The OPI1 gene of Saccharomyces cerevisiae, a negative regulator of phospholipid biosynthesis, encodes a protein containing polyglutamine tracts and a leucine zipper

In Saccharomyces cerevisiae, recessive mutations at the OPI1 locus result in constitutively derepressed expression of inositol 1-phosphate synthase, the product of the INO1 gene. Many of the other enzymes involved in phospholipid biosynthesis are also expressed at high derepressed levels in opi1 mutants. Thus, the OPI1 gene is believed to encode a negative regulator that is required to repress a whole subset of structural genes encoding for phospholipid biosynthetic enzymes. In this study, the OPI1 gene was mapped to chromosome VIII and cloned. When transformed into an opi1 mutant, the cloned DNA was capable of complementing the mutant phenotype and restoring correct regulation to the INO1 structural gene. Construction of two opi1 disruption alleles and subsequent genetic analysis of strains bearing these alleles confirmed that the cloned DNA was homologous to the genomic OPI1 locus. Furthermore, the OPI1 gene was found to be nonessential to the organism since mutants bearing the null allele were viable and exhibited a phenotype similar to that of previously isolated opi1 mutants. Similar to other opi1 mutants, the opi1 disruption mutants accumulated INO1 mRNA constitutively to a level 2-3-fold higher than that observed in wild-type cells. The cloned OPI1 gene was sequenced, and translation of the open reading frame predicted a protein composed of 404 amino acid residues with a molecular weight of 40,036. The predicted Opi1 protein contained a well defined heptad repeat of leucine residues that has been observed in other regulatory proteins. In addition, the predicted protein contained polyglutamine residue stretches which have also been reported in yeast genes having regulatory functions. Sequencing of opi1 mutant alleles, isolated after chemical mutagenesis, revealed that several were the result of a chain termination mutation located within the largest polyglutamine residue stretch.     

Biosynthesis of riboflavin: cloning, sequencing, and expression of the gene coding for 3,4-dihydroxy-2-butanone 4-phosphate synthase of Escherichia coli.

3,4-Dihydroxy-2-butanone 4-phosphate is biosynthesized from ribulose 5-phosphate and serves as the biosynthetic precursor for the xylene ring of riboflavin. The gene coding for 3,4-dihydroxy-2-butanone 4-phosphate synthase of Escherichia coli has been cloned and sequenced. The gene codes for a protein of 217 amino acid residues with a calculated molecular mass of 23,349.6 Da. The enzyme was purified to near homogeneity from a recombinant E. coli strain and had a specific activity of 1,700 nmol mg-1 h-1. The N-terminal amino acid sequence and the amino acid composition of the protein were in agreement with the deduced sequence. The molecular mass as determined by ion spray mass spectrometry was 23,351 +/- 2 Da, which is in agreement with the predicted mass. The previously reported loci htrP, "luxH-like," and ribB at 66 min of the E. coli chromosome are all identical to the gene coding for 3,4-dihydroxy-2-butanone 4-phosphate synthase, but their role had not been hitherto determined. Sequence homology indicates that gene luxH of Vibrio harveyi and the central open reading frame of the Bacillus subtilis riboflavin operon code for 3,4-dihydroxy-2-butanone 4-phosphate synthase.     

The cloning and nucleotide sequence of a Corynebacterium glutamicum 3-deoxy-d- arabinoheptulosonate-7-phosphate synthase gene

Abstract The aro gene of Corynebacterium glutamucum CCRC 18310 encoding 3-deoxy- d -arabinoheptulosonate-7-phosphate (DAHP) synthase was isolated by complementation of a DAHP synthase-deficient mutant of Escherichia coli AB3257. The specific activity of DAHP synthase was increased four-fold in a C. glutamicum strain harboring the cloned aro gene. The complete nucleotide sequence of the aro gene and 5' and 3' flanking regions has been determined. The sequence contained an open reading frame of 368 codons, from which a protein with a molecular mass of 39 340 Da could be predicted. The deduced amino acid sequence shows high identity with the aro gene products of E. coli and Salmonella typhimurium .     

