Yeast sequencing reports. Molecular cloning and sequencing of the hcs gene,which encodes 3-hydroxy-3-methylglutaryl coenzyme a synthase of Schizosaccharomyces pombe

We have cloned and sequenced the hcs gene, which is thought to encode a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase consisting of 447 amino acids, from the fission yeast Schizosaccharomyces pombe. The predicted amino acid sequence of the hcs product of S. pombe has homology with the HMG-CoA synthase of rat (47·8%), chicken (49·2%), hamster (47·1%) and human cells (46·9%). One of the hcs genes was replaced with a marker gene in the diploid cell. No viable hcs-disrupted haploid was isolated after tetrad dissection, suggesting that the hcs gene is essential for growth. However the hcs-defective mutant could be grown on a medium containing 5 mg/ml mevalonate. These results strongly support that the hcs gene encodes HMG-CoA synthase and S. pombe contains a single copy of the hcs gene. The sequence of the hcs gene has been entered into the public data libraries under Accession Number U32187.     

Yeast sequencing reports. Gcs1, a gene encoding γ-glutamylcysteine synthetase in the fission yeast Schizosaccharomyces pombe

By complementation of a mutant resistant to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) we have identified the gcs1 gene, encoding a putative γ-glutamylcysteine synthetase. The gene is possibly interrupted by two introns and has 49% identical and 80% similar amino acids compared with the homologous protein from rat. In comparison with the Saccharomyces cerevisiae homologue it possesses 41% identical and 74% similar amino acids. The gsc1 sequence appears in the EMBL database under Accession Number X 85017.     

Sequence of the genes encoding subunits A and B of the vacuolar H(+)-ATPase of Schizosaccharomyces pombe.

The genes encoding subunits A (vma1) and B (vma2) of the vacuolar H(+)-ATPase from Schizosaccharomyces pombe were cloned by hybridization to cDNAs of the homologous genes in Neurospora crassa. Both genes are interrupted by introns, two in vma1 and four in vma2. Positions of introns do not appear to be conserved when compared to those of N. crassa. The subunit A gene encodes a single product of 619 amino acids and is not interrupted by the coding sequence for a second product as found for Saccharomyces cerevisiae (Kane, P. K., Yamashiro, C. T., Wolczyk, D. F., Neff, N., Goebl, M., and Stevens, T. H. (1990). Science 250, 651-657).     

A genomic sequence of the Schizosaccharomyces pombe 16 kDa vacuolar H(+)-ATPase.

We have isolated the gene encoding the 16 kDa vacuolar H(+)-ATPase from Schizosaccharomyces pombe. On the basis of RNA splicing signals and amino acid sequence homology with other 16 kDa H(+)-ATPases, the genomic DNA sequence indicated the 16 kDa protein is encoded by five exons. The C-terminal 50 amino acids has more than 90% homology with vacuolar H(+)-ATPases of mammalian cells.     

Properties and Heterologous Expression of the Glucose Transporter GHT1 from Schizosaccharomyces pombe

Genomic DNA of the Schizosaccharomyces pombe glucose transporter, GHT1, was obtained by complementation of the glucose transport deficient Sz. pombe strain YGS-5. Here we describe the GHT1 gene that encodes a protein of 565 amino acids with a corresponding molecular mass of 62·5 kDa. This eukaryotic glucose transporter contains 12 putative transmembrane segments and is homologous to the HXT multigene family of S. cerevisiae with several amino acid motifs of this sugar transporter family. It is also homologous to other sugar carriers from human, mouse and Escherichia coli. The function of the Ght1 protein as a glucose transporter was proved both by homologous and heterologous expression in the Sz. pombe mutant YGS-5 and in the S. cerevisiae hxt mutant RE700A, respectively. Both transformed yeast strains transported d -glucose with substrate specificity similar to that in Sz. pombe wild-type cells. Moreover, the cells of the two transformed yeast strains accumulated 2-deoxy-d -glucose, a non-metabolizable d -glucose analogue, with an efficiency similar to Sz. pombe wild-type cells. The ability of the S. cerevisiae mutant RE700A to accumulate 2DG in an Δμdependent manner after transformation with GHT1 provides evidence that the Sz. pombe transporter catalyses an energy-dependent uptake of glucose. The sequence of GHT1 was deposited at EMBL, Outstation EBI, Accession Number X91218. ©1997 John Wiley & Sons, Ltd.     

