Two tightly linked silent cassettes in the mating-type region of Schizosaccharomyces pombe

Summary Genetic evidence is presented for the presence of two silent cassettes mat2-P and mat3- M, which both map to the right of the expressible site mat1 of the mating-type region in Schizosaccharomyces pombe. During a switch of mating type, the resident cassette at mat1 is replaced by a copy of opposite mating-type information from one of the silent loci. Usually the switch becomes effective in one of two daughter cells, thus allowing for efficient sister-cell conjugation. In swi mutants, mating-type switching can be observed as early as for the first division after spore germination, albeit at a lower frequency. Genetically the two silent cassettes are linked so tightly that no crossovers were observed between mat2 and mat3 at a resolution of 10^–3 cM.     

Transient inhibition of histone deacetylase activity overcomes silencing in the mating-type region in fission yeast

We have investigated the effects of inhibition of histone de-acetylase activity on silencing at the silent mating-type loci in fission yeast. Treatment of exponentially growing cells with the histone deacetylase inhibitor, trichostatin A (TSA), resulted in derepression of a marker gene inserted 150 bp distal from the silent mat3-M locus. The natural targets for the silencing mechanism in this region were only partially derepressed and the activation appeared to be asymmetric, i.e. the mat2-P cassette remained silent at concentrations that clearly partially derepressed the mat3-M cassette. We further noted that treatment of wild-type h ⁹⁰ cells resulted in the generation of altered sporulation phenotypes, indicating that the treatment affected the expression of mating-type genes and/or mating-type switching. The results are discussed in the light of recent accumulated data regarding the role of deacetylation for silencing in other species. Received: 8 September 1998 / 5 January 1999     

Trans-acting factors and properly positioned DNA elements repress mating-type genes in fission yeast

Summary Repression of the mating-type P genes at the silent mat2-P locus in fission yeast is dependent on four cis-acting DNA elements, two on each side of the coding sequences. The mechanism by which these elements exert their influence on the mating-type promoter is studied here by insertion of a bacterial antibiotic resistance gene at several positions in the silent region. The behavior of the resistance gene itself, and the changes its insertion causes in mating-type expression, reveal that the repressive elements have a limited range of action and that the four elements have unequal effects on gene expression. Repression of the antibiotic resistance gene inside the silent region leads to an antibiotic-sensitive phenotype and facilitates the selection of resistant mutants. These mutants can de-repress the resistance gene at other positions than the one used for their selection. Strong antibiotic resistance correlates with derepression of the plasmid-borne mating-type cassette. These data argue that mat2-P repression is dependent on trans-acting factors and the positioning of the repressive DNA elements, but less dependent on the nature of the affected promoter.     

Mating configurations in Schizosaccharomyces pombe strains of different geographical origins

In genetic research with Schizosaccharomyces pombe the strains used are almost exclusively descendants of the clones originally isolated by Leupold. In the standard homothallic (h ⁹⁰) strain three closely linked mating-type (MT) genes are present in the MT region: the actual MT locus, mat1, and two silent cassettes, mat2 and mat3, respectively. Various rearrangements are known in the MT region, e.g., heterothallic h ⁺ or h ^- strains arise by duplications or deletions. In the present paper we analysed the mating behavior and the configurations of the MT regions of 19 S. pombe isolates from different parts of the world. In comparison with the Leupold strains several new MT configurations were found.     

Construction of an h +Sstrain of Schizosaccharomyces pombe

Summary By spontaneous in vivo integration of a mat2:1° plasmid, containing a Plus (P) cassette, into an h ^-LMT region of Schizosaccharomyces pombe an h ⁺ strain was obtained which neither mutates to h ^- nor to h ⁹⁰. Southern blotting showed that it possesses the same mating-type (MT) configuration as h ^-S except that P information resides in both cassettes. Therefore the strain was called h ^+S. By crossing h ^+S with the h ^- strain LK42 of Engelke et al. (1987) it was possible to obtain h ^- recombinants with the MT configuration mat1:1(M)smt-o-L-mat2:3(P). Because of the totally defective smt signal (smt-o) in these recombinants no MT switching occurs, so that M information is conserved in mat1:1; furthermore the cassette mat2:3(P) is not expressed like in strains with a K region. This proves that the K region does not cause the silencing of mat2:3(P).     

