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.     

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.     

Sex appeal in fission yeast

Summary Using diploid strains of Schizosaccharomyces pombe, cytological evidence has been obtained which shows that not only h ^– cells (Fukui et al. 1986) but also h ⁺ cells secrete a diffusible mating pheromone that attracts cells of the opposite 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).     

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.     

Switching genes in Schizosaccharomyces pombe: their influence on cell viability and recombination

Summary In Schizosaccharomyces pombe, mutations in 10 swi genes are known which reduce but not abolish mating-type (MT) switching in homothallic (h ⁹⁰) strains. Three classes (Ia, Ib, and II) of swi genes are distinguished. Three swi1 alleles are nonsense mutations. In strains with mutations in two or three swi genes, a cumulative reduction of MT switching only occurs if the genes belong to different classes. The class 1a combinations swi1 swi7 and swi3 swi7 are lethal. Besides its influence on MT switching, swi5 also reduces the frequencies of meiotic intragenic recombination and gene conversion in the ade6 locus.We dedicate this paper to Professor Kaudewitz on the occasion of his retirement     

Sterile mutants of Schizosaccharomyces pombe: Analysis by somatic hybridization

Summary Sixteen sterile mutants of Schizosaccharomyces pombe, isolated from homothallic h ⁹⁰strains, were examined. It was found that they are blocked either in copulation and meiosis or in copulation alone.Protoplasts of the sterile strains were fused with protoplasts of h^+N, h^–S or h ⁹⁰strains. In these somatic crosses, eight of the sterile strains yielded hybrids which were able to form azygotic asci. Tetrad analyses revealed that seven of these sterile strains contain a single mutation; the mutations represent at least five sterility genes (ste2, ste3, ste4, ste5, ste6). One sterile strain contains two unlinked mutations which do not represent sterility genes, but genes the interaction of which results in a sterile phenotype.     

Induction of heterothallic strains and their genetic and physiological characterization in a homothallic strain of the yeast Saccharomyces exiguus

Summary We isolated heterothallic strains from a homothallic strain of S. exiguus by mutagenization with UV or ethylmethanesulfonate (EMS). A gene, not linked to the mating-type locus, was found to control homothallism in the yeast, as in S. cerevisiae. Pheromone of S. exiguus (^se pheromone) induced formation of large pear-shaped cells (shmooing) in a strains of S. exiguus, S. cerevisiae, and S. kluyveri, and sexual agglutinability of an inducible a strain of S. cerevisiae. ^se Pheromone is a peptidyl substance a little different from pheromone of S. cerevisiae. a Pheromone of S. exiguus acts only on a cells of S. exiguus. Contrary to the above results, neither sexual agglutination nor zygote formation occurred among these three Saccharomyces yeasts.     

Thiamin regulates agglutination and zygote formation in Schizosaccharomyces pombe

Summary Nutritional conditions regulate mating of the fission yeast S. pombe. To investigate how nutritional signals are monitored by the cell and translated into appropriate mating behaviour, effects of unique and specific growth factors would be desirable. We show that thiamin can inhibit sexual agglutination and zygote formation in S. pombe. A concentration of 50 nM thiamin in the culture medium is required for full growth of a thiamin auxotrophic strain. At this concentration thiamin starts to inhibit mating of wild-type cells of opposite heterothallic mating type and at a 1M concentration zygote formation is inhibited by more than 95%. Growth conditions modulate the inhibitory effect of thiamin. Thiamin acts only for a restricted period of time and seems to inhibit commitment to zygote formation rather than the cell aggregation and fusion process itself. Pyrithiamin, a thiamin antagonist, inhibits growth as well as mating.     

