Characterization of the gene for fructose-1,6-bisphosphatase from Saccharomyces cerevisiae and Schizosaccharomyces pombe. Sequence, protein homology, and expression during growth on glucose

We have determined the nucleotide sequence of the gene for fructose-1,6-bisphosphatase from both Saccharomyces cerevisiae and Schizosaccharomyces pombe. The predicted protein sequence for fructose-1,6-bisphosphatase from S. cerevisiae contains 347 amino acids and has a molecular weight of 38,100; that from S. pombe, contains 346 amino acids and has a molecular weight of 38,380. Comparison of these amino acid sequences with each other and that of pig kidney fructose-1,6-bisphosphatase shows several regions of strong homology separated by regions of divergence. These homologous regions are likely candidates for functional domains. A gene cassette was constructed for fructose-1,6-bisphosphatase from S. cerevisiae and the gene cassette expressed from the regulated PHO5 and GAL1 promoters of yeast. Yeast cells expressing fructose-1,6-bisphosphatase, while growing on glucose, accumulated large amounts of enzyme intracellularly, suggesting that glucose-regulated proteolytic inactivation does not operate efficiently under these conditions. Growth on glucose was not inhibited by the expression of fructose 1,6-bisphosphatase.     

Analysis of the alpha-amylase gene of Schwanniomyces occidentalis and the secretion of its gene product in transformants of different yeast genera

We have cloned and characterized the alpha-amylase gene (AMY1) of the yeast Schwanniomyces occidentalis. A cosmid gene library of S. occidentalis DNA was screened in Saccharomyces cerevisiae for alpha-amylase secretion. The positive clone contained a DNA fragment harbouring an open reading frame of 1536 nucleotides coding for a 512-amino-acid polypeptide with a calculated Mr of 56,500. The deduced amino acid sequence reveals significant similarity to the sequence of the Saccharomycopsis fibuligera and Aspergillus oryzae alpha-amylases. The AMY l gene was found to be expressed from its original promoter in S. cerevisiae, Kluyveromyces lactis and Schizo-saccharomyces pombe leading to an active secreted gene product and thus enabling the different yeast transformants to grow on starch as a sole carbon source.     

Conservation between human and fungal squalene synthetases: similarities in structure, function, and regulation.

Squalene synthetase (farnesyl diphosphate:farnesyl diphosphate farnesyltransferase; EC 2.5.1.21) is thought to represent a major control point of isoprene and sterol biosynthesis in eukaryotes. We demonstrate structural and functional conservation between the enzymes from humans, a budding yeast (Saccharomyces cerevisiae), and a fission yeast (Schizosaccharomyces pombe). The amino acid sequences of the human and S. pombe proteins deduced from cloned cDNAs were compared to those of the known S. cerevisiae protein. All are predicted to encode C-terminal membrane-spanning proteins of approximately 50 kDa with similar hydropathy profiles. Extensive sequence conservation exists in regions of the enzyme proposed to interact with its prenyl substrates (i.e., two farnesyl diphosphate molecules). Many of the highly conserved regions are also present in phytoene and prephytoene diphosphate synthetases, enzymes which catalyze prenyl substrate condensation reactions analogous to that of squalene synthetase. Expression of cDNA clones encoding S. pombe or hybrid human-S. cerevisiae squalene synthetases reversed the ergosterol requirement of S. cerevisiae cells bearing ERG9 gene disruptions, showing that these enzymes can functionally replace the S. cerevisiae enzyme. Inhibition of sterol synthesis in S. cerevisiae and S. pombe cells or in cultured human fibroblasts by treatment with the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor lovastatin resulted in elevated levels of squalene synthetase mRNA in all three cell types.     

Sequence of the Saccharomyces cerevisiae PHR1 gene and homology of the PHR1 photolyase to E. coli photolyase.

The nucleotide sequence of a 2301 base pair region of Saccharomyces cerevisiae DNA containing the PHR1 gene is reported. Within this region a single open reading frame of 1695 base pairs was found; using the insertional inactivation technique it was shown that part or all of this open reading frame specifies the PHR1-encoded photolyase. The amino acid sequence of the 565 amino acid long polypeptide predicted from the PHR1 nucleotide sequence was compared to the amino acid sequence of E. coli photolyase. Overall the sequence homology was 36.5%; however, two short regions near the amino terminus as well as the carboxy-terminal 150 amino acids display significantly greater sequence homology. The presence of these strongly conserved regions suggests that the yeast and E. coli photolyase possess common structural and functional domains involved in substrate and/or chromophore binding.     