The smt-0 mutation which abolishes mating-type switching in fission yeast is a deletion

Mating-type switching in the fission yeast, S. pombe, is initiated by a DNA double-strand break (DSB) between the mat1 cassette and the H1 homology box. The mat1-cis-acting mutant, smt-0, abolishes mating-type switching and is shown here to be a 263-bp deletion. This deletion starts in the middle of the H1 homology box, 31 bp from the site of the DSB, and extends into the flanking region distal to mat1. The sequence of the region distal to H1 in the wild-type is also presented. In this region we observe a bias in the distribution of purine residues between the two DNA strands.     

Gene activation by copy transposition in mating-type switching of a homothallic fission yeast

Summary Mating-type switching in homothallic clones of the fission yeast, Schizosaccharomyces pombe, appears to follow the same route as previously found for mutations from homothallism to heterothallic strains. A copy of mat2-P is transposed to and inserted at mat1, where it functionally replaces the mat1-M allele, and only the mat1 segment is expressed (!) to determine the actual mating type: mat1-M(!) mat2-P = = mat1-P(!) mat2-P. This phenomenon has hitherto been concealed by the high switch-back rate from to observed in homothallic wild-type strains. It only becomes apparent in the presence of mutant switching genes, which retard the rates of mating-type interconversion and temporarily freeze one or the other state of gene activation at the mat1 segment. Mutations to lowered rates of switching are found to map both inside and outside the mating-type locus. While the internal mutations of this kind exert their effect autonomously in the cis-configuration, the unlinked mutations are recessive to their wild-type alleles.We dedicate this paper to Carsten Bresch. The authors first met in the most stimulating scientific environment C. B. had created at the Southwest Center for Advanced Studies.     

Molecular characterization of h − mutants of Schizosaccharomyces pombe

Summary Meade and Gutz (1976) have described mat2:2 mutants of Schizosaccharomyces pombe having various defects in the Plus (P) function; four different classes were distinguished. Of special interest are the class II mutants in which none of the P mating-type functions is expressed. We made Southern analyses of 23 class II strains. In most of these, the P cassette and the K region are deleted as in h ^–s strains, however, some distinct differences were found as to the intensity of the bands in the blots (classes Ila, Ilb 1, and IIb2). The class Ilb mutants have strong bands characteristic of lethal deletions (h ^–L) and mat2:1 ⁰ plasmids. Two class II mutants turned out to have a typical h⁹⁰ mating-type region with an intact P cassette, but they seem to have a completely defective switching signal at matl:1 (new class V). Mutants of classes I, III, and IV yielded band patterns identical to those of an h ⁹⁰ strain; they obviously have point mutations in the P cassette.     

Rescue of lethal mutations in the mating-type region of Schizosaccharomyces pombe by an extrachromosomal element

Summary In Schizosaccharomyces pombe spontaneous deletions occur in the mating-type (MT) region; the latter comprises closely linked mat loci and intervening L and K regions. The loss of L is lethal; such deletions (e.g., h ^+L ) can only be maintained in diploid strains. In tetrads of h ^+L /h ^-S strains we found some viable h ^+L spores yielding colonies with a weak iodine reaction. These wipI clones contain mat2:1° (M) plasmids derived from h ^-S . The L region of the plasmid can compensate the chromosomal h ^+L deletion. The episomal M cassette is also expressed. Furthermore, MT switching takes place in this cassette. The plasmids can integrate into the chromosomal MT region giving rise to h ⁹⁰ , h ^+R and some other configurations.     