Moc3, a novel Zn finger type protein involved in sexual development, ascus formation, and stress response of Schizosaccharomyces pombe

The cAMP pathway in Schizosaccharomyces pombe is the major nutrient sensing pathway to initiate sexual development when opposite mating type cells exist. We identified moc1–moc4 as genes that overcome a partially sterile S. pombe strain due to an elevation of cAMP. When we compared the strength of inducing ability of sexual development in the same S. pombe strain, Moc1 had highest, Moc2 had lowest, and both Moc3 and Moc4 had intermediate effects. Moc1/Sds23 and Moc2/Ded1 are known to be a potential regulator of M-phase progression and an essential RNA helicase, respectively. While Moc4 was found to be identical with a Zn-finger protein Zfs1, Moc3 (SPAC821.07c) was a novel protein containing a Zn-finger (Zn(2)-Cys(6)) motif. Deletion mutant of the moc3 gene was constructed and its disruptant was found to be lower in mating efficiency and formed aberrant asci. In addition, unexpectedly, a moc3 disruptant was sensitive to CaCl₂ and DNA damaging agents such as MMS and UV. Those phenotypes were opposite to the phenotypes observed in a zfs1 disruptant, and quite different from the ones in a moc1 disruptant. Moc3 localized in the nucleus as observed for Zfs1. Moc3 bound with Moc4/Zfs1 weakly in the two hybrid system, but no other combination of Moc(s) bound each other in the same analysis. Thus, Moc3 is not only involved in sexual development, but also in ascus formation and DNA integrity in an independent manner with Moc1 and Moc2 in S. pombe.     

Pheromone response in fission yeast: modulation by pat1

Summary The pat1 gene, which encodes an essential protein kinase, was identified in temperature-sensitive mutants in which, at a nonpermissive temperature, copious sporulation occurred irrespective of mating type and ploidy. In addition, at a semi-permissive temperature, conjugation was uncoupled from nutritional control, although it still required both mating types. To further characterize this intermediate effect on conjugation, we studied pheromone production and response as influenced by a pat1 mutation. Using microscopic pheromone assay conducted under starvation conditions, we observed that the pat1-114 mutant allele exerted a strong sensitizing effect towards the complementary pheromone in both mating types even at the low temperature of 23°C. The production of the pheromone was affected as well, being strongest in the P cells. Presumably the pat1 protein kinase can be tuned in to various levels of activity and thereby is able to function as a universal inhibitor of sexual differentiation in S. pombe.     

Meiosis-dependent mRNA splicing of the fission yeast Schizosaccharomyces pombe mes1 + gene

The mes1 ⁺ gene of the fission yeast Schizosaccharomyces pombe is essential for the second meiotic division. We have cloned a 1.1-kb HindIII fragment containing mes1 ⁺ by complementation from an S. pombe genomic library. Sequencing of the genomic and cDNA fragments indicates the existence of one small intron of 75 nucleotides, although both the 5 (G/GTTAGT) and 3 (CAG/T) intron-exon junctions deviate from the consensus sequences proposed for S. pombe. The putative translation product of the mature mes1 ⁺ mRNA is a 11-kDa protein of 101 amino acids which has no significant homology to any previously-reported proteins. Disruption of mes1 has no effect on cell growth but causes an arrest of meiosis before the second meiotic division. Northern-blot analysis revealed that mes1 ⁺ was preferentially transcribed under conditions of nitrogen starvation. When a h ⁹⁰ homothallic strain was shifted to a nitrogen-deficient medium, a pre-mRNA accumulated and then was gradually processed to generate a mature mRNA. This splicing did not occur in either a heterothallic haploid strain or in a homothallic mei2 mutant strain which was defective in the initiation of meiosis. Expression of the first exon alone was not able to suppress the mes1 null allele. These results indicate that mes1 ⁺ is required for the completion of meiosis, that splicing is required for the function of the mes1 ⁺ gene, and that this splicing requires the function of the mei2 ⁺ product.     

Mapping of rRNA genes by integration of hybrid plasmids in Schizosaccharomyces pombe

Summary The major rRNA genes of the fission yeast Schizosaccharomyces pombe were mapped on chromosome III by plasmid integration. The integration vector YIp33 containing S. cerevisiae LEU2 gene was combined with the S. pombe rDNA. Since LEU2 complements S. pombe leu1 deficiency, it could be used as the genetic marker for integration. The 10.4 kb rDNA repeat contained ARS sequence, and therefore 2.4 kb and 0.7 kb subfragments not containing ARS were subcloned into YIp33 and transformed leu1 S. pombe cells to Leu⁺. Genetic analyses of the transformants indicated that the integrated rDNA resides in the long arm of the shortest chromosome III, tightly linked to ade5 (1.4 cM). This result is consistent with our previous finding that the DAPI-stained smallest chromosomes were associated with the nucleolus (Umesono et al. 1983).Abbreviations ARS autonomously replicating sequence - DAPI 4,6-diamidino-2-phenylindole - kb kilo base pairs - rDNA DNA segment containing ribosomal RNA genes - rRNA ribosomal RNA     

