Pheromone production and response in sterile mutants of fission yeast

Summary Genetically heterothallic strains of various sterile mutants were assayed for residual production of the corresponding mating pheromone as well as responsivenes towards the opposite pheromone. No sexual activities were detected in ste11 strains (previously referred to as aff1 or steX, which we show are allelic), whilst the production of M factor was unaffected by ste1 to ste10 mutations. P factor production was still possible in class I ste mutants (ste5, ste6 and ste10), which also allow meiosis in diploid strains. With the exception of the leaky ste10-F23 mutant, no changes in cell morphology were induced by exposure to the opposite pheromone in the ste mutant strains.     

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.     

Synchronized meiosis and recombination in fission yeast: observations with pat1-114 diploid cells

Summary The mutation pat1-114 has been used to synchronize meiosis in the fission yeast Schizosaccharomyces pombe. We have investigated several aspects of such synchronized meiotic cultures. In both pat1-114 and pat1 ⁺ diploids, meiotic landmark events are initiated at the same time after meiosis induction, but synchrony is much more pronounced in the pat1-114-driven meiosis. Commitment to recombination and to meiosis have been timed at 2 h after meiotic induction. Due to a seven-fold reduction of intragenic recombination frequency in the ade6 region of pat1-114 diploids, physical analysis of recombination has not been possible. We have distinguished three factors that influence intragenic recombination frequencies: temperature, azygotic versus zygotic meiosis, and the nature of the pat1 allele. Differences and similarities in the timing of meiotic landmarks in S. cerevisiae and S. pombe are discussed.     

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.     

Characterization of a partially fertile ras1-like ste10-UGA nonsense mutant of fission yeast

Summary Mutants which carry a leaky UGA nonsense mutation in the fertility gene ste10 are characterized by a deformed cell morphology which resembles that described in the literature for sterile ras1 ^- (ste5) and ral1 to ral4 mutant cells. Although frequent conjugation attempts are observed in combinations of two ste10 mutant strains of opposite heterothallic mating type, zygotes and asci are formed only rarely and the fertility of such crosses remains low (not more than 1% of the fertility of comparable crosses of two ste ⁺ wild-type strains). The fertility is considerably increased, however, in combinations of the ste10 mutant with ste ⁺ wild-type strains (up to 10% if the h ^– partner, and more than 30% if the h ⁺ partner, carries the ste10 mutation).     

RAN1 + controls the transition from mitotic division to meiosis in fission yeast

Summary We have investigated the genetic and physiological control of meiosis in fission yeast. Nutritionally depleted h ⁺/h ^– diploid cells become irreversibly commited to meiosis immediately prior to the initiation of premeiotic S phase. Premeiotic DNA synthesis requires matP ⁺, matM ⁺, mei2 ⁺ and mei3 ⁺ but not the mitotic cell cycle control gene, cdc2 ⁺, ran1 ⁺ is an essential gene, loss of which provokes sexual conjugation, premeiotic DNA synthesis, pseudo-meiosis and the sporulation of haploid cells. Our experiments suggest that sexual differentiation is achieved physiologically by the inhibition of ran1 ⁺ activity in a two-step process. In the first step, partial inhibition of ran1 ⁺ in starved haploid cells, leads to cell cycle arrest in G₁ followed by sexual conjugation. In the second step, a pathway requiring the matP ⁺, matM ⁺ and mei3 ⁺ genes of the newly-formed zygote, further inhibits ran1 ⁺ and thereby commits the cell to meiosis. mei2 ⁺ is required for meiotic commitment after full inhibition of ran1 ⁺. ran1 ⁺ is normally essential for vegetative cell reproduction but is inessential in cells which have abnormally high levels of cAMP-dependent protein kinase. We propose that the ran1 ⁺ gene encodes a highly controlled protein kinase which shares key substrates with cAMP-dependent protein kinase.     

