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.     

Physical mapping of the Schizosaccharomyces pombe histone genes

The histone-encoding genes in Schizosaccharomyces pombe were physically mapped by hybridisation to filters containing cosmid and P1 genomic libraries. The H2A.2 gene and the H2A.1-H2B.1 gene pair mapped between the ade6 and rikl genes on chromosome III. The three H4–H3 gene pairs were mapped to three different regions by a H4.1 probe. Southern analysis of clones from each region revealed the positions of the three H4–H3 gene paris. H4.1–H3.1 was localised to chromosome I between the mei2 and rad1 genes; H4.2–H3.2 mapped between rad3 and cdc2 on chromosome II; H4.3–H3.3 was localised to a region between the nuc1 and puc1 genes on chromosome II.     

Prospects for functional genomics in Schizosaccharomyces pombe

Schizosaccharomyces pombe is well established as an experimental organism for basic research, with well developed technologies for molecular biology, genetics and cell biology. Its full genome sequence has recently been published. Here, the prerequisites for functional genomics studies in Sch. pombe are examined and compared with those of some established prominent functional genomics model organisms, especially Saccharomyces cerevisiae. It is argued that functional genomics studies in certain areas of cellular and molecular biology could potentially be more efficiently performed in Sch. pombe than in most other experimental organisms.     

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.     

Cloning the Schizosaccharomyces pombe lys2 + gene and construction of new molecular genetic tools

Molecular genetic analyses in Schizosaccharomyces pombe rely on selectable markers that are used in cloning vectors or to mark targeted gene deletions and other integrated constructs. In this study, we used genetic mapping data and genomic sequence information to predict the identity of the S. pombe lys2 ⁺ gene, which is homologous to Saccharomyces cerevisiae LYS4 ⁺. We confirmed this prediction, showing that the cloned SPAC343.16 gene can complement a lys2-97 mutant allele, and constructed the lys2 ⁺-based cloning vector pRH3. In addition, we deleted the S. pombe his7 ⁺ gene with a lys2 ⁺-marked polymerase chain reaction (PCR) product and the S. pombe lys2 ⁺ gene with a his7 ⁺-marked PCR product. Strains carrying these deletions of lys2 ⁺ or his7 ⁺ serve as relatively efficient hosts for the deletion of the ade6 ⁺ gene by lys2 ⁺- or his7 ⁺-marked PCR products when compared with hosts carrying lys2 or his7 point mutations. Therefore, these studies provide plasmids and strains allowing the use of lys2 ⁺ as a selectable marker, along with improved strains for the use of his7 ⁺ to mark gene deletions.     

Construction of an h +Sstrain of Schizosaccharomyces pombe

Summary By spontaneous in vivo integration of a mat2:1° plasmid, containing a Plus (P) cassette, into an h ^-LMT region of Schizosaccharomyces pombe an h ⁺ strain was obtained which neither mutates to h ^- nor to h ⁹⁰. Southern blotting showed that it possesses the same mating-type (MT) configuration as h ^-S except that P information resides in both cassettes. Therefore the strain was called h ^+S. By crossing h ^+S with the h ^- strain LK42 of Engelke et al. (1987) it was possible to obtain h ^- recombinants with the MT configuration mat1:1(M)smt-o-L-mat2:3(P). Because of the totally defective smt signal (smt-o) in these recombinants no MT switching occurs, so that M information is conserved in mat1:1; furthermore the cassette mat2:3(P) is not expressed like in strains with a K region. This proves that the K region does not cause the silencing of mat2:3(P).     

Molecular characterization of h − mutants of Schizosaccharomyces pombe

Summary Meade and Gutz (1976) have described mat2:2 mutants of Schizosaccharomyces pombe having various defects in the Plus (P) function; four different classes were distinguished. Of special interest are the class II mutants in which none of the P mating-type functions is expressed. We made Southern analyses of 23 class II strains. In most of these, the P cassette and the K region are deleted as in h ^–s strains, however, some distinct differences were found as to the intensity of the bands in the blots (classes Ila, Ilb 1, and IIb2). The class Ilb mutants have strong bands characteristic of lethal deletions (h ^–L) and mat2:1 ⁰ plasmids. Two class II mutants turned out to have a typical h⁹⁰ mating-type region with an intact P cassette, but they seem to have a completely defective switching signal at matl:1 (new class V). Mutants of classes I, III, and IV yielded band patterns identical to those of an h ⁹⁰ strain; they obviously have point mutations in the P cassette.     