Physiological and biochemical analysis of cytochrome P-450 in the yeast Schizosaccharomyces pombe

The yeast Schizosacchromyces pombe has been shown to contain a microsomal cytochrome P-450 (cyt. P-450) inducible under conditions of glucose repression. Under these conditions the enzyme has maximal expression of 0.43 nmol g^?1 wet wt at the end of the exponential phase of growth. Substrate and inhibitor affinity was examined using studies of spectral changes on binding and revealed a type II spectrum with ketoconazole (K_s = 23 μM ) and a type I spectrum with benzo(a)pyrene (K_s = 77 μM ). A K_m of 112 μM was found in the aryl hydrocarbon hydroxylas assay. These properties show broad comparability with the cyt. P-450 of Saccharomyces cerevisiae.     

Molecular,functional and evolutionary characterization of the gene encoding HMG-CoA reductase in the fission yeast,Schizosaccharomyces pombe

The synthesis of mevalonate, a molecule required for both sterol and isoprene biosynthesis in eukaryotes, is catalysed by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Using a gene dosage approach, we have isolated the gene encoding HMG-CoA reductase, hmg1+, from the fission yeast Schizosaccharomyces pombe (Accession Number L76979). Specifically, hmg1+ was isolated on the basis of its ability to confer resistance to lovastatin, a competitive inhibitor of HMG-CoA reductase. Gene disruption analysis showed that hmg1+ was an essential gene. This result provided evidence that, unlike Saccharomyces cerevisiae, S. pombe contained only a single functional HMG-CoA reductase gene. The presence of a single HMG-CoA reductase gene was confirmed by genomic hybridization analysis. As observed for the S. cerevisiae HMG1p, the hmg1+ protein induced membrane proliferations known as karmellae. A previously undescribed ‘feed-forward’ regulation was observed in which elevated levels of HMG-CoA synthase, the enzyme catalysing the synthesis of the HMG-CoA reductase substrate, induced elevated levels of hmg1+ protein in the cell and conferred partial resistance to lovastatin. The amino acid sequences of yeast and human HMG-CoA reductase were highly divergent in the membrane domains, but were extensively conserved in the catalytic domains. We tested whether the gene duplication that produced the two functional genes in S. cerevisiae occurred before or after S. pombe and S. cerevisiae diverged by comparing the log likelihoods of trees specified by these hypotheses. We found that the tree specifying post-divergence duplication had significantly higher likelihood. Moreover, phylogenetic analyses of available HMG-CoA reductase sequences also suggested that the lineages of S. pombe and S. cerevisiae diverged approximately 420 million years ago but that the duplication event that produced two HMG-CoA reductase genes in the budding yeast occurred only approximately 56 million years ago. To date, S. pombe is the only unicellular eukaryote that has been found to contain a single HMG-CoA reductase gene. Consequently, S. pombe may provide important opportunities to study aspects of the regulation of sterol biosynthesis that have been difficult to address in other organisms and serve as a test organism to identify novel therapies for modulating cholesterol synthesis.     

A truncated derivative of nmt 1 promoter exhibits temperature-dependent induction of gene expression in Schizosaccharomyces pombe

Despite increasing exploitation of Schizosaccharomyces pombe as a model system there is a lack of convenient vectors for research and application. Expression with the commonly used promoter, nmt 1, requires a laborious regime involving the removal of repressor, thiamine, from a growing culture and further growth for 18 h to achieve maximum expression, thus underlining the need for more user-friendly promoters. We report here the isolation and characterization of a truncated derivative of the nmt 1 promoter having novel induction characteristics: it is induced by shift of growth temperature from 36 degrees C to 25 degrees C, achieving maximum expression within 3 h. Similar features of expression were observed with the reporter genes GFP and beta-galactosidase, a native gene, cdc 18, and a commercially important foreign therapeutic protein, streptokinase. The new promoter element offers additional advantages, such as lack of deleterious effect on cell viability and potential ability to express toxic proteins. These features make the new promoter a potentially better alternative to nmt 1, both as a research tool and for expression of commercially important proteins in Sz. pombe, and suggest the possibility of using similar approaches to design promoters with novel and useful properties.     

