Identification of thermostable glyoxalase I in the fission yeast <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

Glyoxalase I is a ubiquitous enzyme that detoxifies methylglyoxal, which is derived from glycolysis but inhibits the growth of cells from microorganisms to mammals. Here, the structural gene for glyoxalase I (glo1⁺) from the fission yeast Schizosaccharomyces pombe was identified. Disruption of glo1⁺ enhanced susceptibility to methylglyoxal, while expression of glo1⁺ in a glo1 mutant of Saccharomyces cerevisiae restored tolerance to this aldehyde. The glo1⁺ gene product was purified. The glyoxalase I of S. pombe was a monomeric enzyme with a molecular weight of 34,000 and the k_cat/K_m value for methylglyoxal was 4.3×10⁷ M^–1 min^–1. Treatment of purified enzyme with EDTA in imidazole buffer completely abolished enzyme activity, whereas the EDTA-treated enzyme was reactivated by several divalent metal ions, such as Zn²⁺, Co²⁺, Ni²⁺ and Mn²⁺. The glyoxalase I of S. pombe exhibited fairly high thermal stability, and almost 100% activity was retained after incubating the enzyme at 60°C for 4 h.     

The gld1 + gene encoding glycerol dehydrogenase is required for glycerol metabolism in Schizosaccharomyces pombe

The budding yeast Saccharomyces cerevisiae is able to utilize glycerol as the sole carbon source via two pathways (glycerol 3-phosphate pathway and dihydroxyacetone [DHA] pathway). In contrast, the fission yeast Schizosaccharomyces pombe does not grow on media containing glycerol as the sole carbon source. However, in the presence of other carbon sources such as galactose and ethanol, S. pombe could assimilate glycerol and glycerol was preferentially utilized over ethanol and galactose. No equivalent of S. cerevisiae Gcy1/glycerol dehydrogenase has been identified in S. pombe. However, we identified a gene in S. pombe, SPAC13F5.03c (gld1 ⁺), that is homologous to bacterial glycerol dehydrogenase. Deletion of gld1 caused a reduction in glycerol dehydrogenase activity and prevented glycerol assimilation. The gld1Δ cells grew on 50 mM DHA as the sole carbon source, indicating that the glycerol dehydrogenase encoded by gld1 ⁺ is essential for glycerol assimilation in S. pombe. Strains of S. pombe deleted for dak1 ⁺ and dak2 ⁺ encoding DHA kinases could not grow on glycerol and showed sensitivity to a higher concentration of DHA. The dak1Δ strain showed a more severe reduction of growth on glycerol and DHA than the dak2Δ strain because the expression of dak1 ⁺ mRNA was higher than that of dak2 ⁺. In wild-type S. pombe, expression of the gld1 ⁺, dak1 ⁺, and dak2 ⁺ genes was repressed at a high concentration of glucose and was derepressed during glucose starvation. We found that gld1 ⁺ was regulated by glucose repression and that it was derepressed in scr1Δ and tup12Δ strains.     

Mating of the fission yeast occurs independently of pmd1 + gene product,a structural homologue of budding yeast STE6 and mammalian P-glycoproteins

The pmd1 ⁺, a multidrug resistance gene of the fission yeast Schizosaccharomyces pombe, encodes a protein similar to the budding yeast Saccharomyces cerevisiae STE6 gene product and mammalian P-glycoproteins. The STE6 protein is a membrane transporter of a-factor, a mating pheromone of a-type S. cerevisiae, which is structurally related to M-factor of the fission yeast. However, heterothallic or homothallic pmd1 null mutant cells of S. pombe, which were constructed by means of gene disruption, showed no significant decrease in the mating abilities. On the other hand, the multidrug resistance conferred by the pmd1 ⁺ was overcome by the treatment with verapamil, a typical inhibitor of mammalian P-glycoproteins. These results indicate that the pmd1 ⁺ gene product is functionally similar to mammalian P-glycoproteins, rather than to the budding yeast STE6.     

Relationship among the genes, enzymes, and intermediates of the biosynthetic pathway of lysine in Saccharomyces

Summary Genetic lesions of 10 of the lysine loci in Saccharomyces have been provisionally correlated with the various enzymatic steps of the homocitric acid pathway for the biosynthesis of lysine. The characterization of mutational blocks in different mutants was made on the basis of the accumulation of appropriate intermediates, lack of enzymatic activity, and feeding experiments. Genetic lesions in mutants lys ₄, lys₇, lys₈, and lys ₁₂ correspond to the biochemical steps between homocitric acid and -amino-adipic acid. Similarly the genetic lesions in lys ₁, lys₂, lys₅, lys₉, lys₁₃, and lys ₁₄ correspond to the intermediate steps between -aminoadipic acid and lysine. Lysine-genes are not clustered, instead are mapped on different chromosomes of Saccharomyces. Some of the intermediate steps in the biosynthesis of lysine correspond to two or more unlinked genes.Supported by NSF Grant GB 28558X, Lilly Research Foundation, and Faculty Research Committee, Miami University.     