Complementation of the DNA repair-deficient swi10 mutant of fission yeast by the human ERCC1 gene.

In human cells DNA damage caused by UV light is mainly repaired by the nucleotide excision repair pathway. This mechanism involves dual incisions on both sides of the damage catalyzed by two nucleases. In mammalian cells XPG cleaves 3' of the DNA lesion while the ERCC1-XPF complex makes the 5' incision. The amino acid sequence of the human excision repair protein ERCC1 is homologous with the fission yeast Swi10 protein. In order to test whether these proteins are functional homologues, we overexpressed the human gene in a Schizosaccharomyces pombe swi10 mutant. A swi10 mutation has a pleiotropic effect: it reduces the frequency of mating type switching (a mitotic transposition event from a silent cassette into the expression site) and causes increased UV sensitivity. We found that the full-length ERCC1 gene only complements the transposition defect of the fission yeast mutant, while a C-terminal truncated ERCC1 protein also restores the DNA repair capacity of the yeast cells. Using the two-hybrid system of Saccharomyces cerevisiae we show that only the truncated human ERCC1 protein is able to interact with the S . pombe Rad16 protein, which is the fission yeast homologue of human XPF. This is the first example yet known that a human gene can correct a yeast mutation in nucleotide excision repair.     

Histone gene organization of fission yeast: a common upstream sequence.

Histone genes of the fission yeast Schizosaccharomyces pombe were cloned from Charon 4A and cosmid gene libraries by hybridization, and their nucleotide sequences were determined. The genome of S. pombe has a single, isolated H2A, a pair of H2A-H2B and three pairs of H3-H4 (one H2B, two H2A and three each of H3 and H4). This non-assorted histone gene organization is distinct from that of the budding yeast which has two pairs of H2A-H2B and H3-H4. The predicted amino acid sequences of S. pombe histone H2As, H3s and H4s were identical except for three residue changes in H2As. Compared with those os S. cerevisiae and human, variable residues were clustered near the NH2- and COOH-terminal regions of H2A and H2B. Sequence homologies to the two organisms were roughly the same in H2A (79-83%), H3 (92-93%) and H4 (91%), but differed in H2B (82% to S. cerevisiae and 68% to human). The coding sequences in pairs of S. pombe histone genes were divergently directed. A 17-bp long highly homologous sequence (AACCCT box) that had internal 6-bp direct repeats was present in the intergene spacer sequences or in the 5' upstream region of all the cloned histone genes. A possible regulatory role of the common upstream sequence for histone gene expression is discussed.     

A chicken RAD51 homologue is expressed at high levels in lymphoid and reproductive organs.

Comparisons of the amino acid sequences of three yeast RecA-like proteins, Rad51 and DMC1 from S.cerevisiae and Rad51 from S.pombe, revealed several highly conserved regions. Degenerated oligonucleotides encoding two of these regions were used for the polymerase chain reaction to clone a chicken RecA-like gene. The encoded protein shares 68% and 49% identical amino acids with the Rad51 and DMC1 proteins. The strong sequence conservation between the yeast and chicken genes indicates that RecA homologues are conserved throughout evolution from prokaryotes to higher eukaryotes. High expression of the chicken Rad51 gene was found within the organs of lymphoid and germ cell development suggesting its involvement in lymphoid and meiotic recombination.     

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 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.     

Subunits of the Schizosaccharomyces pombe RNA polymerase II: enzyme purification and structure of the subunit 3 gene.

To improve our understanding of the structure and function of eukaryotic RNA polymerase II, we purified the enzyme from the fission yeast Schizosaccharomyces pombe. The highly purified RNA polymerase II contained more than eleven polypeptides. The sizes of the largest the second-, and the third-largest polypeptides as measured by SDS-polyacrylamide gel electrophoresis were about 210, 150, and 40 kilodaltons (kDa), respectively, and are similar to those of RPB1, 2, and 3 subunits of Saccharomyces cerevisiae RNA polymerase II. Using the degenerated primers designed after amino acid micro-sequencing of the 40 kDa third-largest polypeptide (subunit 3), we cloned the subunit 3 gene (rpb3) and determined its DNA sequence. Taken together with the sequence of parts of PCR-amplified cDNA, the predicted coding sequence of rpb3, interrupted by two introns, was found to encode a polypeptide of 297 amino acid residues in length with a molecular weight of 34 kDa. The S. pombe subunit 3 contains four structural domains conserved for the alpha-subunit family of RNA polymerase from both eukaryotes and prokaryotes. A putative leucine zipper motif was found to exist in the C-terminal proximal conserved region (domain D). Possible functions of the conserved domains are discussed.     