The Schizosaccharomyces pombe cfr1+ gene participates in mating through a new pathway that is independent of fus1+

Conjugation is a complex event directed to ensure the transfer of genetic material, which is achieved by the union of two cells. In fungi, success of this relevant process requires digestion of the cell wall at the point where both cells have agglutinated and, later, the union of the plasma membranes and nuclei from the mating partners. In order to gain information about cell fusion, we have cloned and disrupted the cfr1+ gene from the fission yeast Schizosaccharomyces pombe. cfr1+ gene is slightly induced at the beginning of mating but Cfr1p protein is degraded soon after the cells are transferred to nitrogen-lacking medium. cfr1Delta mutants present a defect in cell fusion owing to a failure in the digestion of the cell walls between the two parental cells. Finally, cytological and genetic analyses show that cfr1+ acts in a new pathway involved in conjugation that is independent of fus1+, the only gene that has been found to be specifically required for cell fusion during mating in the fission yeast.     

Expression and secretion of a prokaryotic protein streptokinase without glycosylation and degradation in Schizosaccharomyces pombe

Streptokinase (SK) is an important thrombolytic protein that is secreted by pathogenic strains of Streptococcus. Expression of streptokinase has been so far attempted in Pichia pastoris, Escherichia coli and Bacillus subtilis and shown to yield protein that was either highly glycosylated or degraded. Since the fission yeast, Schizosaccharomyces pombe, shares several molecular characteristics with higher eukaryotes, we decided to express the streptokinase gene in this yeast. A chimeric gene comprising the signal sequence of the Plus pheromone of Sz. pombe fused in-frame with the mature streptokinase from Streptococcus sp. was constructed and inserted into the expression vector containing the thiamine-regulated promoter. We obtained a high level of expression of streptokinase comparable to that in E. coli and P. pastoris, with 50-100% processing of the signal sequence and secretion of the mature streptokinase into the periplasmic fraction. The mature enzyme co-migrates with the authentic mature SK in SDS gels, lacks any major modification and is functional. Importantly, a higher level of expression under stationary phase conditions and improved extractability of the mature and undegraded streptokinase was achieved in a novel mutant of Sz. pombe defective for a potent extracellular protease activity. We suggest that the unique vector/strain system developed here could be advantageous for large-scale production of prokaryotic proteins without significant modification or degradation in Sz. pombe.     

The ade6 gene of the fission yeast Schizosaccharomyces pombe has the same chromatin structure in the chromosome and in plasmids.

We have analysed the chromatin structure of the ade6 gene of Schizosaccharomyces pombe and its flanking regions both in the chromosome and in plasmids. The chromatin structure is independent of the chromosomal or extrachromosomal location. The ade6 gene contains eight precisely positioned nucleosomes on the 5' half, 'not positioned' nucleosomes around the 3' end and a nuclease-sensitive promoter region. Precisely positioned nucleosomes, but no nuclease-sensitive region were also detected on the ura4 gene in the chromosome and on a plasmid. The results show that S. pombe chromosomal and extrachromosomal genes have chromatin structures similar to those of S. cerevisiae and higher eukaryotes.     

Cloning,sequencing and disruption of the ARG8 gene encoding acetylornithine aminotransferase in the petite-negative yeast Kluyveromyces lactis

A recombinant plasmid was isolated from a Kluyveromyces lactis genomic DNA library which complements a Saccharomyces cerevisiae arg8 mutant defective in the gene encoding acetylornithine aminotransferase. The complementation activity was found to reside within a 2.0 kb DNA fragment. Nucleotide sequence analysis revealed an open reading frame able to encode a 423-residue protein sharing 68·1% and 35·0% sequence identities with the products of the ARG8 and argD genes of S. cerevisiae and Escherichia coli. That the cloned gene, KlARG8, is the functional equivalent of S. cerevisiae ARG8 was supported by a gene disruption experiment which showed that K. lactis strains carrying a deleted chromosomal copy of KlARG8 are auxotrophic for arginine. The nucleotide sequence of KlARG8 has been submitted to GenBank under Accession Number U93209.     

