The structural gene coding for thiamin-repressible acid phosphatase in Schizosaccharomyces pombe

Summary The pho4 gene of the fission yeast Schizosaccharomyces pombe is regulated by thiamin. The nucleotide sequence of this gene is given here and it is shown that it matches the amino acid sequence of thiamin-repressible acid phosphatase, corroborating genetic evidence that pho4 represents the structural gene of this enzyme. The gene codes for a protein of 463 amino acids in length and shows regions of strong similarity with the phosphate-repressible acid phosphatase of Schizosaccharomyces pombe. The enzyme has a cleavable signal sequence 18 amino acids long and carries nine potential N-glycosylation sites.     

Molecular cloning of the ARG7 gene of Schizosaccharomyces pombe encoding argininosuccinate lyase

Summary A gene bank of Sau3A partially restricted Schizosaccharomyces pombe DNA in YEp13 was used to transform an arg4 mutant of Saccharomyces cerevisiae. One colony was recovered which contained the YEp13 plasmid bearing a large insert complementing the argininosuccinate lyase (ASL) mutation. As shown by restriction mapping and subcloning experiments, the DNA sequence required for complementation is localized on a 2 kb BamHI-BamHI fragment. The plasmid complemented several S. cerevisiae arg4 mutants of independent origin and a S. pombe arg7 mutant lacking ASL. Low but significant ASL activities were detected in crude extracts of these transformants. No complementation of the E. coli argH mutant was observed. Southern blot hybridizations showed that the insert originates from the S. pombe genome. No cross-hybridization was found between this sequence and S. cerevisiae DNA. It can be concluded that the cloned DNA fragment bears the S. pombe ARG7 gene coding for ASL.Abbreviations ASL argininosuccinate lyase - bp base pair - kb kilobase pair     

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.     

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.     

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.     

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.     

The recombinational hot spot mutation ade6-M26 of Schizosaccharomyces pombe stimulates recombination at sites in a nearby interval

Summary With the help of in vitro constructed intragenic double mutants, we investigated the influence of the recombinational hot spot mutation ade6-M26 on meiotic recombination between two additional ade6 mutations proximal to it. Recombination was stimulated four-fold when M26 was present in a heterozygous condition and ten-fold when homozygous. M26 itself remained unaffected in a substantial number of these events. This indicates that the stimulation can not only be due to a preferred conversion of M26 to wild-type with co-conversion of the second mutation in cis. A model is proposed in which M26 acts as an entry site for recombinational enzymes.     

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.     

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.     

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.     

pH sensitivity of Schizosaccharomyces pombe: effect on the cellular phenotype associated with lacZ gene expression

We report on a series of experiments inSchizosaccharomyces pombe to detect the blue-colour colony phenotype associated with expression of theEscherichia coli lacZ gene. Increasing the pH in solid minimal medium to optimize blue colony colour revealed a pH-sensitive phenotype in auxotrophic strains requiring uracil and leucine as external supplements. This phenotype was observed among commonS. pombe stock strains, 5-fluoroorotic acid (5-FOA)-selected strains, and random genetic segregants. Growth of prototrophicS. pombe strains 972 and 975 or the adenine auxotrophic strain NCYC 1860 were unaffected by an increase in external pH. Analysis of genetic segregants from three independent crosses indicated that a single auxotrophic marker (ura4 ^- orleu1-32) was sufficient for yeast cell-growth inhibition when the medium pH was increased above 6.6. In contrast, growth of aSaccharomyces cerevisiae strain isogenic to AH22, requiring uracil, leucine and histidine, was unaffected by changes in the pH of the medium. These observations suggest that uptake of uracil and leucine intoS. pombe cells is compromised by alterations in external pH. Our results have implications for detection of thelacZ gene-encoded bluecolour colony phenotype inS. pombe, which is optimized by growth in the presence of 5-bromo-4-chloro-3-indolyl--D-galactoside (Xgal) at pH 7.0. We discuss the conditions under which this blue-colour phenotype can be routinely observed inS. pombe.     

A mating deficient and temperature-sensitive lethal mutant of Schizosaccharomyces pombe defines a new fertility locus

Summary A recessive mutant allele, mef1-84, of a novel locus mapping on the left arm of chromosome I, between ade3 and ura1, 5 cM apart from lys5, confers temperature-sensitive growth and mating deficiency at the nonrestrictive temperatures for growth. Two other mutations suppress the phenotype conferred by mef1-84: sts1-1 suppresses the temperature-sensitive growth only, and smd1-35 suppresses both temperature-sensitive growth and mating deficiency.     

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.     

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).     

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.     

The Saccharomyces cerevisiae RAD2 gene complements a Schizosaccharomyces pombe repair mutation

Summary Two Saccharomyces cerevisiae genes necessary for excision repair of UV damage in DNA, RAD1 and RAD2, were introduced individually, on a yeast shuttle vector, into seven Schizosaccharomyces pombe mutants — rads1, 2, 5, 13, 15,16 and 17. The presence of the cloned RAD1 gene did not affect survival of any of the S. pombe mutants. The RAD2 gene increased survival of S. pombe rad13 to near the wild-type level after UV irradiation and had no effect on any of the other mutants tested. S. pombe rad13 mutants are somewhat defective in removal of pyrimidine dimers so complementation by the S. cerevisiae RAD2 gene suggests that the genes may code for equivalent proteins in the two yeasts.     

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.     

Caffeine-resistance in Schizosaccharomyces pombe: a pleiotropic mutation affecting UV-sensitivity,fertility, and cell cycle

Summary A stable mutant having increased resistance to growth inhibition by caffeine was obtained from Schizosaccharomyces pombe after UV-irradiation of vegetative cells. The caf1-21 mutation confers increased UV-sensitivity, impaired fertility and sporulation, lengthened cell cycle and a slightly abnormal cell morphology, but it does not affect ectopic recombination in vegetative cells. The mutation is partially dominant and defines a gene mapping on chromosome II. Various possible mechanisms of resistance are discussed.     

Comparison of Tetrahymena ARS sequence function in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe

Summary We have isolated several Tetrahymena thermophila chromosomal DNA fragments which function as autonomously replicating sequences (ARS) in the heterologous Saccharomyces cerevisiae and Schizosaccharomyces pombe selection systems. The Tetrahymena ARS sequences were first isolated in S. cerevisiae and were derived from non-ribosomal micro- and macronuclear DNA. Sequence analysis of the ARS elements identified either perfect or close matches with the 11 by S. cerevisiae ARS core consensus sequence. Subcloning studies of two Tetrahymena ARS elements defined functional regions ranging in size from 50 to 300 bp. Testing of the ARS elements in S. pombe revealed that most of the T. thermophila inserts confer ARS function in both yeasts, at least in the sense of promoting a high transformation frequency to plasmids which contain them. However, the actual sequences responsible for ARS activity were not always identical in the two yeasts.