Functional analysis of the ABF1-binding sites within the Ya regions of the MATa and HMRa loci of Saccharomyces cerevisiae

Cell type in the yeast Saccharomyces cerevisiae is determined by information present at the MAT locus. Cells can switch mating types when cell-type information located at a silent locus, HML or HMR, is transposed to the MAT locus. The HML and HMR loci are kept silent through the action of a number of proteins, one of which is the DNA-binding protein, ABF1. We have identified a binding site for ABF1 within the Ya region of MAT a and HMR a. In order to examine the function of this ABF1-binding site, we have constructed strains that lack the site in the MAT a or HMR a loci. Consistent with the idea that ABF1 plays a redundant role in silencing, it was found that a triple deletion of the ABF1-binding sites at HMRE, Ya and I did not permit the expresion of HMR a. We have also shown that chromosomal deletion of the binding site at MATY a had no effect on the level of cutting by the HO endonuclease nor on the amount of mating-type switching observed. Similarly, chromosomal deletion of all three ABF1-binding sites at HMR a had no effect on the directionality of mating-type switching.     

Mating-type differentiation and mate selection in the homothallic Chlamydomonas monoica

By using combinations of phenotypically-distinct — but sexually-compatible — mutant strains of C. monoica (zym-1, zym-27, and nit-2) and assaying for zygote genotypes in genetically-mixed mating populations (where gametes of the two parental homothallic strains were present at similar frequencies), we have found that matings occur preferentially between cells of the same genotype. Additional support for an hypothesis of non-random mate selection was provided by using an easily-selectable genetic marker (sup-1) to verify the frequent occurrence of matings between cells of identical genotype in populations where the selectable genotype was present at very low relative frequency (10^-2 or 10^-3) in a mixed mating population. Direct evidence for non-random mate selection was obtained by presenting nitrogen-starved cells with limiting nitrate to synchronize gametic differentiation in wild-type strains. Under these conditions, the four, eight, or 16 mitotic daughters released from the same mother sporangium often immediately established mating pairs within the group. Thus successive mitotic divisions of a single mother cell yielded progeny of opposite expressed mating-type.     

Switching genes in Schizosaccharomyces pombe

Summary In homothallic (h ⁹⁰) Schizosaccharomyces pombe strains mutants occur which exhibit reduced frequencies of mating-type switching. The colonies of such mutants show a mottled iodine reaction. The underlying mutations map either in a switching signal at matl or in switching (swi) genes which are not linked to the mating-type region. Forty-nine swi mutants were examined. They map in ten different swi genes, swi1 to swi10. Seven swi genes were assigned to chromosomes I and II, respectively. Two classes of swi genes can be distinguished: when plated, class I mutants yield only mottled colonies, whereas class Il mutants yield mottled and iodine-negative colonies (most of the latter are h ¹).     

Genetic and cytological characterization of the RecA-homologous proteins Rad51 and Dmc1 of Schizosaccharomyces pombe

The Schizosaccharomyces pombe rad51⁺ and dmc1⁺ genes code for homologues of the Escherichia coli recombination protein RecA. Deletion of rad51⁺ causes slow growth, retardation of cell division and a decrease in viability. rad51 cells have a defect in mating-type switching. The DNA modification at the mating-type locus required for mating-type switching contributes to slow growth in the rad51 mutant. Cell mating is reduced in crosses homozygous for rad51. Ectopic expression of the dmc1⁺ gene allowed us to demonstrate that the reduction in meiotic recombination in dmc1 mutants is not caused by a disturbance of rad24 expression from the dmc1-rad24 bicistronic RNA. We describe the functional defects of terminally epitope-tagged Dmc1 and Rad51 and discuss it in terms of protein interaction. Presumptive Rad51 and Dmc1 foci were detected on spreads of meiotic chromatin.     

Transmission patterns of chloroplast genes after polyethylene glycol-induced fusion of gametes in non-mating mutants of Chlamydomonas reinhardtii

Summary Polyethylene glycol-induced artificial fusions were made between gametes of opposite mating-type defective in sexual copulation capacity, and the inheritance patterns of chloroplast genes were analysed. Gametogenesis in non-agglutinating mutant strains was verified by a recently developed assay system (Saito et al. 1988) which distinguishes between the vegetative cell and the gamete. Although sexual crosses leading to the formation of vegetative zygotes (diploids) occurred very rarely in non-fusing mutant strain imp-1, gametes of non-agglutinating mutant strains, imp-2 and agl-1, did not fuse to form vegetative zygotes. The artificial fusions gave rise to diploid products which preferentially transmitted the chloroplast genes from the mating-type plus gamete. Non-motile cells grown on nitrogen-free agar plates — a cell type used as gametes by Matagne (1981) in artificial fusion experiments which exhibited patterns of chloroplast gene transmission very different from ours (Matsuda et al. 1983; this paper) — can be classified as vegetative, but not as gametic, as defined by the assay system. We demonstrate that even gametes, when placed on solid nitrogen-free medium, de-differentiate into vegetative cells and, after suspension in a liquid medium, differentiate once again into gametes.     

