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.     

Human RNA lariat debranching enzyme cDNA complements the phenotypes of Saccharomyces cerevisiae dbr1 and Schizosaccharomyces pombe dbr1 mutants

The cDNA encoding the human RNA lariat debranching enzyme (hDBR1) was identified and cloned by searching the Expressed Sequence Tag (EST) database and screening a HeLa cDNA library, based on predicted amino acid sequence homologies with the Saccharomyces cerevisiae, Schizosaccharomyces pombe and Caenorhabditis elegans debranching enzymes. The hDBR1 cDNA expressed in Escherichia coli showed debranching activity in vitro and was also shown to be functional in an interspecies specific complementation experiment. hDBR1 cDNA in a S.cerevisiae expression vector complemented the intron accumulation phenotype of a S.cerevisiae dbr1 null mutant. Integration of the cDNA for hDBR1 into the ura4 locus of S.pombe also complemented both the intron accumulation and slow growth phenotypes of a S.pombe dbr1 null mutant strain. Comparison of the amino acid sequence of hDBR1 with the other DBR protein sequences showed several conserved regions, with 40, 44 and 43% identity to the S.cerevisiae, S.pombe and C.elegans debranching enzymes, respectively.     

Isolation and characterization of the gene encoding yeast debranching enzyme.

Using a genetic screen aimed at identifying cellular factors involved in Ty1 transposition, we have identified a mutation in a host gene that reduces Ty1 transposition frequency. The mutant, dbr1, is also defective in the process of intron turnover. In dbr1 cells, excised introns derived from a variety of pre-mRNAs are remarkably stable and accumulate to levels exceeding that of the corresponding mRNA. The stable excised introns accumulate in the form of a lariat that is missing the linear sequences 3' of the branchpoint. The DBR1 gene has been isolated by complementation of the transposition phenotype. DBR1 is shown to encode debranching enzyme, an RNA processing activity that hydrolyzes the 2'-5' phosphodiester linkage at the branchpoint of excised intron lariats. In Saccharomyces cerevisiae, debranching enzyme plays a requisite role in the rapid turnover of excised introns, yet its function is not essential for viability.     

Cloning and sequencing of a human cDNA coding for dihydroorotate dehydrogenase by complementation of the corresponding yeast mutant.

Dihydroorotate dehydrogenase (DHOdehase, EC 1.3.3.1) catalyses the fourth enzymatic step in de novo pyrimidine biosynthesis. A truncated human cDNA encoding this enzyme was isolated from a HeLa cell cDNA library by functional complementation of a corresponding deletion mutant from the yeast, Saccharomyces cerevisiae. The complementing clone contained a 1.5-kb poly(A)(+)-tailed insert with a 1191-bp open reading frame, hybridising with a unique human mRNA of 1.6 kb. The deduced amino acid sequence has 54%, 46% and 42% identity with Arabidopsis thaliana, Schizosaccharomyces pombe and Escherichia coli DHOdehases, respectively. In contrast, it has only 21% identity with the S. cerevisiae enzyme, which probably reflects the cytosolic location of the enzyme in the latter organism.     

Relationship between RNA lariat debranching and Ty1 element retrotransposition

The Saccharomyces cerevisiae DBR1 gene encodes a 2'-5' phosphodiesterase that debranches intron RNA lariats following splicing. Yeast dbr1 mutants accumulate intron lariats and are also defective for mobility of the retrotransposons Ty1 and Ty3. We used a mutagenic PCR method to generate a collection of dbr1 mutant alleles to explore the relationship between the roles of DBR1 in transposition and debranching. Eight mutants defective for Ty1 transposition contained single amino acid changes in Dbr1p. Two mutations, G84A and N85D, are in a conserved phosphoesterase motif that is believed to be part of the active site of the enzyme, supporting a connection between enzymatic activity and Ty1 transposition. Two other mutations, Y68F and Y68D, occur at a potential phosphorylation site, and we have shown that Dbr1p is phosphorylated on tyrosine. We have developed an RNase protection assay to quantitate intron RNA accumulation in cells. The assay uses RNA probes that hybridize to ACT1 intron RNA. Protection patterns confirm that sequences from the 5' end of the intron to the lariat branch point accumulate in dbr1 mutants in a branched (lariat) conformation. RNase protection assays indicate that all of the newly generated dbr1 mutant alleles are also deficient for debranching, further supporting a role for 2'-5' phosphodiesterase activity in Ty1 transposition. A Ty1 element lacking most of its internal sequences transposes independently of DBR1. The existence of Dbr1p-dependent Ty1 sequences raises the possibility that Dbr1p acts on Ty1 RNA.     

