Cloning and sequence determination of the gene encoding the largest subunit of the fission yeast Schizosaccharomyces pombe RNA polymerase I

The gene encoding the largest subunit of RNA polymerase I (SPRPA190) was cloned from the fission yeast Schizosaccharomyces pombe by cross-hybridization with a probe containing part of the corresponding Saccharomyces cerevisiae gene RPA190. The SPRPA190 gene is present in a single copy per haploid genome and is essential for cell growth. The polypeptide encoded by this gene, as deduced from the nucleotide sequence of the uninterrupted coding frame, consists of 1689 amino acids and its calculated Mr is 189,300. The amino acid identity between the subunits of the two yeast species is 50%. Amino acid sequence conservation covers the regions previously suggested to be functionally important for the S. cerevisiae enzyme. In addition, two markedly hydrophilic regions recognized in the S. cerevisiae polypeptide can also be recognized in the S. pombe polypeptide in approximately the same positions, even though the amino acid sequences in these regions are diverged from each other. In the 5'-flanking region of the gene, several nucleotide sequence elements are detected which are also found in the two S. pombe ribosomal protein genes so far sequenced.     

Isolation and characterization of a second protein tyrosine phosphatase gene, PTP2, from Saccharomyces cerevisiae.

A putative protein tyrosine phosphatase (PTPase) gene, PTP2, was cloned from Saccharomyces cerevisiae. The complete yeast PTP2 gene encodes a 750-amino acid residue protein with a predicted mass of 86 kDa. The conserved PTPase domain was localized in the C-terminal half of the protein. Amino acid sequence alignment of the yeast PTPase domain with other phosphatases indicated approximately 20-25% sequence identity with the mammalian PTPase and a similar degree of identity with the PTPase encoded by the yeast PTP1 gene. The PTP2 gene is closely linked to the yeast RET1 and STE4 genes and is localized on the right arm of chromosome 15. Gene disruption experiments demonstrated that neither PTP2 alone nor PTP2 in combination with PTP1 was essential for growth under the conditions tested. The ability of PTP2 to complement the cdc25-22 mutant of Schizosaccharomyces pombe was also examined, and unlike the human T-cell PTPase, which was able to complement the cdc25-22 mutant, the S. cerevisiae PTP2 was unable to complement the cdc25-22 mutant of S. pombe.     

Motifs in Schizosaccharomyces pombe ars3002 important for replication origin activity in Saccharomyces cerevisiae

Ars3002 is an efficient single-copy replication origin in the fission yeast, Schizosaccharomyces pombe. In a previous study, we tested the effects of consecutive approximately 50-bp deletions throughout ars3002 on the replication efficiency of those origins in S. pombe. Here we report the results of our use of the same approximately 50-bp deletions to test the hypothesis that some of the cis-acting sequences important for replication origin activity in fission yeast might be conserved in the evolutionarily distant budding yeast, Saccharomyces cerevisiae. We found that in most cases there was no correlation between the effects of particular mutations in S. pombe and in S. cerevisiae. We conclude that it is unlikely that any of the cis-acting sequences recognised by homologous replication proteins is conserved between these two yeast species.     

Severe growth defect in a Schizosaccharomyces pombe mutant defective in intron lariat degradation.

The cDNAs and genes encoding the intron lariat-debranching enzyme were isolated from the nematode Caenorhabditis elegans and the fission yeast Schizosaccharomyces pombe based on their homology with the Saccharomyces cerevisiae gene. The cDNAs were shown to be functional in an interspecific complementation experiment; they can complement an S. cerevisiae dbr1 null mutant. About 2.5% of budding yeast S. cerevisiae genes have introns, and the accumulation of excised introns in a dbr1 null mutant has little effect on cell growth. In contrast, many S. pombe genes contain introns, and often multiple introns per gene, so that S. pombe is estimated to contain approximately 40 times as many introns as S. cerevisiae. The S. pombe dbr1 gene was disrupted and shown to be nonessential. Like the S. cerevisiae mutant, the S. pombe null mutant accumulated introns to high levels, indicating that intron lariat debranching represents a rate-limiting step in intron degradation in both species. Unlike the S. cerevisiae mutant, the S. pombe dbr1::leu1+ mutant had a severe growth defect and exhibited an aberrant elongated cell shape in addition to an intron accumulation phenotype. The growth defect of the S. pombe dbr1::leu1+ strain suggests that debranching activity is critical for efficient intron RNA degradation and that blocking this pathway interferes with cell growth.     