The cloning and characterization of a RAS gene fromSchizosaccharomyces pombe

We have cloned and determined the complete nucleotide sequence of a RAS gene from the yeast Schizosaccharomyces pombe (SP-RAS). The putative RAS protein of 214 amino acids is encoded by two noncontiguous reading frames separated by an intron of 86 bp. The SP-RAS gene product shares extensive homology with the proteins of the Saccharomyces cerevisiae (SC), Dictyostelium, Drosophila, and human RAS genes in its N-terminal region but not in its C-terminal region. The extended C-terminal regions found in the SC-RAS genes have no counterpart in the SP-RAS gene. Thus the RAS genes of these two yeasts are structurally quite distinct. The SP-RAS sequence was expressed in vivo.     

The nucleotide sequence of the yeast PHO5 gene: a putative precursor of repressible acid phosphatase contains a signal peptide.

The nucleotide sequence of the PHO5 gene of the yeast, Saccharomyces cerevisiae, which encodes repressible acid phosphatase (APase) was determined. Comparison of N-terminal amino acid sequence deduced from the nucleotide sequence with that of the purified repressible APase revealed the existence of a putative signal peptide in the precursor protein. The signal peptide was shown to contain 17 amino acid residues and its structural features were quite similar to those of higher eukaryotic and prokaryotic signal peptides. The nucleotide sequence of 5' and 3' noncoding flanking regions of the PHO5 gene are also discussed.     

Characterization of a second gene encoding gamma-glutamyl transpeptidase from Schizosaccharomyces pombe

The first gene encoding gamma-glutamyl transpeptidase (GGTI) of the fission yeast has previously been characterized, and its expression was found to be regulated by various oxidative stress-inducing agents. In this work, a second gene, encoding GGTII, was cloned and characterized from the fission yeast Schizosaccharomyces pombe. The structural gene encoding GGTII was amplified from the genomic DNA of the fission yeast and ligated into the shuttle vector pRS316 to generate the recombinant plasmid pPHJ02. The determined sequence contains 3040 bp and is able to encode the putative 611 amino acid sequence of GGTII, which resembles the counterparts of Saccharomyces cerevisiae, Homo sapiens, Rattus norvegicus, and Escherichia coli. The DNA sequence also contains 940-bp upstream and 289-bp downstream regions of the GGTII gene. The Schizosaccharomyces pombe cells harboring plasmid pPHJ02 showed about 4-fold higher GGT activity in the exponential phase than the cells harboring the vector only, indicating that the cloned GGTII gene is functional. The S. pombe cells containing the cloned GGTII gene were found to contain higher levels of both intracellular glutathione (GSH) content and GSH uptake. The S. pombe cells harboring plasmid pPHJ02 showed increased survival on solid media containing hydrogen peroxide, diethylmaleate, aluminum chloride, cadmium chloride, or mercuric chloride. The GGTII mRNA level was significantly elevated by treatment with GSH-depleting diethylmaleate. These results imply that the S. pombe GGTII gene produces functional GGTII protein and is involved in the response to oxidative stresses in S. pombe cells.     

The nucleotide sequence of the yeast MEL1 gene.

The complete nucleotide sequence of the MEL1 gene of the yeast, Saccharomyces cerevisiae, encoding alpha-galactosidase was determined. The nucleotide sequence contains an open reading frame of 1413 bp encoding a protein of 471 amino acids. Comparison with the known N-terminal amino acid sequence of the mature secreted protein indicated that alpha-galactosidase is synthesized as a precursor with an N-terminal signal sequence of 18 amino acids. The general features of this signal peptide resemble those of other yeast signal peptides. Molecular weight of the mature alpha-galactosidase polypeptide deduced from the nucleotide sequence is 50.049 kd. The 5' regulatory region has sequences in common with other yeast genes regulated by the GAL4-protein.