Dolichol phosphate mannose synthase from the filamentous fungus Trichoderma reesei belongs to the human and Schizosaccharomyces pombe class of the enzyme

Dolichol phosphate mannose (DPM) synthase activity, which is required in N:-glycosylation, O-mannosylation, and glycosylphosphatidylinositol membrane anchoring of protein, has been postulated to regulate the Trichoderma reesei secretory pathway. We have cloned a T.reesei cDNA that encodes a 243 amino acid protein whose amino acid sequence shows 67% and 65% identity, respectively, to the Schizosaccharomyces pombe and human DPM synthases, and which lacks the COOH-terminal hydrophobic domain characteristic of the Saccharomyces cerevisiae class of synthase. The Trichoderma dpm1 (Trdpm1) gene complements a lethal null mutation in the S.pombe dpm1(+) gene, but neither restores viability of a S.cerevisiae dpm1-disruptant nor complements the temperature-sensitivity of the S. cerevisiae dpm1-6 mutant. The T.reesei DPM synthase is therefore a member of the "human" class of enzyme. Overexpression of Trdpm1 in a dpm1(+)::his7/dpm1(+) S.pombe diploid resulted in a 4-fold increase in specific DPM synthase activity. However, neither the wild type T. reesei DPM synthase, nor a chimera consisting of this protein and the hydrophobic COOH terminus of the S.cerevisiae DPM synthase, complemented an S.cerevisiae dpm1 null mutant or gave active enzyme when expressed in E.coli. The level of the Trdpm1 mRNA in T.reesei QM9414 strain was dependent on the composition of the culture medium. Expression levels of Trdpm1 were directly correlated with the protein secretory capacity of the fungus.     

Cloning and sequencing of the gene encoding the large subunit of glutathione synthetase of Schizosaccharomyces pombe

The gene for the large subunit of glutathione synthetase (EC 6.3.2.3) of Schizosaccharomyces pombe was cloned from a S. pombe genomic DNA library by complementation of cadmium hypersensitivity of a glutathione synthetase deficient mutant of S. pombe. A long open reading frame was found in the cloned DNA sequence. Amino acid sequence predicted from the long open reading frame coincided with amino acid sequences of peptides obtained by V8 protease digestion of the large subunit of the purified glutathione synthetase. The glutathione synthetase deficient mutant which harbored plasmids containing the glutathione synthetase large subunit gene exhibited glutathione synthetase activity higher than the activity in the wild type strain, though the plasmid did not contain the gene for the small subunit of the enzyme.     

Characterisation of the gptA gene,encoding UDP N-acetylglucosamine: dolichol phosphate N-acetylglucosaminylphosphoryl transferase,from the filamentous fungus,Aspergillus niger

The production of asparagine (N)-linked oligosaccharides is of vital importance in the formation of glycosylated proteins in eukaryotes and is mediated by the dolichol pathway. As part of studies to allow manipulation of this pathway, the gene coding for the production of the enzyme UDP N-acetylglucosamine: dolichol phosphate N-acetylglucosaminylphosphoryl transferase (GPT), catalysing the first step in the assembly of dolichol-linked oligosaccharides, was cloned from the filamentous fungus Aspergillus niger. Degenerate-PCR was used to amplify a 470-bp fragment of the gene, which was labelled as a probe to obtain a full-length clone from a genomic library of A. niger. This contained a 1557-bp open reading frame encoding a highly hydrophobic protein of 468 amino acids with a predicted molecular weight of 51.4 kDa. The gene contained two intron sequences and putative dolichol recognition sites (PDRSs) were present in the deduced amino acid sequence. Comparison with other eukaryotic GPTs revealed the A. niger GPT to share 45-47% identity with yeasts (Saccharomyces cerevisiae and Schizosaccharomyces pombe) and 41-42% identity with mammals (mouse, hamster, human). Nested-PCR of a cDNA library was used to confirm the position of an intron. A complete cDNA clone of A. niger gpt was obtained by employing a recombinant PCR approach. This was used to rescue a conditional lethal mutant of S. cerevisiae carrying a dysfunctional gpt gene by heterologous expression, confirming that the gpt genes from A. niger and S. cerevisiae are functionally equivalent.     