絮凝特性对自絮凝颗粒酵母耐酒精能力的影响及作用机制

首次报道絮凝特性提高酵母菌耐酒精能力的现象及其机制。融合株SPSC与其两亲本粟酒裂殖酵母变异株和酿酒酵母变异株于 30℃经 18% (V/V)酒精冲击 7h的存活率分别为 52%、37%和 9%。细胞膜磷脂脂肪酸组成分析表明 ,两絮凝酵母 (融合株SPSC和粟酒裂殖酵母变异株 )的棕榈酸含量均约为非絮凝酵母 (酿酒酵母变异株 )的两倍 ,而棕榈油酸和油酸的含量明显低于后者。研究表明 ,当两絮凝酵母在培养中由于柠檬酸钠的作用 (抑制絮凝体的形成 )而以游离细胞生长存在时 ,其细胞膜磷脂棕榈酸含量显著下降 ,而棕榈油酸和油酸的含量明显增加 ,结果细胞膜磷脂脂肪酸组成特点与酿酒酵母变异株相似 ;而且实验表明 ,絮凝特性的消失伴随菌体耐酒精能力的急剧下降 ,变得与酿酒酵母变异株的水平相当。这些结果提示两絮凝酵母具有较强的耐酒精能力与其细胞膜磷脂脂肪酸组成中含有更高比例的棕榈酸有关。     

Cytoplasmic ribosomal protein genes of the fission yeast Schizosaccharomyces pombe display a unique promoter type: a suggestion for nomenclature of cytoplasmic ribosomal proteins in databases.

We identified 34 new ribosomal protein genes in the Schizosaccharomyces pombe database at the Sanger Centre coding for 30 different ribosomal proteins. All contain the Homol D-box in their promoter. We have shown that Homol D is, in this promoter type, the TATA-analogue. Many promoters contain the Homol E-box, which serves as a proximal activation sequence. Furthermore, comparative sequence analysis revealed a ribosomal protein gene encoding a protein which is the equivalent of the mammalian ribosomal protein L28. The budding yeast Saccharomyces cerevisiae has no L28 equivalent. Over the past 10 years we have isolated and characterized nine ribosomal protein (rp) genes from the fission yeast S.pombe . This endeavor yielded promoters which we have used to investigate the regulation of rp genes. Since eukaryotic ribosomal proteins are remarkably conserved and several rp genes of the budding yeast S.cerevisiae were sequenced in 1985, we probed DNA fragments encoding S.cerevisiae ribosomal proteins with genomic libraries of S.pombe . The deduced amino acid sequence of the different isolated rp genes of fission yeast share between 65 and 85% identical amino acids with their counterparts of budding yeast.     

The primary structure of the alcohol dehydrogenase gene from the fission yeast Schizosaccharomyces pombe

We have cloned and sequenced the alcohol dehydrogenase gene of the fission yeast Schizosaccharomyces pombe. The gene was isolated by transformation and complementation of a Saccharomyces cerevisiae strain which lacked functional alcohol dehydrogenase with an S. pombe gene bank constructed in the autonomously replicating yeast plasmid YEp13. Southern hybridization analysis indicates that S. pombe contains only one alcohol dehydrogenase gene. The structural region of the gene is 50% homologous to the alcohol dehydrogenase encoding genes of the budding yeast S. cerevisiae. The gene exhibits a very strong codon usage bias; with the set of predominantly used codons generally resembling that which S. cerevisiae employs preferentially. All of the differences in codon usage bias between S. pombe and S. cerevisiae are in the direction of greater G + C content in S. pombe codons. It is argued that this observation supports the hypothesis that selection toward uniform codon-anticodon binding energies contributes to codon usage bias and that the optimum binding energy is, on the average, higher in S. pombe than S. cerevisiae.     