Spatial regulation of cytokinesis by the Kin1 and Pom1 kinases in fission yeast

Cytokinesis requires a tight spatio-temporal coordination with mitosis to ensure proper segregation of the genetic information during cell division. In fission yeast, an actomyosin contractile ring is assembled in mitosis and dictates the site of cytokinesis. Here we investigated the functions of Kin1 and Pom1, two conserved fission yeast kinases, in cell division. We found that kin1Δ is synthetically lethal with pom1Δ because double mutant cells fail to spatially organize the actomyosin ring during mitosis, leading to aberrant septum synthesis and accumulation of post-mitotic nuclei in the same cell compartment. Assembly of an Rlc1-GFP ring in the cell center at mitosis is also compromised. Similar cytokinetic defects are observed in a tea1Δ kin1Δ mutant. Furthermore, aberrant septation and nuclear accumulation are observed in a pom1Δ strain in which the Kin1 level is either down or up-regulated. Thus, a tight control of Kin1 level is critical for ensuring accurate cell division in a pom1Δ background. Since none of the kinases can substitute for each other, Kin1 and Pom1 have distinct complementary functions. We show that Kin1 is required for F-actin polarization in interphase and after completion of mitosis and this function may be essential for cytokinesis in a pom1Δ background.     

Mapping of additional markers in fission yeast,especially fus1 and three mfm genes

The following genes of the fission yeast Schizosaccharomyces pombe have been mapped by tetrad analysis — chromosome arm I-L: mfm2, rad24, rad25; I-R: abc1, fus1, mfm1; II-L: mfm3; II-R: mam1, rad13. A hotspot of meiotic recombination although not quite so active as suggested by previous maps, may be located between rad25 and aro5 on I-L.     

Reorientation of the distal region in linkage group IIR of fission yeast

The genetic map of the fission yeast Schizosaccharomyces pombe has been revised in the distal region of chromosome arm IIR. The spo4 locus, hitherto considered the outermost marker, has been moved to an intermediate position. As a result, and in accordance with recent physical mapping data, the order of the entire distal subgroup of some 12 genetic markers is reversed relative to previously published gene maps.     

Expression and analysis of the green fluorescent protein gene in the fission yeast Schizosaccharomyces pombe

This report demonstrates that the Aequorea victoria green fluorescence protein (gfp) gene product will fluoresce in the fission yeast Schizosaccharomyces pombe when expressed from an episomal expression vector. Fluorescence was readily detectable at both the colony and single cell level. Application of fluorescence-activated cell sorting (FACS) techniques showed that gfp-expressing cells could be detected when they were as rare as 1% of a total yeast population. Quantitative analysis of gfp-expressing cells constituting as little as 5% of a total population was possible. These observations establish the suitability of the gfp gene for use in S. pombe and, in combination with FACS, offers an experimental strategy for quantitative analysis of gene expression in yeast populations.     

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.     

Sterile UGA nonsense mutants of fission yeast

Summary Eight sterile mutants, which regain their fertility upon reactivation of an inactivated UGA suppressor allele of the serine tRNA gene sup3, are shown to carry UGA nonsense alleles of two established ste genes, ste1 (one mutant) and ste6 (two mutants), and of two novel genes, ste9 (four mutants) and ste10 (one leaky mutant of ras1 ^-/ste5-like cell morphology). The mutant alleles of ste1 and ste9 lead to a defect in both conjugation and meiosis, whereas those of ste6 and ste10 affect mating only. Two of the four genes map to chromosome I, ste1 in the left arm 6 cM distal of ura1, and ste9 in the right arm 3 cM distal of ade2. The ste10 and ste6 genes are located in the right arms of chromosomes II and III, respectively, the former 4 cM distal of trp1 and the latter 1 cM proximal or distal of trp3.     