Isolation and characterization of the structural gene for secreted acid phosphatase from Schizosaccharomyces pombe

The Schizosaccharomyces pombe acid phosphatase structural gene (PHO 1) was isolated by complementation of an S. pombe acid phosphatase mutant with a wild type S. pombe DNA recombinant plasmid library. Northern analysis indicates that acid phosphatase is encoded by a 1.4-kilobase mRNA of which approximately 100 bases are 3'-poly(A). The gene contains no introns and the 3' and 5' untranslated regions are short. According to DNA and amino acid sequence data, the S. pombe acid phosphatase has a molecular weight of 50,600. An 18-amino acid sequence at the N terminus was found that is similar to previously identified signal peptides in other eukaryotic secretory proteins. This signal peptide is apparently removed during secretion, since it is absent in the mature secreted acid phosphatase. The gene can be induced 2--3-fold by starvation for phosphate. The signals required for this induction are contained on the isolated DNA clone. Although the gene can be expressed in Saccharomyces cerevisiae, secretion is abnormal.     

Cloning and sequencing of arg3 and arg11 genes of Schizosaccharomyces pombe on a 10-kb DNA fragment. Heterologous expression and mitochondrial targeting of their translation products.

The Schizosaccharomyces pombe arginine anabolic genes encoding ornithine carbamoyltransferase (arg3) and acetylglutamate kinase/acetylglutamyl-phosphate reductase (arg11) were cloned by functional complementation of S. pombe arg3 and arg11 mutant strains from S. pombe DNA genomic libraries. Restriction analysis and sequencing of the two clones showed that both genes are located on a common DNA fragment. The arg3 gene encodes a 327-amino-acid polypeptide presenting a strong identity to Saccharomyces cerevisiae and human ornithine carbamoyltransferases. The arg11 gene encodes a 884-amino-acid polypeptide. The acetylglutamate kinase and acetylglutamate-phosphate reductase domains have been defined by their identity with the S. cerevisiae ARG5,6 protein. The cloned arg11 gene from S. pombe does not complement an arg5,6 mutation in S. cerevisiae, nor does the ARG5,6 gene complement the S. pombe arg11- mutation. In contrast, both ornithine-carbamoyltransferase-encoding genes function in S. pombe. However, the S. pombe arg3 gene complements only weakly an arg3 S. cerevisiae strain, which is in agreement with the low level of expression of the S. pombe gene in S. cerevisiae. The subcellular localization of both ornithine carbamoyltransferases in the two yeasts indicates that, in contrast to the S. pombe enzyme, more than 95% of the S. cerevisiae enzyme remains in the S. pombe cytoplasm. The low expression of S. pombe ornithine carbamoyltransferases in S. cerevisiae did not allow its localization. The promoters of S. pombe arg3 and arg11 genes do not present striking similarities among themselves nor with the promoters of the equivalent genes of S. cerevisiae.     

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.     

The fission yeast rad22 gene, having a function in mating-type switching and repair of DNA damages, encodes a protein homolog to Rad52 of Saccharomyces cerevisiae.

The gene rad22 of the fission yeast Schizosaccharomyces pombe has a function in DNA repair and mating-type switching. We have cloned the rad22 gene from a genomic gene bank by functional complementation of the switching defect. An open reading frame coding for a putative protein of 469 amino acids was found by sequence analyses. The rad22 gene contains no intron. A region of 126 amino acids in the N-terminal half of the Rad22 protein has significant homologies (56% identity and 36% similarity) to the Rad52 protein of Saccharomyces cerevisiae. A rad22 disruption strain was constructed which seems to be inviable in a homothallic background. Southern blot analyses have shown that the rad22-67 mutant frequently gives rise to deletions in the mating-type region. These data indicate that the Rad22 protein has a function in the repair of DNA double-strand breaks.     