Intracellular pH inSchizosaccharomyces pombe — Comparison withSaccharomyces cerevisiae

We examined cytoplasmic pH regulation inSchizosaccharomyces pombe andSaccharomyces cerevisiae using pH-sensitive fluorescent dyes. Of several different fluorescent compounds tested, carboxy-seminaphthorhodafluor-1 (C.SNARF-1) was the most effective. Leakage of C.SNARF-1 fromS. pombe was much slower than leakage fromC. cerevisiae. Using the pH-dependent fluorescence of C.SNARF-1 we showed that at an external pH of 7, mean resting internal pH was 7.0 forS. pombe and 6.6 forS. cerevisiae. We found that internal pH inS. pombe was maintained over a much narrower range in response to changes in external pH, especially at acidic pH. The addition of external glucose caused an intracellular alkalinization in both species, although the effect was much greater inS. cerevisiae than inS. pombe. The plasma membrane H⁺-ATPase inhibitor diethylstilbestrol reduced both the rate and extent of alkalinisation, with an IC₅₀ of approximately 35 M in both species. Amiloride also inhibited internal alkalinisation with IC₅₀'s of 745 M forS. cerevisiae and 490 M forS. pombe.Abbreviations C.SNARF-1 carboxy-seminaphthorhodafluor-1 (-AM-acetoxy-methylester) - DES diethylstilbestrol - IC₅₀ apparent inhibitory constant - BCECF 2,7-bis-(carboxyethyl)-5(6)-carboxyfluorescein (-AM--pentaacetoxymethyl ester) - FDA fluorescein diacetate     

Intrachromosomal recombination between direct repeats in Penicillium chrysogenum : gene conversion and deletion events

Recombination between direct repeats has been studied in Penicillium chrysogenum using strain TD7-88 (lys⁻ pyr⁺), which contains two inactive copies of the lys2 gene separated by 4.5 kb of DNA (including the pyrG gene) in its genome. Gene conversion leading to products with the lys⁺ pyr⁺ phenotype was observed at a frequency of 1 in 3.2 × 10³ viable spores. Two types of deletion events giving rise to lys⁺ pyr⁻ and lys⁻ pyr⁻ phenotypes were obtained with different frequencies. Southern analysis revealed that gene conversion occurs mainly as a result of crossing over events that remove the BamHI frameshift mutation present in one of the repeats. In lys⁻ pyr⁻ recombinants, the deletion events do not affect the frameshift mutation in the BamHI site, while lys⁺ pyr⁻ recombinants showed repair of the BamHI frameshift mutation and the genotype of the parental non-disrupted strain was restored. In summary, deletion events in P. chrysogenum tend to favor the restoration of the phenotype and genotype characteristic of the parental non-disrupted strain. Received: 9 November 1998 / Accepted: 14 April 1999     

The N-terminal region of the <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> RecQ helicase,Rqh1p,physically interacts with Topoisomerase III and is required for Rqh1p function

The Schizosaccharomyces pombe rqh1⁺ gene encodes a member of the RecQ DNA helicase family. Members of this protein family are essential for the maintenance of genetic integrity. Thus, mutations in the genes encoding the human RecQ homologues Blm, Wrn and RecQ4 cause Bloom syndrome, Werner syndrome and Rothmund–Thomson syndrome, respectively—diseases which result from genome instability. S. pombe cells that lack a functional rqh1⁺ gene show reduced viability and display defective chromosome segregation, particularly after UV irradiation or S-phase arrest. In this study we used an rqh1⁺ deletion series to show that the N-terminal portion of Rqh1 is essential for Rqh1 function. Moreover, the conserved Helicase and RNaseD C-terminal (HRDC) domain of Rqh1 also plays a role in allowing cells to tolerate exposure to DNA damaging agents and the S-phase inhibitor hydroxyurea (HU). We also demonstrate that Topoisomerase III (Top3) binds to a site within the first 322 N-terminal amino acids of Rqh1 and that this binding correlates with Rqh1 function. Genetic analysis of rqh1^– top3 mutants reveals that, in the presence of functional or partially functional Rqh1 protein, Top3 is required to maintain genome integrity and cell viability.     