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     

Comparison of Dam tagging and chromatin immunoprecipitation as tools for the identification of the binding sites for <Emphasis Type="Italic">S. pombe</Emphasis> CENP-C

We have established the identity of the Schizosaccharomyces pombe homologue of vertebrate CENP-C and Saccharomyces cerevisiae MIF2p and have used it to compare Dam tagging and chromatin immunoprecipitation (ChiP)as tools for the mapping of protein binding sites on DNA. ChiP shows that S. pombe CENP-C binds to the central core and inner repeats of the S. pombe centromere. It binds weakly, however, to the outer repeats. The binding pattern is thus similar to that of S. pombe CENP-A. Dam-tagged S. pombe CENP-C, however, methylates the entire centromere and 5 kb of flanking DNA. This comparison suggests that Dam tagging is less precise as a tool for mapping DNA binding sites than ChiP. We have also used the Dam tagging technique to address the question of whether there is any CENP-C binding to the ribosomal DNA in S. pombe and find none.     

Pheromone-induced meiosis in P-specific mutants of fission yeast

Summary Diploid strains of Schizosaccharomyces pombe defective in P-specific pheromone production and response provided cytological evidence that the induction of meiosis as such (and not only conjugation) depends upon an initial stimulation by sexual pheromones.     

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.     

Genetic interactions in somatic inter-mating type hybrids of the zygomycete Absidia glauca

Summary Protoplasts of auxotrophic mutants of the heterothallic zygomycete Absidia glauca were efficiently fused by electrofusion. Fusion heterokaryons between the complementary mating types, which grew prototrophically, were selected. Among 72 fusion colonies which were further analysed, 11 did not only grow prototrophically but also combined both mating types as they formed zygotes within the same mycelium. These homothallic hybrids possessed two sets of rDNA repeats originating from the complementary mating types. Those fusion colonies which gave only a (+) type mating reaction, synthesized a 15 kDa (+) type-specific surface protein; fusion colonies which mated as (-) type, did not produce this surface protein. In homothallic fusion products no expression of the (+) type-specific protein could be detected. Single spore derivatives of fusion colonies in general show the phenotype of only one fusion partner, but in some cases prototrophic recombinants were obtained. This system opens up the possibility for genetic analysis of an organism with inefficient meiotic system, lacking a natural parasexual cycle.     

Nucleotide sequence of the Schizosaccharomyces japonicus var. versatilis ribosomal RNA gene cluster and its phylogenetic implications

Fission yeasts form a small but heterogeneous group of ascomycetes and it is still unclear whether they should be subdivided into three genera (Schizosaccharomyces, Octosporomyces, Hasegawaea) or remain a single genus (Schizosaccharomyces). In order to decide whether a new genus Hasegawaea should be established for the species Schizosaccharomyces japonicus and Schizosaccharomyces versatilis, we have characterized the entire rDNA cluster in Schizosaccharomyces japonicus var. versatilis and compared it with the homologous region from Schizosaccharomyces pombe and with complete rRNA gene sequences from other yeast genera. From a phage genomic library a recombinant lambda phage containing the entire rDNA repeat unit was isolated. In this paper we report the primary sequence of the 18s, 5.8s and 25s rRNA coding regions. The S. japonicus var. versatilis rRNA genes are 1823 (18s), 158 (5.8s) and 3422 (25s) nucleotides long. The two sequences of the larger rRNA genes exhibit 95.7% (18s) and 93% (25s) similarity with the homologous genes from S. pombe. The differences between the rRNA genes of S. japonicus and S. pombe, however, are much smaller than the intrageneric differences within the rDNA sequences of other yeast genera. Therefore, subdivision of fission yeasts into the genera Schizosaccharomyces and Hasegawaea does not to seem to be justified. The sequence has been deposited in the EMBL data bank under the accession number Z 32848.