The Rpb4 Subunit of Fission Yeast Schizosaccharomyces pombe RNA Polymerase II Is Essential for Cell Viability and Similar in Structure to the Corresponding Subunits of Higher Eukaryotes

Both the gene and the cDNA encoding the Rpb4 subunit of RNA polymerase II were cloned from the fission yeast Schizosaccharomyces pombe. The cDNA sequence indicates that Rpb4 consists of 135 amino acid residues with a molecular weight of 15,362. As in the case of the corresponding subunits from higher eukaryotes such as humans and the plant Arabidopsis thaliana, Rpb4 is smaller than RPB4 from the budding yeast Saccharomyces cerevisiae and lacks several segments, which are present in the S. cerevisiae RPB4 subunit, including the highly charged sequence in the central portion. The RPB4 subunit of S. cerevisiae is not essential for normal cell growth but is required for cell viability under stress conditions. In contrast, S. pombe Rpb4 was found to be essential even under normal growth conditions. The fraction of RNA polymerase II containing RPB4 in exponentially growing cells of S. cerevisiae is about 20%, but S. pombe RNA polymerase II contains the stoichiometric amount of Rpb4 even at the exponential growth phase. In contrast to the RPB4 homologues from higher eukaryotes, however, S. pombe Rpb4 formed stable hybrid heterodimers with S. cerevisiae RPB7, suggesting that S. pombe Rpb4 is similar, in its structure and essential role in cell viability, to the corresponding subunits from higher eukaryotes. However, S. pombe Rpb4 is closer in certain molecular functions to S. cerevisiae RPB4 than the eukaryotic RPB4 homologues.     

A member of the sugar transporter family, Stl1p is the glycerol/H+ symporter in Saccharomyces cerevisiae

Glycerol and other polyols are used as osmoprotectants by many organisms. Several yeasts and other fungi can take up glycerol by proton symport. To identify genes involved in active glycerol uptake in Saccharomyces cerevisiae we screened a deletion mutant collection comprising 321 genes encoding proteins with 6 or more predicted transmembrane domains for impaired growth on glycerol medium. Deletion of STL1, which encodes a member of the sugar transporter family, eliminates active glycerol transport. Stl1p is present in the plasma membrane in S. cerevisiae during conditions where glycerol symport is functional. Both the Stl1 protein and the active glycerol transport are subject to glucose-induced inactivation, following identical patterns. Furthermore, the Stl1 protein and the glycerol symporter activity are strongly but transiently induced when cells are subjected to osmotic shock. STL1 was heterologously expressed in Schizosaccharomyces pombe, a yeast that does not contain its own active glycerol transport system. In S. pombe, STL1 conferred the ability to take up glycerol against a concentration gradient in a proton motive force-dependent manner. We conclude that the glycerol proton symporter in S. cerevisiae is encoded by STL1.     

Identification of sds21 in fission yeast in an inhibitor-resistant high molecular mass protein phosphatase-1 complex.