Cloning human pyrroline-5-carboxylate reductase cDNA by complementation in Saccharomyces cerevisiae.

Pyrroline-5-carboxylate reductase (EC 1.5.1.2) catalyzes the NAD(P)H-dependent conversion of pyrroline-5-carboxylate to proline. We cloned a human pyrroline-5-carboxylate reductase cDNA by complementation of proline auxotrophy in a Saccharomyces cerevisiae mutant strain, DT1100. Using a HepG2 cDNA library in a yeast expression vector, we screened 10(5) transformants, two of which gained proline prototrophy. The plasmids in both contained similar 1.8-kilobase inserts, which when reintroduced into strain DT1100, conferred proline prototrophy. The pyrroline-5-carboxylate reductase activity in these prototrophs was 1-3% that of wild type yeast, in contrast to the activity in strain DT1100 which was undetectable. The 1810-base pair pyrroline-5-carboxylate reductase cDNA hybridizes to a 1.85-kilobase mRNA in samples from human cell lines and predicts a 319-amino acid, 33.4-kDa protein. The derived amino acid sequence is 32% identical with that of S. cerevisiae. By genomic DNA hybridization analysis, the human reductase appears to be encoded by a single copy gene which maps to chromosome 17.     

The Schizosaccharomyces pombe cho1+ gene encodes a phospholipid methyltransferase.

The isolation of mutants of Schizosaccharomyces pombe defective in the synthesis of phosphatidylcholine via the methylation of phosphatidylethanolamine is reported. These mutants are choline auxotrophs and fall into two unlinked complementation groups, cho1 and cho2. We also report the analysis of the cho1+ gene, the first structural gene encoding a phospholipid biosynthetic enzyme from S. pombe to be cloned and characterized. The cho1+ gene disruption mutant (cho1Delta) is viable if choline is supplied and resembles the cho1 mutants isolated after mutagenesis. Sequence analysis of the cho1+ gene indicates that it encodes a protein closely related to phospholipid methyltransferases from Saccharomyces cerevisiae and rat. Phospholipid methyltransferases encoded by a rat liver cDNA and the S. cerevisiae OPI3 gene are both able to complement the choline auxotrophy of the S. pombe cho1 mutants. These results suggest that both the structure and function of the phospholipid N-methyltransferases are broadly conserved among eukaryotic organisms.     

The mitochondrial genome of the fission yeast Schizosaccharomyces pombe: highly homologous introns are inserted at the same position of the otherwise less conserved cox1 genes in Schizosaccharomyces pombe and Aspergillus nidulans.

The DNA sequence of the second intron in the mitochondrial gene for subunit 1 of cytochrome oxidase (cox1), and the 3'' part of the structural gene have been determined in Schizosaccharomyces pombe. Comparing the presumptive amino acid sequence of the 3'' regions of the cox1 genes in fungi reveals similarly large evolutionary distances between Aspergillus nidulans, Saccharomyces cerevisiae and S. pombe. The comparison of exon sequences also reveals a stretch of only low homology and of general size variation among the fungal and mammalian genes, close to the 3'' ends of the cox1 genes. The second intron in the cox1 gene of S. pombe contains an open reading frame, which is contiguous with the upstream exon and displays all characteristics common to class I introns. Three findings suggest a recent horizontal gene transfer of this intron from an Aspergillus type fungus to S. pombe. (i) The intron is inserted at exactly the same position of the cox1 gene, where an intron is also found in A. nidulans. (ii) Both introns contain the highest amino acid homology between the intronic unassigned reading frames of all fungi identified so far (70% identity over a stretch of 253 amino acids). However, in the most homologous region, a GC-rich sequence is inserted in the A. nidulans intron, flanked by two direct repeats of 5 bp. The 37-bp insert plus 5 bp of direct repeat amounts to an extra 42 bp in the A. nidulans intron. (iii) TGA codons are the preferred tryptophan codons compared with TGG in all mitochondrial protein coding sequences of fungi and mammalia.(ABSTRACT TRUNCATED AT 250 WORDS)