Cloning, sequence analysis, and expression in Escherichia coli of a streptococcal plasmin receptor.

Plasmin(ogen) receptors are expressed by many gram-positive and gram-negative bacteria. We previously isolated a plasmin receptor from a pathogenic group A streptococcal strain (C. C. Broder, R. Lottenberg, G. O. von Mering, K. H. Johnston, and M. D. P. Boyle, J. Biol. Chem. 266:4922-4928, 1991). The gene encoding this plasmin receptor, plr, was isolated from a lambda gt11 library of chromosomal DNA from group A streptococcal strain 64/14 by screening plaques with antibodies raised against the purified streptococcal plasmin receptor protein. The gene was subcloned by using a low-copy-number plasmid and stably expressed in Escherichia coli, resulting in the production of an immunoreactive and functional receptor protein. The DNA sequence of the gene contained an open reading frame encoding 335 amino acids with a predicted molecular weight of 35,787. Upstream of the open reading frame, putative promoter and ribosomal binding site sequences were identified. The experimentally derived amino acid sequences of the N terminus and three cyanogen bromide fragments of the purified streptococcal plasmin receptor protein corresponded to the predicted sequence encoded by plr. The deduced amino acid sequence for the plasmin receptor protein revealed significant similarity (39 to 54% identical amino acid residues) to glyceraldehyde 3-phosphate dehydrogenases.     

Cloning and nucleotide sequence of the aroA gene of Bordetella pertussis.

The aroA locus of Bordetella pertussis, encoding 5-enolpyruvylshikimate 3-phosphate synthase, has been cloned into Escherichia coli by using a cosmid vector. The gene is expressed in E. coli and complemented an E. coli aroA mutant. The nucleotide sequence of the B. pertussis aroA gene was determined and contains an open reading frame encoding 442 amino acids, with a calculated molecular weight for 5-enolpyruvylshikimate 3-phosphate synthase of 46,688. The amino acid sequence derived from the nucleotide sequence shows homology with the published amino acid sequences of aroA gene products of other microorganisms.     

Cloning and sequence analysis of the gene encoding 3-hexulose-6-phosphate synthase from the methylotrophic bacterium, Methylomonas aminofaciens 77a, and its expression in Escherichia coli

Abstract A DNA fragment of 550 bp was specifically amplified by PCR with primers based on the N-terminal sequence of the purified 3-hexulose-6-phosphate synthase from Methylomonas aminofaciens 77a and on that of a lysyl endopep(idase-derived peptide. Using this PCR product as a probe, a gene coding for 3-hexulose-6-phosphate synthase in M. aminofaciens 77a chromosomal DNA was cloned in Escherichia coli JM109. Sequencing analysis revealed that the gene encoding 3-hexulose-6-phosphate synthase contained a 624-bp open reading frame, encoding a protein composed of 208 amino acid residues with a calculated relative molecular mass of 21 224.     

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.     

The product of the UL31 gene of herpes simplex virus 1 is a nuclear phosphoprotein which partitions with the nuclear matrix.

The nucleotide sequence of the UL31 open reading frame is predicted to encode a basic protein with a hydrophilic amino terminus and a nuclear localization signal. To identify its gene product, we constructed a viral genome in which the thymidine kinase gene was inserted between the UL31 and UL32 open reading frames. The thymidine kinase gene was then deleted, and in the process, the 5' terminus of the UL31 open reading frame was replaced with a 64-bp sequence in frame with the complete, authentic sequence of the UL31 open reading frame. The inserted sequence encoded a hydrophilic epitope derived from glycoprotein B of human cytomegalovirus and for which a monoclonal antibody is available. We report that in infected cells, the tagged protein localized in and was dispersed throughout the nucleus. Nuclear fractionation studies revealed that the UL31 protein partitions with the nuclear matrix. The protein is phosphorylated in infected cells maintained in medium containing 32Pi.     