Evidence for a nucleotide-dependent topoisomerase activity from yeast mitochondria

Yeast mitochondria were found to contain a novel topoisomerase-like activity which required nucleoside di- or tri-phosphates as a cofactor. ADP supported activity as effectively as ATP and the optimal concentration for each was approximately 20 M. None of the other standard ribo- or deoxyrib-onucleotides could fully substitute for either ADP or ATP. The non-hydrolyzable ATP analogs, adenosine-5-0-(3-thiotriphosphate) (ATP--S), adenylyl (, -methylene) (AMP-PCP), and andenyl-imidodiphosphate (AMP-PNP) also supported activity suggesting that the nucleotide cofactor regulated topoisomerase activity rather than serving as an energy donor in the reaction. The mitochondrial topoisomerase activity relaxed both positively and negatively supercoiled DNA. It was not inhibited by concentrations of ethidium bromide up to 2 g/ml nor by either nalidixic or oxolinic acids; novobiocin, coumermycin, and berenil inhibited the activity. Genetic and biochemical analysis of the mitochondrial topoisomerase activity indicated that it was not encoded by the nuclear TOP1, TOP2, and TOP3 genes.     

A revised chromosome map of the fission yeast Schizosaccharomyces pombe

Summary The genetic map of the nuclear genome of the fission yeast Schizosaccharomyces pombe has been extended by mitotic and meiotic mapping data. A total of 158 markers are now assigned to the three linkage groups known in this organism, and 118 of them have been located on the corresponding chromosome map. Chromosome II and III each consist of one linkage group. There is some indication that the two large fragments which define chromosome I are meiotically linked, but the linkage observed is significant at the P = 0.05 level only. The length of the map is at least 1,700 map units, corresponding to an average of about 8 kilobases per map unit. The latter figure is comparable to the one obtained for intragenic recombination in the sup3 gene (Hofer et al. 1979). The basic frequency of gene conversion as measured for 21 genes varies according to a distribution of Poisson (with a modal value of 0.6% conversion per meiosis and per gene), in sharp contrast with Saccharomyces cerevisiae (Fogel et al. 1980) and Ascobolus immersus (Nicolas 1979). This may reflect the rarity of gene or region-specific rec alleles in S. pombe and may be related to the homothallism of this organism.     

On the nature and specificity of replicating instability in fission yeast

Summary A strain of fission yeast carrying replicating instability (RI) will segregate mitotically three types of cells: unstable (still RI-carrying) cells, stable identical mutants and stable non-mutants. RI in fission yeast has previously been considered as a specific type of premutational lesion capable of (1) being replicated as such and (2) reverting at an appreciable rate to the normal state as well as changing into a stable mutation. In the present work genetic analysis of a previously studied RI-carrying strain showed this strain to be a diploid, most probably heterozygous for a recessive mutation. It was possible to construct other unstable heterozygous strains segregating predetermined mutants but not to induce RI by UV-irradiation of haploid cells. Thus evidence is presented against the premutational nature of RI in fission yeast.     

Unblocking of meiotic crossing-over between the silent mating-type cassettes of fission yeast,conditioned by the recessive,pleiotropic mutant rik1

Summary The mating-type region of Schizosaccharomyces pombe consists of three subloci: the expressed cassette at mat1, and the silent cassettes at mat2-P and mat3-M. Previous work has shown that the genetically inert spacer region of 15 kb between mat2 and mat3 is completely devoid of meiotic recombination. This crossover blockage is lifted in the recessive mutant rik1. Other properties such as mating-type switching, sporulation efficiency and spore viability are also affected in this pleiotropic mutant. Presumably the wild-type rik1 product is responsible for heterochromatinization throughout the silent domain of the mating-type region.     

Genetic mapping of eleven spo genes essential for ascospore formation in the fission yeast Schizosaccharomyces pombe

Summary Sporulation-deficient mutants of the fission yeast Schizosaccharomyces pombe were isolated from a homothallic strain mutagenized with ethyl methanesulfonate. Complementation tests defined two new genetic loci (spo19 and spo20) essential for ascospore formation, in addition to the 18 known spo loci (Bresch et al. 1968). A novel mapping procedure using random spore analysis prior to tetrad analysis allowed us to map 11 spo genes. Four genes (spo3, spo15, spo19 and spo20) were mapped on chromosome I, 6 genes (spo2, spo4, spoS, spo6, spo14 and spo18) on chromosome II and 1 gene (spo13) on chromosome III. Although there was no noticeable clustering of spo genes on the chromosomes, three pairs of linked genes (spo15-spo20, spo3-spo19 and spo2-spo18) were found.