Induction control of purine catabolism in Schizosaccharomyces pombe

Summary The purine catabolic enzymes uricase, allantoinase, allantoicase and ureidoglycollase are highly induced by purines in wild-type Schizosaccharomyces pombe cells. In contrast, urease activity is constitutive. Attempts have been made to identify the inducer or inducers involved. There appears to be a sequential form of induction, uricase being induced by uric acid, allantoinase by allantoic acid (and possibly allantoin), allantoicase by allantoic acid, ureidoglycollase by ureidoglycollic acid. This sequential control of purine breakdown is compared with that found in other fungi.     

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.     

Gene database for the fission yeast Schizosaccharomyces pombe

Summary As an aid to the fission yeast genome project, we describe a database for Schizosaccharomyces pombe consisting of both genetic and physical information. As presented, it is therefore both an updated gene list of all the nuclear genes of the fission yeast, and provides an estimate of the physical distance between two mapped genes. Additionally, a field indicates whether the sequence of the gene is available. Currently, sequence information is available for 135 of the 501 known 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.     

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.     

Putative frameshift suppressors in Schizosaccharomyces pombe

Summary Nine genetically distinct suppressors of ICR-170-induced ade6 and ade7 mutations have been identified in Schizosaccharomyces pombe. The nine suppressors of ICR-170-induced and spontaneous origin have been assigned to the three chromosomes by haploidization and meiotic analysis. They do not suppress missense or nonsense mutations and are therefore likely to be frameshift suppressors. Based on the spectrum of suppression, the nine suppressors fall into two mutually exclusive groups. Group I comprises the two dominant suppressors sufl and suf11. Group II consists of the seven dominant suppressors suf2 through suf8. The suppressors of both groups are inefficient and all lead to a marked reduction of growth rate. Within suppressor groups, combinations of suppressors lead to drastic reductions of growth rates and to an increased efficiency of suppression. Freely segregating modifiers of suppression increasing and decreasing the efficiency of supression have been found for all the suppressors. The two omnipotent suppressors sup1 and sup2 increase the efficiency of suppression of some frameshift suppressors. The suf5 locus is unstable and reverts at very high frequency both meiotically and mitotically.     

Use of the Tn903 neomycin-resistance gene for promoter analysis in the fission yeast Schizosaccharomyces pombe

Summary The bacterial neo gene from transposon Tn903 (Tn601) was used for dominant transformation of the fission yeast Schizosaccharomyces pombe. It was found that high transformation efficiency was dependent on a high level of promoter activity, mediated by the strong promoter of the Schizosaccharomyces pombe alcohol dehydrogenase gene (adh1), as shown by comparing the efficiency of transformation to G418-resistance, the resistance levels of transformed cells, and the in vitro aminoglycoside phosphotransferase activity. On the other hand, the heterologous promoter of the Saccharomyces cerevisiae alcohol dehydrogenase I gene (adc1) is shown to be a weak promoter in Schizosaccharomyces pombe, though its activity is significantly enhanced in cells grown on glycerol as a carbon source. This system for selection and detection of promoter-active sequences may provide a useful basis for the analysis of promoter elements in fission yeast.     

Biochemical and genetical studies of NADP-specific glutamate dehydrogenase in the fission yeast Schizosaccharomyces pombe

The initial velocity, pH and temperature optima, and Km values of Schizosaccharomyces pombe NADP-glutamate dehydrogenase (NADP-GDH: EC 1.4.1.4) have been determined. NADP-GDH was found to be specific for the substrates used in the reaction mixtures. NADP-GDH activity showed a sigmoidal response to changes in -ketoglutarate concentrations, following Hill kinetics with a coefficient n_H=2. A two-fold and a three-fold increase in activity was found in extracts of cells grown on a medium containing cytosine or histidine as a sole nitrogen source, respectively, relative to the activity found in cells grown on other sole nitrogen sources including ammonium, adenine, arginine, aspartate, asparagine, glutamate, glutamine, leucine, lysine, proline, uridine and urea. Five NADP-GDH-defective mutants were isolated on the basis of no growth on ammonium plus allantoin as sole nitrogen sources. The mutants also failed to grow on allantion alone but, in contrast, they were phenotypically indistinguishable from the wild-type growing on solid minimal medium with ammonium. Additionally, the mutants were found to grow as wild-type on minimal medium with alanine, arginine, asparagine, aspartate, glutamate, glutamine, leucine, ornithine and proline in the absence or presence of allantoin. In liquid minimal medium with ammonium as sole nitrogen source they had a slower growth than the wild-type. Normal growth was observed in cells grown on alanine, arginine, asparagine, aspartate, glutamate, glutamine, leucine, ornithine and proline. The mutants had undetectable levels of NADP-GDH activity, but retained wild-type levels of NAD-GDH, glutame synthase (GOGAT) and glutamine synthetase (GS). Mutants were found to map in two unlinked genetic loci by recombinational analysis. One locus was designated as gdh1 and was represented by four mutant alleles (gdh1-1, gdh1-2, gdh1-3 and gdh1-4), whilst the second locus, gdh2, had one mutant allele (gdh2-1). No differences were observed in growth tests or enzymic studies between these loci.     