Cloning and sequencing of the yeast gene for dolichol phosphate mannose synthase, an essential protein

Dolichol phosphate mannose (Dol-P-Man) synthase (EC 2.4.1.83) catalyzes the formation of Dol-P-Man from Dol-P and GDP-Man. The structural gene for yeast Dol-P-Man synthase (DPM1) was isolated by screening a yeast genomic DNA library for colonies that overexpressed Dol-P-Man synthase activity. This approach relied on a method to screen for Dol-P-Man synthase activity in lysed yeast colonies and used a yeast mutant with very low Dol-P-Man synthase activity in colony lysates. Transformants isolated using this technique expressed Dol-P-Man synthase activity 9-14-fold higher than that of a wild type strain, and all seven plasmids conferring this overproduction had a common region in their yeast genomic DNA insert. DPM1 is the structural gene for yeast Dol-P-Man synthase since Escherichia coli transformants harboring this gene express Dol-P-Man synthase activity in vitro. DNA sequencing of the DPM1 gene revealed an open reading frame of 801 bases. The 30-kDa size of the predicted protein is in excellent agreement with the size of the purified yeast enzyme (Haselbeck, A., and Tanner, W. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 1520-1524). Analysis of the predicted amino acid sequence reveals the protein has a potential membrane spanning domain of 25 amino acids at its COOH terminus. The protein's NH2 terminus, though not hydrophobic, meets existing criteria for yeast signal sequences, but there is no site for cleavage by signal peptidase. If the NH2 terminus is a functional signal sequence, the protein is predicted to be oriented toward the lumen of the endoplasmic reticulum with both NH2 and COOH termini serving as membrane anchors. If there is no signal sequence, the enzyme is predicted to face the cytoplasm and be anchored only by its COOH terminus. The DPM1 gene is essential for viability in yeast since disruption of the gene is lethal. We suspect Dol-P-Man synthase is not an essential protein due to its role in N-glycosylation since mutations in other genes that affect the late steps in lipid-linked oligosaccharide synthesis do not affect cell growth. Instead, DPM1 may be an essential gene because its product is required for O-glycosylation in yeast or because Dol-P-Man synthase is needed in some unidentified pathway.     

Cloning, expression, and functional characterization of the Dunaliella salina 5-enolpyruvylshikimate-3-phosphate synthase gene in Escherichia coli

5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase, EC 2.5.1.19) is the sixth enzyme in the shikimate pathway which is essential for the synthesis of aromatic amino acids and many secondary metabolites. The enzyme is widely involved in glyphosate tolerant transgenic plants because it is the primary target of the nonselective herbicide glyphosate. In this study, the Dunaliella salina EPSP synthase gene was cloned by RT-PCR approach. It contains an open reading frame encoding a protein of 514 amino acids with a calculated molecular weight of 54.6 KDa. The derived amino acid sequence showed high homology with other EPSP synthases. The Dunaliella salina EPSP synthase gene was expressed in Escherichia coli and the recombinant EPSP synthase were identified by functional complementation assay.     