The mating type in fission yeast is switched independently of its expression

Summary The mating type of fission yeast is determined by the mat1 locus on chromosome II. The sequence content of this locus, and hence the mating type, is switched in a strictly regular pattern by transposition from one of two unexpressed mating type sequences. The expressed and the two silent sequences are located on the same chromosome. It is not understood how one of the two donor sequences is selected in this reaction. Here I test the possibility that the selection is governed by gene expression from the mat1 locus. Such a mechanism could favor transposition of a donor sequence of opposite mating type to the one present at mat1. Alternatively it could disfavor transposition of a synonymous sequence. The present data argue strongly against any type of participation of mat1 gene products in the choice of donor during the mating type switch. Alternative steering mechanisms are discussed.     

Deletion of the <Emphasis Type="Italic">MAT-</Emphasis>2 mating-type gene during uni-directional mating-type switching in <Emphasis Type="Italic">Ceratocystis</Emphasis>

 Ceratocystis eucalypti is strictly heterothallic, with single ascospore strains representing one of two opposite mating types. Most other Ceratocystis species, including C. virescens, C. pinicola, and C. fimbriata, are homothallic. In the homothallic species, the MAT-2 strains are self-fertile, while MAT-1 strains are self-sterile and grow more slowly than MAT-2 strains. The current hypothesis is that self-fertility of MAT-2 strains is due to the deletion of the MAT-2 mating-type gene, resulting in the expression of the MAT-1 mating type. These mutant MAT-1 strains are able to cross with MAT-2 strains. Part of the MAT-2 mating-type gene in C. eucalypti, C. pinicola, and C. fimbriata was amplified using degenerate primers designed from the conserved MAT-2 HMG DNA-binding motif. The expected approximately 300-bp PCR products were cloned and sequenced. Specific primers were designed that amplified 210-bp fragments only in MAT-2 isolates of C. eucalypti, C. virescens, C. pinicola, and C. fimbriata. These fragments were present in self-fertile field isolates and self-fertile progeny but were absent in the self-sterile (MAT-1) progeny from selfings of C. virescens, C. pinicola, and C. fimbriata, thus supporting the hypothesis that the MAT-2 mating-type gene is deleted during uni-directional mating-type switching. A Southern-blot analysis was performed to confirm the deletion of MAT-2 gene in self-sterile progeny. The DNA sequence data for the C. eucalypti MAT-2 mating-type gene was increased to 1371-bp using TAIL-PCR and uneven PCR, representing a portion of the complete MAT-2 gene DNA sequence. Received: 5 November 1999 / 25 February 2000     

Alteration of the cytochrome c oxidase subunit 2 gene in the [exn-5] mutant of Neurospora crassa

Summary The maternally inherited [exn-5] mutant of Neurospora crassa is characterized by its slow-growth rate and deficiency of cytochrome aa ₃ relative to wildtype strains. We have determined the DNA sequence of the COXI and COXII genes of the mutant, which encode subunits 1 and 2 of cytochrome c oxidase, respectively. No changes in the DNA sequence of the COXI gene relative to the corresponding wild-type gene were found. In the region of the COXII gene we found two alterations, one a C to T transition eight base pairs upstream of the coding sequence and the second within the coding sequence for subunit 2 affecting amino acid 27 of the precursor polypeptide (amino acid 15 of the mature polypeptide). The altered codon in [exn-5] specifies an isoleucine residue rather than the wild-type threonine residue. The corresponding position in subunit 2 sequences of all other organisms examined is conserved either as a threonine or a serine residue. Thus, we consider it likely that the mutation directly affecting the coding sequence of the polypeptide is responsible for the [exn-5] phenotype. Analysis of serially passaged heterokaryons constructed between wild-type and [exn-5] shows that both mutations segregate with the [exn-5] phenotype. Examination of mitochondrial translation products in [exn-5] revealed a deficiency of subunit 2, as well as the presence of a polypeptide that corresponds to a previously described precursor of subunit 1 that accumulates in a COXI mutant of N. crassa, [mi-3]. We propose possible relationships between [exn-5], [mi-3], and the nuclear su-1 ^[mi-3] allele, which suppresses both mutations.     

