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.     

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.     

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.     

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.     

Genetic and molecular analysis of REC114, an early meiotic recombination gene in yeast

Four new meiotic recombination genes were previously isolated by selecting for mutations that rescue the meiotic lethality of rad52 spo13 strains. One of these genes, REC114, is described here, and the data confirm that REC114 is a meiosis-specific recombination gene with no detectable function in mitosis. REC114 is located on chromosome XIII approximately 4,9 cM from CIN4. The nucleotide sequence reveals an open reading frame of 1262 bp, consensus intron splice sites close to the 3 end, and indicates that the second exon codes for only seven amino acids. In the promoter region, a URS1 consensus sequence (TGGGCGGCTA), identical to the URS1 found in the promoter of SPO16, is present 93 bp upstream of the translation start site. Northern-blot hybridization demonstrates that REC114 is transcribed only during meiosis and that it is not expressed in the absence of the IME1 gene product, even when IME2 is constitutively expressed.     

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.     

Triploid meiosis and aneuploidy in Schizosaccharomyces pombe: an unstable aneuploid disomic for chromosome III

Summary Triploid meiosis in crosses between haploid and diploid strains of the fission yeast Schizosaccharomyces-pombe was investigated by tetrad analyses. Viability of the segregants was low. Only about 10% of the total tetrads contained four colony-forniing spores and most of these segregated into two haploids and two diploids. The remaining tetrads were rather normal in germination but were defective in colony formation. More than 50% of the total tetrads had no colony-forming spores while about 30% contained 1–3 colony-forming spores. Among the colony-forming segregants from the defective tetrads, a class of aneuploids disomic for the shortest chromosome III was obtained. In such aneuploid cells combined with a cold-sensitive ß-tubulin mutation, an additional short chromosome corresponding to chromosome III was observed by DAPI staining when the cells were incubated at a restrictive temperature. The other classes of aneuploids appeared able to be to germinate but not to enter into vegetative growth. Detailed genetical analyses indicated that recombination frequencies near the centromere were strikingly different between triploid and normal diploid meiosis.     

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.     

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.     

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.     

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.     

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.     

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.     

Construction and characterization of centric circular and acentric linear chromosomes in fission yeast

Summary Gene disruption and gap repair of chromosomal DNA have been frequently employed techniques in yeast genetics. To extend the possibility of using these gene manipulations for larger genomic regions, we have examined the maximal sizes of chromosomal DNA disrupted or repaired in vivo. Here we report a simple, potentially general, method for selectively deleting a 150 kb region, or gap-filling a 100 kb region, in the fission yeast genome. This enables the generation of acentric linear chromosomes by deletion, or the cloning of large functional centromeric DNAs into circular minichromosomes by gap-filling. The fidelity of the resulting gap-filling is high, judging from partial-digestion mapping of gap-repaired DNAs. By analysing a series of such circular minichromosomes, we conclude that only a part of the repetitive centromeric region, including the central domain, is essential for mitotic and meiotic chromosome segregation. Acentric linear chromosomes, although unstable, could be maintained, indicating that it may be possible to construct an acentric vector for large DNA fragments in this organism.     

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).     

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.     

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.