Cloning and overexpression of a maltase gene from Schizosaccharomyces pombe in Escherichia coli and characterization of the recombinant maltase

The Schizosaccharomyces pombe maltase structural gene (SPMAL1⁺) was amplified from genomic DNA of S. pombe by PCR. An open reading frame of 1740 bp, encoding a putative 579 amino-acid protein with a calculated molecular mass of 67.7 kDa was characterized in the genomic DNA insert of plasmid pQE30. The specific maltase activity in the induced transformants was 21 times higher than that in wild-type. However, the estimated molecular mass of the purified recombinant maltase was 44.3 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The optimal temperature and pH of the purified recombinant maltase were 40 °C and 6, respectively. The recombinant maltase was weakly activated by Mg²⁺, Ca²⁺, Na⁺, and Ba²⁺, but was strongly inhibited by Hg²⁺, Ag⁺ and Cu²⁺, EDTA, and PMSF. The purified maltase could actively hydrolyse ρ-nitrophenyl glucoside (PNPG), maltose, dextrin, and soluble starch. The results demonstrate that maltase from S. pombe was different from that from other yeasts, and might be usefully exploited in the future by the biotechnology industry or lead to the development of new molecular genetic tools.     

Isolation of novel pre-mRNA splicing mutants of Schizosaccharomyces pombe

 New prp (pre-mRNA processing) mutants of the fission yeast Schizosaccharomyces pombe were isolated from a bank of 700 mutants that were either temperature sensitive (ts^-) or cold sensitive (cs^-) for growth. The bank was screened by Northern blot analysis with probes complementary to S. pombe U6 small nuclear RNA (sn RNA), the gene for which has a splicesomal (mRNA-type) intron. We identified 12 prp mutants that accumulated the U6 snRNA precursor at the nonpermissive temperature. All such mutants were also found to have defects in an early step of TFIID pre-mRNA splicing at the nonpermissive temperature. Complementation analyses showed that seven of the mutants belong to six new complementation groups designated as prp8 and prp10-prp14, whereas the five other mutants were classified into the known complementation groups prp1, prp2 and prp3. Interestingly, some of the isolated prp mutants produced elongated cells at the nonpermissive temperature, which is a phenotype typical of cell division cycle (cdc) mutants. Based on these findings, we propose that some of the wild-type products from these prp ⁺ genes play important roles in the cellular processes of pre-mRNA splicing and cell cycle progression. Received: 15 April 1996/Accepted: 9 July 1996     

Control of basidiospore production by a nuclear gene in the fungus Coprinus congregatus

Summary The genetical control of basidiospore production by sporophores of the fungus Coprinus congregatus was studied. This species is characterized by a bipolar compatibility control, and homokaryons with complementary alleles A1 and A2 can be distinguished apart. We confirmed that the pale mushroom phenotype of the fungus is determined by a nuclear gene symbolized pal. This gene also controls a sporeless character and segregates independently of the mating-type locus. Dikaryons homoallelic for the pal ^– allele produce typical pale and sporeless sporophores, while heteroallelic (pal ⁺, pal ^–) and homoallelic (pal ⁺, pal ⁺) dikaryons produce normal or almost normal sporulating sporophores. In order to segregate homokaryons homoallelic for the pal gene (A1, pal ^–; A1, pal ⁺, A2, pal ^–; A2, pal ⁺), the following protocols were used: (a) the dikaryotization of stock homokaryons containing the pal ⁺ allele and of each mating type, A1 or A2, by dikaryotic mycelia homoallelic for the pal ^– allele; (b) the culturing of homokaryotic mycelia issuing from the germination of basidiospores from sporophores produced by dikaryotic mycelia heterokaryotic for the pal gene; (c) the culturing of mycelia grown from protoplasts obtained from dikaryons homoallelic for the pal ^– allele (D6 strain), and from homokaryons heteroallelic for the pal gene (H8), or homoallelic for pal ^#x002B;+ allele (H7). These techniques enabled us to segregate homokaryons of the four types defined above and were indispensable in the segregation of the pal ^– homoallelic homokaryons as no basidiospores were produced by typical pale mushrooms.     

