor locus.     

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.