Concerning the map of chromosome I of Schizosaccharomyces pombe

Summary The correct orientation of a large segment (at least 200 cM) on the left arm of chromosome I of S. pombe is inverted relative to the one given in a recent mapping paper.     

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.     

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.     

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).     

The mitochondrial genome of Schizosaccharomyces pombe

Summary The open reading frame of the first intron of the mitochondrial cox1 gene (cox1I1) was expressed in Escherichia coli. The putative intron-encoded protein stimulated the formation of intra-chromosomal lac ⁺-recombinants about threefold. No stimulation was found when the reading frame was inserted in the opposite direction, or when it was interrupted by a deletion. The intronic open reading frame did not complement recA ^– or recB ^– mutants of E. coli. In S. pombe, elimination of this intron did not abolish homologous recombination in mitochondria. A possible role of the recombinase activity in yeast mitochondria will be discussed.     

Two genes control three alkaline phosphatases in Schizosaccharomyces pombe

Summary Mutants of Schizosaccharomyces pombe defective in alkaline phosphatase activity have been selected and analyzed. The mutations map in two different loci, pho2 and pho3, both located on the right arm of chromosome II. Pho2-mutants lack the activity of a soluble alkaline phosphatase specific for nitrophenyl-phosphate. Its activity requires Mg ions and it is abolished with Zn ions. The pho3-mutations block the activities of a soluble and of a membrane-bound nonspecific alkaline phosphatase. Both pho3-controlled enzyme forms exhibit similar substrate specificities, pH-optima and Mg ions are essential for both activities. Zn ions can partially replace the Mg ions. The three enzyme forms migrate differently on nondenaturing polyacrylamide gels. We speculate that pho2 and pho3 represent the structural genes for a substrate-specific and two forms of a nonspecific alkaline phosphatase.     

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.     

The primary structure of the leul + gene of Schizosaccharomyces pombe

Summary A DNA fragment which carries the leul gene encoding beta-isopropylmalate dehydrogenase in Schizosaccharomyces pombe has been isolated by complementation of an E. coli leuB mutation. This 1.5 kb DNA fragment complements not only the S. pombe leul mutation, but also the S. cerevisiae leu2 mutation. The nucleotide sequence of the essential part of the leul gene and its flanking regions was determined. This sequence contains an open reading frame of 371 codons, from which a protein having a Mr = 39,732 can be predicted. The deduced amino acid sequence and its codon usage were compared with those of the S. cerevisiae LEU2 protein. The cloned DNA will be a useful marker when transforming S pombe.     

Acid phosphatase deficient mutants of Schizosaccharomyces pombe are defective in tyrosine uptake

Summary The uptake of tyrosine and arginine into wild type and acid phosphatase deficient mutants (pho 1) of Schizosaccharomyces pombe was investigated. All 11 pho 1-alleles tested exhibited a reduced tyrosine uptake and impaired uptake cosegregated with the lack of acid phosphatase activity. Kinetic analyses using wild type cells grown in high phosphate medium (acid phosphatase repressed) and low phosphate medium (acid phosphatase derepressed) showed staturation kinetics for tyrosine with a K_M of about 2 × 10^–4 M for both media and a V of about 5 nmol min^–1mg^–1 and 2 nmol min^–1mg^–1 for derepressed and repressed cells respectively. The pho 1–118 strain completely lacked this saturable uptake system for tyrosine. Preliminary evidence suggests that tyrosine uptake may be via a general amino acid permease system and we conclude that mutations in the structural gene of acid phosphatase which abolish enzyme activity lead to a loss of this uptake system. In contrast to tyrosine, arginine uptake seems not to be significantly affected either by different acid phosphatase levels in wild type cells or by the pho 1–118 mutation.