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.     

The in vivo effect of acriflavine on mitochondrial functions in the petite negative yeast Hansenula saturnus

Summary In the petite negative yeast Hansenula saturnus, acriflavine determined a decrease of cell yield and of the total Q_O2,the disappearance of the cytochromes aa ₃ and b and the inhibition of in vivo mitochondrial protein synthesis without affecting the cell survival. The restriction enzymes analysis of mitDNA shows that no specific fragmentation occurred after acriflavine treatment.     

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.     

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.     

Effect of ethidium bromide on transmission of mitochondrial genomes and DNA synthesis in the petite negative yeast Schizosaccharomyces pomhe

Summary Treatment of haploid strains of the petite negative yeast Schizosaccharomyces pomhe with ethidium bromide prior to mating with untreated cells reduces transmission of mitochondrial markers from the treated strains. This effect is fully reversible after 20 generations of growth in drug free medium before mating. In contrast to the petite positive yeast Saccharomyces cerevisiae, where nuclear DNA synthesis is not affected but mitochondrial DNA is degraded in the presence of 20 g/ml ethidium bromide, the same concentration decreases both nuclear and mitochondrial DNA synthesis in Schizosaccharomyces pomhe. After removal of the drug, nuclear DNA synthesis increases faster than its mitochondrial counterpart in Schizosaccharomyces pomhe.     

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.     

An abnormal cell division cycle in an AIR carboxylase-deficient mutant of the fission yeast Schizosaccharomyces pombe

Summary Adenine-requiring mutant strains of S. pombe enter the stationary phase after depleting a culture medium of adenine or its analogues. Stationary phase cells of six mutants defective at different stages of the purine nucleotide synthetic pathway were examined for cell volume and DNA content, and then compared in these respects with those of a prototrophic wild-type strain. The cell cycle of the wild-type strain was arrested in the G₂ phase (2C state) in the nitrogen rich medium, as is evident from DNA content per cell (0.0425 pg) and cell volume (47.7 m³). An AIR carboxylase-deficient (ade6) mutant strain was found to have an unusual cell volume (307.4 m³) and DNA content (0.1187 pg). By DAPI fluorescence microscopy, each mutant cell was seen to contain only one enlarged nucleus, which indicates the absence of cell populations containing cells in the 4C state of the S phase following nuclear division. It then follows that in ade6 mutant cells, DNA synthesis occurs in the absence of a completed nuclear division. Thus in S. pombe cells, the completion of nuclear division is not necessarily required for the next cycle initiation of DNA synthesis under certain physiological conditions.Abbreviation AIR aminoimidazole ribonucleotide - DAPI 4,6-diamidino-2-phenylindole - PCA perchloric acid - DABA 3,5-diaminobenzoic acid     

Sporulation and mitochondrial activity in the dimorphic yeast Endomycopsis capsularis

Summary E. capsularis transferred to sporulation medium undergoes two different differentiation processes: formation of a true mycelium and sporulation. Formation of a true mycelium, which occurs during mitotic division, can occur in the absence of 1 st respiration and in the presence of a very low level of mitochondrial translation. Sporulation occurring within both specialized and unspecialized structures of the mycelium, which involves meiosis, requires a full level of both mitochondrial protein synthesis and 1st respiration.     

The organization of repeating units in mitochondrial DNA from yeast petite mutants

Summary We have reinvestigated the linkage orientation of repeating units in mtDNAs of yeast ^– petite mutants containing an inverted duplication. All five petite mtDNAs studied contain a continuous segment of wild-type mtDNA, part of which is duplicated and present in inverted form in the repeat. We show by restriction enzyme analysis that the non-duplicated segments between the inverted duplications are present in random orientation in all five petite mtDNAs. There is no segregation of sub-types with unique orientation. We attribute this to the high rate of intramolecular recombination between the inverted duplications. The results provide additional evidence for the high rate of recombination of yeast mtDNA even in haploid ^– petite cells.We conclude that only two types of stable sequence organization exist in petite mtDNA: petites without an inverted duplication have repeats linked in straight head-to-tail arrangement (abcabc); petites with an inverted duplication have repeats in which the non-duplicated segments are present in random orientation.     

Identifying regulators of pheromone signalling in the fission yeast Schizosaccharomyces pombe

The rate and extent of a cell's response to an extracellular stimulus is influenced by regulators that act on the intracellular signalling machinery. Although not directly involved in propagating the intracellular signal, regulators control the activity of the proteins that transmit the signals. To understand this aspect of cell signalling, we studied the pheromone-response pathway in the fission yeast Schizosaccharomyces pombe, a relatively simple signalling system in a genetically tractable organism. Here, we describe the development of yeast strains containing ura4 and lacZ reporter genes under the control of the pheromone-regulated sxa2 promoter and the use of these strains to isolate mutants defective in their ability to regulate signalling. Several different types of mutant were identified. Some mutants were defective in proteins already known to regulate the pheromone-signalling pathway (Rgs1, Map1, Map2). Our approach also identified the MAP kinase phosphatase Pmp1 as a regulator of the pheromone-response pathway. Although previously shown to regulate other MAP kinase pathways in Sz. pombe, this is the first demonstration of a role for Pmp1 in pheromone signalling.     