Molecular cloning of the plc1+ gene of Schizosaccharomyces pombe,which encodes a putative phosphoinositide-specific phospholipase C

Exploiting the polymerase chain reaction, we have isolated a gene that encodes a putative phosphoinositide-specific phospholipase C (PLC) of the fission yeast Schizosaccharomyces pombe. Inspection of the nucleotide sequence of the gene revealed an open reading frame that can encode a polypeptide of 899 amino acid residues with a calculated molecular mass of 102 kDa. This putative polypeptide contains both the X and Y regions that are conserved among three classes of mammalian PLC, and also contains a presumptive Ca²⁺-binding site (an E-F hand motif). The structure of the putative protein is most similar to that of the δ class of PLC isozymes. To investigate the role of this gene, designated plc1⁺, gene disruption was carried out by interrupting the coding region with the ura4⁺ marker. Growth of plc1 cells was temperature-sensitive in rich medium, and cells could not grow in synthetic medium. Expression of the PLC1 gene of Saccharomyces cerevisiae suppressed the growth defect phenotype of plc1^? cells, a strong suggestion that the plc1⁺ gene encodes PLC. The PLC1 sequence appears in the public data libraries, DDBJ GenBank, EMBL under the following Accession Number: D38309.     

The mae1 gene of Schizosaccharomyces pombe encodes a permease for malate and other C4 dicarboxylic acids

The mae1 gene of the yeast Schizosaccharomyces pombe was identified on the basis of its ability to complement a mutant defective in the transport of malic acid. Analysis of the DNA sequence revealed an open reading frame of 1314 base pairs, encoding a polypeptide of 438 amino acids with a predicted molecular weight of 49 kDa. A hydropathy profile of the predicted amino acid sequence revealed a protein with ten membrane-spanning or associated domains and hydrophilic N- and C- termini. The predicted secondary structure of the protein is similar to models proposed for other integral mambrane proteins from both prokaryotes and eukaryotes. The S. pombe mae1 gene encodes a single mRNA of 1·5 kb. The mae1 gene is expressed constitutively and is not subject to catabolite repression as was previously reported for the malate permease systems of Candida utilis and Hansenula anomala. The mae1 gene was mapped 2842 bp 5′ to the MFm1 gene on chromosome I. Transport assays revealed that the mae1 gene encodes a permease involved in the uptake of L-malate, succinate and malonic acid. The sequence of the S. pombe mae1 gene is available in GenBank under Accession Number U21002.     

Characterization of vps33+, a gene required for vacuolar biogenesis and protein sorting in Schizosaccharomyces pombe

From the fission yeast Schizosaccharomyces pombe we have identified and deleted vps33, a gene encoding a homologue of VPS33, which is required for vacuolar biogenesis in S. cerevisiae cells. When the vps33(+) gene is disrupted, Sz. pombe strains are temperature-sensitive for growth and contain numerous small vesicular structures stained with FM4-64 in the cells. Deletion of the Sz. pombe vps33(+) gene results in pleiotropic phenotypes consistent with the absence of normal vacuoles, including missorting of vacuolar carboxypeptidase Y, various ion- and drug-sensitivities, and sporulation defects. These results are consistent with Vps33p being necessary for the morphogenesis of vacuoles and subsequent expression of vacuolar functions in Sz. pombe cells.     

Mutational analysis of the gene for Schizosaccharomyces pombe RNase MRP RNA,mrp1, using plasmid shuffle by counterselection on canavanine

Reverse genetics in fission yeast is hindered by the lack of a versatile established plasmid shuffle system. In order to screen efficiently and accurately through plasmid-borne mutations in the essential gene for the RNA component of RNase MRP, mrp1, we have developed a system for plasmid shuffling in fission yeast using counterselection on canavanine. The system takes advantage of the ability of the Saccharomyces cerevisiae CAN1 gene to complement a Schizosaccharomyces pombe can1-1 mutation. Two general use plasmids were constructed that allow directional cloning and initial selection for histidine before counterselection by canavanine. The strain constructed for plasmid shuffling carries auxotrophic markers for ade6, leu1, ura4 and his3 along with the can1-1 mutation. Using this system we examined several partial deletions and point mutations in conserved nucleotides of Schizosaccharomyces pombe RNase MRP RNA for their ability to complement a chromosomal deletion of the mrp1 gene. The degree of background canavanine resistance as well as plasmid–plasmid recombination encountered in these experiments was sufficiently low to suggest that the system we have set up for counterselection by canavanine in fission yeast using multicopy plasmids will be widely useful.