The nucleotide and partial amino acid sequence of toxic shock syndrome toxin-1

The nucleotide sequence of toxic shock syndrome toxin-1 (TSST-1) has been determined. In addition, one-third of the predicted amino acid sequence was confirmed by amino acid sequence analysis of cyanogen bromide-generated TSST-1 protein fragments. The DNA sequencing results identified a 708-base pair open reading frame starting with an ATG, 7 base pairs downstream from a Shine-Dalgarno sequence, and terminating at a UAA stop codon. Amino acid analysis of the intact protein defined the NH2 terminus of the mature protein and located the cleavage point for the signal peptide (Ala/Ser). The signal peptide contained the first 40 amino acids and had characteristic structural similarities with other bacterial signal peptides. The coding sequence of the mature protein was 585 base pairs (194 amino acids) in length, and the molecular weight of the predicted protein was 22,049. This is in good agreement with the previously reported molecular weight of TSST-1 (22,000), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NH2-terminal amino acid sequence analysis performed on isolated TSST-1 CNBr fragments determined the position of the peptides in the TSST-1 sequence and verified the predicted amino acid sequence in those positions. Computer analyses of the amino acid sequence showed that TSST-1 has little or no sequence homology with biologically related toxins, streptococcal pyrogenic exotoxin A, and staphylococcal enterotoxins B and C.     

Nucleotide sequence of the glucoamylase gene GLU1 in the yeast Saccharomycopsis fibuligera.

The complete nucleotide sequence of the glucoamylase gene GLU1 from the yeast Saccharomycopsis fibuligera has been determined. The GLU1 DNA hybridized to a polyadenylated RNA of 2.1 kilobases. A single open reading frame codes for a 519-amino-acid protein which contains four potential N-glycosylation sites. The putative precursor begins with a hydrophobic segment that presumably acts as a signal sequence for secretion. Glucoamylase was purified from a culture fluid of the yeast Saccharomyces cerevisiae which had been transformed with a plasmid carrying GLU1. The molecular weight of the protein was 57,000 by both gel filtration and acrylamide gel electrophoresis. The protein was glycosylated with asparagine-linked glycosides whose molecular weight was 2,000. The amino-terminal sequence of the protein began from the 28th amino acid residue from the first methionine of the putative precursor. The amino acid composition of the purified protein matched the predicted amino acid composition. These results confirmed that GLU1 encodes glucoamylase. A comparison of the amino acid sequence of glucoamylases from several fungi and yeast shows five highly conserved regions. One homology region is absent from the yeast enzyme and so may not be essential to glucoamylase function.     

Cloning and expression of bovine brain inositol monophosphatase.

Inositol monophosphatase is a key enzyme of the inositol phosphate second messenger signaling pathway. It is responsible for the provision of inositol required for synthesis of phosphatidylinositol and polyphosphoinositides and has been implicated as the pharmacological target for lithium action in brain. Using oligonucleotide probes based on partial amino acid sequence data for the bovine brain enzyme, several overlapping cDNA clones of 2-3 kilobases in length have been isolated. All contain an open reading frame encoding a 277-amino acid protein. No significant sequence homology was found with any known protein. The open reading frame was inserted into a bacterial expression vector in order to confirm the presumed identity of the protein. The expressed protein reacted with an anti-inositol monophosphatase monoclonal antibody. In addition, the protein was enzymically active and indistinguishable from the bovine brain enzyme with respect to Km values for substrate and Li+ sensitivity of inositol 1-phosphate hydrolysis.     

Nucleotide sequence and expression of a Streptomyces griseosporeus proteinaceous alpha-amylase inhibitor (HaimII) gene.

The coding region of the alpha-amylase inhibitor (HaimII) gene from the producing strain Streptomyces griseosporeus YM-25 was localized on an 800-base-pair DNA segment. The nucleotide sequence of a 1,191-base-pair region including the HaimII gene was determined by the dideoxy-chain termination method. The nucleotide sequence data predicted an open reading frame of 363 base pairs starting with an ATG initiation codon and ending with a TGA translational stop codon. The amino acid sequence deduced from the nucleotide sequence indicated that the presumptive pre-HaimII protein extends 37 amino acids to the amino terminus and 6 amino acids to the carboxyl terminus of the mature HaimII protein. The pre-HaimII protein is believed to be processed both during and after secretion. Two forms of the inhibitor, which have a higher molecular weight than that of the HaimII protein isolated from S. griseosporeus, were partially purified from the culture filtrate of Streptomyces lividans containing the cloned HaimII gene.