Genetic studies of purine breakdown in the fission yeast Schizosaccharomyces pombe

Summary Purines such as hypoxanthine, xanthine, uric acid, allantoin and allantoic acid serve as sole nitrogen sources for the yeast Schizosaccharomyces pombe. A number of classes of mutants unable to use purines have been isolated and genetically analysed. Mutants in the urol gene lack uricase, all1 lack allantoinase, ala1 lack allantoicase whilst in ure1, ure2, ure3 and ure4 genes lack urease activity. Mutants in four hyp genes are unable to convert hypoxanthine to uric acid whilst mutation in xan1 results in impaired growth with xanthine. hyp5 strains are unable to convert both hypoxanthine and xanthine to uric acid. The mutations are recessive and none of the loci are linked to each other. The possible catalytic steps involved are discussed.     

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.     

Studies on HIV/AIDS using the fission yeast Schizosaccharomyces pombe

Human immunodeficiency virus (HIV) is a causative agent of acquired immunodeficiency syndrome (AIDS) and a member of Retrovirus family. The name of "retrovirus" is said to be derived from "reverse-transcribing oncogenic virus." Living up to its name, retrovirus has contributed to oncology, especially in the field of cancer pathogenesis. Retrovirus research has led to discovery of a number of oncogenes as well. Since the discovery of HIV in 1983, however, retrovirus has also been considered as an important etiologic agent that could incapacitate the cells involved in immune responses. As of the end of 2004, the number of people living with HIV/AIDS is estimated to be as large as 40 million. Every year, nearly 5 million people are newly infected with HIV, and 3 million people die of AIDS in the world, mainly in Africa and southeastern and southern Asia. Despite extensive studies, detailed mechanisms of HIV pathogenesis are still unclear, and efforts are being made to clarify functions of various HIV proteins and identify the cellular factors that could interact with the HIV proteins. One of the HIV accessory proteins, Vpr, causes the host cell cycle arrest at G2 phase, which may play an important pathogenic role in AIDS induction. Exploiting the fission yeast Schizosaccharomyces pombe useful for cell cycle studies, I've been trying to elucidate the mechanism by which Vpr induces the G2 arrest as presented in this review.     

Characterization of a novel open reading frame,urf a,in the mitochondrial genome of fission yeast: Correlation of urf a mutations with a mitochondrial mutator phenotype and a possible role of frameshifting in urf a expression

Summary Between the genes for tRNA^gln and tRNA^ile an open reading frame of 227 amino acids has been identified which is unique among known mitochondrial genomes and which has been termed urf a (Lang et al. 1983; Kornrumpf et al. 1984). It uses the mitochondrial genetic code, i.e., it contains a TGA codon, whereas all other protein-encoding genes, and all but one intronic open reading frame, use the standard genetic code (UGG for tryptophan). A previous paper has demonstrated that mutator strains show an increased formation of mitochondrial drug-resistant and respiration-deficient mutants (including deletions). In this paper we show that the mutator activity is correlated with mutations in urf a. A detailed analysis of one urf a mutant is presented (ana ^r -6), where the deletion of an A residue leads to a frameshift mutation and consequently to premature termination of the putative protein. The phenotype of colonies originating from a single mutant clone varies from no growth up to full growth on non-fermentable substrate. This phenomenon of phenotypic segregation can be explained by the ability of the cell to perform translational frameshifting. A detailed analysis of the DNA sequence and the putative urf a protein will be presented and a possible function of the protein will be discussed.Dedicated to Professor Fritz Kaudewitz on the occasion of his 70th birthday on March 11, 1991.     

Drug resistance in the fission yeast Schizosaccharomyces pombe: pleiotropic mutations affecting the oleic acid and sterol composition of cell membranes

Summary The whole cell lipid and sterol content of the drug resistant strains cyh1, cyh3 and cyh4 was compared with that of wild type by thin layer and gas liquid chromatography and by UV spectrophotometric analysis. The cyh3 and cyh4 strains had a decreased content of the unsaturated 18:1 fatty acid oleic acid, a decreased content of ergosterol and an increased content of 24,28 dehydroergosterol with respect to wild type. The cyh1 strain, however, only showed a decreased content of ergosterol and an increased content of 24,28 dehydro-ergosterol when compared to wild type.     

A controversy concerning the structure of the mitochondrial genome of a yeast petite mutant: resolution by sequence analysis

Summary Sequence analysis was used to define the repeat unit that constitutes the mitochondrial genome of a petite (rho ^–) mutant of the yeast Saccharomyces cerevisiae. This mutant has retained and amplified in tandem a 2,547 by segment encompassing the second exon of the oxi3 gene excised from wild-type mtDNA between two direct repeats of 11 nucleotides. The identity of the mtDNA segment retained in this petite has recently been questioned (van der Veen et al., 1988). The results presented here confirm the identity of this mtDNA segment to be that determined previously by restriction mapping (Carignani et al., 1983).     

An approach to identify functional homologues and suppressors of genes in fission yeast

We have developed a procedure using a bank of temperature-sensitive (ts) mutants of fission yeast to identify mutants which can be rescued by expression of a plasmid-borne gene of interest. The procedure has been used to identify new ts alleles of cdc2 and swi7/pol1, a ts mutant rescued by actin, and to identify a ts allele of cdc11 which can be rescued by combined mammalian Myc and Max expression. The procedure should also be useful as an alternative approach to identify genes in fission yeast which are functionally homologous to genes of interest from other organisms. Received: 28 February 1997