Functional analysis of theDrosophila CDC2 Dm gene in fission yeast

Thecdc2 ⁺ gene product (p34^cdc2) is a protein kinase that regulates entry into mitosis in all eukaryotic cells. The role that p34^cdc2 plays in the cell cycle has been extensively investigated in a number of organisms, including the fission yeastSchizosaccharomyces pombe. To study the degree of functional conservation among evolutionarily distant p34^cdc2 proteins, we have constructed aS. pombe strain in which the yeastcdc2 ⁺ gene has been replaced by itsDrosophila homologue CDC2Dm (theCDC2Dm strain). ThisCDC2Dm S. pombe strain is viable, capable of mating and producing four viable meiotic products, indicating that the fly p34^CDC2Dm recognizes all the essentialS. pombe cdc2 ⁺ substrates, and that it is recognized by cyclin partners and other elements required for its activity. The p34^CDC2Dm protein yields a lethal phenotype in combination with the mutant B-type cyclin p56^cdc13-117, suggesting that thisS. pombe cyclin might interact less efficiently with theDrosophila protein than with its native p34^cdc2 counterpart. ThisCDC2Dm strain also responds to nutritional starvation and to incomplete DNA synthesis, indicating that proteins involved in these signal transduction pathways, interact properly with p34^CDC2Dm (and/or that p34^cdc2-independent pathways are used). TheCDC2Dm gene produces a ‘wee’ phenotype, and it is largely insensitive to the action of theS. pombe weel ⁺ mitotic inhibitor, suggesting thatDrosophila weel ⁺ homologue might not be functionally conserved. ThisCDC2Dm strain is hypersensitive to UV irradiation, to the same degree asweel-deficient mutants. A strain which co-expresses theDrosophila and yeastcdc2⁺ genes shows a dominantwee phenotype, but displays a wild-type sensitivity to UV irradiation, suggesting that p34^cdc2 triggers mitosis and influences the UV sensitivity by independent mechanisms. Communicated by B. J. Kilbey     

Nonsense suppression in Schizosaccharomyces pombe: the S. pombe Sup3-e tRNASerUGA gene is active in S. cerevisiae

Summary The gene encoding the efficient UGA suppressor sup3-e of Schizosaccharomyces pombe was isolated by in vivo transformation of Saccharomyces cerevisiae UGA mutants with S. pombe sup3-e DNA. DNA from a clone bank of EcoRI fragments from a S. pombe sup3-e strain in the hybrid yeast vector YRp17 was used to transform the S. cerevisiae multiple auxotroph his4-260 leu2-2 trp1-1 to prototrophy. Transformants were isolated at a low frequency; they lost the ability to grow in minimal medium after passaging in non-selective media. This suggested the presence of the suppressor gene on the non-integrative plasmid. Plasmid DNA, isolated from the transformed S. cerevisiae cells and subsequently amplified in E. coli, transformed S. cerevisiae his4-260 leu2-2 trp1-1 to prototrophy. In this way a 2.4 kb S. pombe DNA fragment carrying the sup3-e gene was isolated. Sequence analysis revealed the presence of two tRNA coding regions separated by a spacer of only seven nucleotides. The sup3-e tRNA _Ser ^UGA tRNA gene is followed by a sequence coding for the initiator tRNA^Met. The transformation results demonstrate that the cloned S. pombe UGA suppressor is active in S. cerevisiae UGA mutant strains.     

The Fission Yeast php2 Mutant Displays a Lengthened Chronological Lifespan

The Schizosaccharomyces pombe php2 ⁺ gene encodes a subunit of the CCAAT-binding factor complex. We found that disruption of the php2 ⁺ gene extended the chronological lifespan of the fission yeast. Moreover, the lifespan of the Δphp2 mutant was barely extended under calorie restricted (CR) conditions. Many other phenotypes of the Δphp2 mutant resembled those of wild-type cells grown under CR conditions, suggesting that the Δphp2 mutant might undergo CR. The mutant also showed low respiratory activity concomitant with decreased expression of the cyc1 ⁺ and rip1 ⁺ genes, both of which are involved in mitochondrial electron transport. On the basis of a chromatin immunoprecipitation assay, we determined that Php2 binds to a DNA region upstream of cyc1 ⁺ and rip1 ⁺ in S. pombe. Here we discuss the possible mechanisms by which the chronological lifespan of Δphp2 mutant is extended.     

Yeast as a model system for understanding the control of DNA replication in eukaryotes

In the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the initiation of DNA replication is controlled at a point called START. At this point, the cellular environment is assessed; only if conditions are appropriate do cells traverse START, thus becoming committed to initiate DNA replication and complete the remainder of the cell cycle. The cdc2⁺ / CDC28⁺ gene, encoding the protein kinase p34, is a key element in this complex control. The identification of structural and functional homologues of p34 suggests that it has a role in the control of DNA replication in all eukaryotes. The WHI1⁺, CLN1⁺ and CLN2⁺ gene products, identified in S. cerevisiae, are positive regulators that function at START and may interact with p34. Determining how passing the START control point leads to the initiation of DNA replication is a major outstanding challenge in cell cycle studies.