While characterizing the type-1 protein phosphatases sds21 and dis2 in fission yeast (Schizosaccharomyces pombe) a novel high molecular mass protein was identified with serine/threonine phosphatase activity (referred to as PP-R) that was resistant to a panel of characteristic inhibitors of protein phosphatases. Purification of the native sds21 catalytic isoform of protein phosphatase-1 (PP-1) from an S. pombe knockout strain lacking dis2 (deltadis2) resulted predominantly in identification of PP-R. To test the hypothesis that the catalytic activity of PP-R comprised sds21, a parallel purification was performed of PP-1 activity from an S. pombe knockout strain lacking sds21 (deltasds21). Both deltasds21 and deltadis2 strains exhibited similar protein phosphatase activity profiles as determined by DEAE-sepharose, Mono-Q and Superdex gel filtration chromatography. However, the peak of protein phosphatase activity from deltasds21 S. pombe that co-migrated with PP-R from deltadis2 S. pombe exhibited the sensitivity to a panel of inhibitors that was characteristic of a type-1 protein phosphatase. These data suggest that the catalytic subunit of PP-R comprises sds21 and that the resistance to inhibitors may originate from structural differences between dis2 and sds21 isoforms. A key structural feature present in sds21, but lacking in dis2, is a classical phosphorylation consensus sequence surrounding serine-145 of sds21. The previous hypothesis was that PP-1 activity among several lower eukaryotes may be regulated directly by cAMP-dependent protein kinase (PKA) phosphorylation. However, this study demonstrated that recombinant sds21 is not a target for PKA in vitro. The constrained configuration of the putative PKA site on the PP-1 holoenzyme may restrict its ability to be targeted by PKA.     

Post-translational processing of Schizosaccharomyces pombe YPT proteins.

ras proteins are post-translationally processed at their carboxyl-terminal CAAX motif by a triplet of modifications: prenylation of C with farnesyl, proteolytic trimming of AAX, and carboxyl-methylation. These modifications co-operate with palmitoylation of nearby sites or a polybasic region to target plasma membrane localization. The related YPT/rab proteins in contrast are localized to compartments of the endo-membrane system and may be involved in directing membrane traffic. These proteins end in XCC or CXC motifs. We have analyzed the processing of members of this subfamily form the fission yeast Schizosaccharomyces pombe. We find using in vitro translation in reticulocyte lysates that YPT1, -3, and -5 are prenylated with geranylgeranyl and that they incorporate label from [3H]mevalonic acid when expressed in transfected COS cells in vivo. Furthermore, prenylation was necessary for membrane binding in vivo. The CXC protein YPT5, but neither of the two XCC proteins YPT1 and YPT3, was carboxyl-methylated in S. pombe and in COS cells in vivo. However, YPT5 was not carboxyl-methylated in vitro in lysates which were able to methylate ras protein. YPT3 was detectably palmitoylated when expressed in COS cells, though at a much lower level than ras.     

裂殖酵母作为外源基因表达系统

虽然裂殖酵母与酿酒酵母同属于子囊真菌,但比其它的酵母相比,裂殖酵母与更高等的真核细胞有许多相似的性质,使得裂殖酵母在分子生物学研究中成为一种提供信息的、准确的真核实验模型.它在外源基因表达方面同样具有前景.主要介绍了裂殖酵母的优点,其表达载体的性质,以及外源蛋白表达的例子.     

PCR-mediated direct gene disruption in Schizosaccharomyces pombe.

We have examined the feasibility and efficiency of PCR-mediated direct gene disruptions in the fission yeast Schizosaccharomyces pombe. In the present study, the S.pombe ura4+ gene was amplified by PCR with oligonucleotides that had short flanking regions ( approximately 40 bp) to the target gene. Using this purified PCR product we were able to disrupt genes in an S. pombe strain bearing aura4 deletion, with an efficiency ranging between 1 and 3% among selected transformants. The results indicated that despite S.pombe's preference for non-homologous or illegitimate recombination, even very short stretches of homologous regions could be used to target genes at a defined frequency in this organism. The successful disruption of four independent genes (sts1+, gcs1+, gsh2+and hmt1+) by this method further demonstrates that, despite the relatively low efficiency, the method is very feasible, and it's simplicity, especially when coupled to phenotype-based screening, should greatly facilitate disruption of genes in S.pombe.