A mutated swi4 gene causes duplications in the mating-type region of Schizosaccharomyces pombe

Summary Efficient mating-type (MT) switching in homothallic strains of Schizosaccharomyces pombe is significantly reduced if they have a mutation in any of the eleven known swi genes. The swi4 mutation causes heterothallic as well as homothallic segregants, both of which have duplications in the MT region. In contrast to homothallic strains, h ⁺ swi4 strains yield only a few duplications. The duplications originate in the process of MT switching, presumably by mistakes in the resolution of DNA intermediates. They always consist of one cassette and one of the intervening sequences, L and K respectively. Strains with up to seven cassettes in the MT region were found. The possible modes of their origins are discussed.     

Phylogeny and evolution of mating-type genes from <Emphasis Type="Italic">Pyrenophora teres</Emphasis>, the causal agent of barley “net blotch” disease

The main aim of this study was to test the patterns of sequence divergence and haplotype structure at the MAT locus of Pyrenophora teres, the causal agent of barley ‘net blotch’ disease. P. teres is a heterothallic ascomycete that co-occurs in two symptomatological forms, the net form (NF) and the spot form (SF). The mating-type genes MAT1-1-1 and MAT1-2-1 were sequenced from 22 NF isolates (12 MAT1-1-1 and 10 MAT1-2-1 sequences) and 17 SF isolates (10 MAT1-1-1 and seven MAT1-2-1 sequences) collected from Sardinian barley landrace populations and worldwide. On the basis of a parsimony network analysis, the two forms of P. teres are phylogenetically separated. More than 85% of the total nucleotide variation was found between formae speciales. The two forms do not share any polymorphisms. Six diagnostic nucleotide polymorphisms were found in the MAT1-1-1 intron (1) and in the MAT1-1-1 (3) and MAT1-2-1 (2) exons. Three diagnostic non-synonymous mutations were found, one in MAT1-1-1 and two in MAT1-2-1. For comparison with P. teres sequence data, the mating-type genes from Pyrenophora graminea were also isolated and sequenced. Divergence between P. graminea and P. teres is of a similar magnitude to that between NF and SF of P. teres. The MAT genes of P. graminea were closer to those of SF than to NF, with the MAT1-2-1 SF peptide not different from the MAT1-2-1 peptide of P. graminea. Overall, these data suggest long genetic isolation between the two forms of P. teres and that hybridization is rare or absent under field conditions, with each form having some particular niche specialization. This indicates that research on resistance to P. teres should consider the two forms separately, as different species.     

Heterozygosity at the b mating-type locus attenuates fusion in Ustilago maydis

Mating and pathogenesis of the corn smut fungus, Ustilago maydis, are controlled by two unlinked mating-type loci, a and b. Yeast-like haploids that differ at both loci are compatible and fuse to establish a pathogenic dikaryon. Mating is assayed in vitro by co-inoculation on culture medium containing activated charcoal; compatible combinations have a characteristic fuzzy appearance caused by the growth of aerial hyphae. In general, this test has not been useful for assaying the mating ability of strains that are already mycelial (e.g., those heterozygous at b or at both mating-type loci). Using an assay for cytoduction involving transfer of a mitochondrial marker during transient cell fusion, and engineered strains with defined genotypes, we examined the mating abilities of strains heterozygous or hemizygous at the mating-type loci. The data (which have not been available from conventional pathogenicity or plate mating tests) show that heterozygosity at b attenuates fusion in haploid and diploid strains, whereas strains heterozygous at a retain the ability to fuse with a compatible haploid partner. It appears, therefore, that subsequent fusion events are attenuated once fusion has occurred to establish the U. maydis dikaryon.