Genetic nomenclature and gene list of the fission yeast Schizosaccharomyces pombe

The nomenclature rules for the genetics of the fission yeast Schizosaccharomyces pombe have been fixed for the first time, after discussion among scientists working with this organism. Conventions are proposed for the naming of genes and alleles that are obtained by classical means or by reverse genetics. In addition a list has been compiled of 460 known genes of S. pombe. It includes genes defined both by classical mutation analysis and by molecular cloning. 270 genes have been assigned either to one of the three nuclear chromosomes or the mitochondrial genome.     

Isolation and characterization of the aureobasidin A-resistant gene, aur1R, on Schizosaccharomyces pombe: roles of Aur1p+ in cell morphogenesis

To study the mechanism of action of the antibiotic aureobasidin A (AbA) on yeasts, we isolated a dominant mutant of Schizosaccharomyces pombe which gave high resistance to AbA. From a genomic library of the mutant, an aur1 ^R mutant gene conferring AbA resistance was isolated. One amino-acid mutation, a substitution of glycine with cysteine at residue 240, was responsible for the acquisition of AbA resistance. The wild-type aur1 ⁺ gene was essential for viability, and its over-expression enhanced significant resistance to AbA. The predicted protein of S. pombe aur1 ^R was highly homologous in primary structure and hydropathy profile with that of Saccharomyces cerevisiae AUR1 ^R isolated as an AbA-resistance gene. To analyze a role in cell growth of S. pombe aur1 ⁺, temperature-sensitive mutants (aur1 ^ts ) were obtained by random mutagenesis procedures using a modified PCR. The aur1 ^ts mutation caused a defect in cell elongation at the non-permissive temperature and finally led to cell death. These results suggest that Aur1p was a target of the antibiotic AbA and was required in the cell elongation of cell-end tips and in the viability of S. pombe. Received: 10 August / 8 September 1997     

Cloning and manipulation of the Schizosaccharomyces pombe his7 +gene as a new selectable marker for molecular genetic studies

We have cloned the his7 ⁺ gene of the fission yeast Schizosaccharomyces pombe by complementation of the recessive mutant allele his7-366. The his7 ⁺ gene is able to complement a mutation of the Escherichia coli hisI gene, suggesting that his7 ⁺ encodes a phosphoribosyl-AMP cyclohydrase. Subcloning experiments localize the gene to a 1.9-kb XbaI-BglII fragment. We describe the construction of plasmids to facilitate the use of his7 ⁺ as a selectable marker in S. pombe studies. Plasmid pEA2 carries his7 ⁺ cloned into the pUC18 polylinker. From either pEA2 or the original his7 ⁺ clone, pMN1, fragments carrying his7 ⁺ can be isolated using a variety of restriction enzymes for the construction of gene disruptions. Plasmid pEA500 is a cloning vector that carries his7 ⁺ and ars1, yet retains the ability to use the blue/white color screen to identify recombinants.     

Molecular cloning of a ribosomal protein gene from the fission yeast Schizosaccharomyces pombe

Summary Using the structural gene for the ribosomal protein L3 from Saccharomyces cerevisiae as a probe, we isolated a homologous fragment from genomic DNA of Schizosaccharomyces pombe. Analysis of the plasmid carrying this fragment by hybridization selection and 2D-electrophoresis revealed a 31 kDa ribosomal protein. Transformation of the vector pDB248x containing this fragment into Schizosaccharomyces pombe leads to an increased level of mRNA suggesting that we have cloned the entire and actively transcribed gene.     

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     

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 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.     

Genetical evidence of carbamoylphosphate compartmentation in Schizosaccharomyces pombe and Schizosaccharomyces japonicus

Summary Mutants defective in carbamoylphosphate synthetase have been isolated in the fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces japonicus. Their growth properties indicate a compartmentation of the carbamoylphosphate pools between arginine and pyrimidine biosyntheses, as in Neurospora crassa but unlike Saccharomyces cerevisiae. Ornithine carbamoyltransferase-minus mutants, arg3 ^– , were also isolated in both Schizosaccharomyces species. In Schiz. pombe, a very close linkage was observed between arg3 and arg11, a gene putatively coding for acetylglutamylphosphate reductase. Arg4 and arg5, the two genes encoding the carbamoylphosphate synthetase of the arginine pathway, are also closely linked but not adjacent.