Complete nucleotide sequence of the metapyrocatechase gene on the TOI plasmid of Pseudomonas putida mt-2

Metapyrocatechase which catalyzes the oxygenative ring cleavage of catechol to form alpha-hydroxymuconic epsilon-semialdehyde is encoded by the xylE gene on the TOL plasmid of Pseudomonas putida mt-2. We have cloned the xylE region in Escherichia coli and determined the nucleotide sequence of the DNA fragment of 985 base pairs around the gene. The fragment included only one open translational frame of sufficient length to accommodate the enzyme. The predicted amino acid sequence consisted of 307 residues, and its NH2- and COOH-terminal sequences were in perfect agreement with those of the enzyme recently determined (Nakai, C., Hori, K., Kagamiyama, H., Nakazawa, T., and Nozaki, M. (1983) J. Biol. Chem. 258, 2916-2922). A mutant plasmid was isolated which did not direct the synthesis of the active enzyme. This plasmid had a DNA region corresponding to the NH2-terminal two-thirds of the polypeptide. From the deduced amino acid sequence, the secondary structure was predicted. Around 10 base pairs upstream from the initiator codon for metapyrocatechase, there was a base sequence which was complementary to the 3'-end of 16 S rRNAs from both E.coli and Pseudomonas aeruginosa. A preferential usage of C- and G-terminated codons was found in the coding region xylE, which contributed to the relatively high G + C content (57%) of this gene.     

Nucleotide sequence of the 2,3-dihydroxybiphenyl dioxygenase gene of Pseudomonas pseudoalcaligenes.

2,3-Dihydroxybiphenyl dioxygenase, which catalyzes ring metacleavage of 2,3-dihydroxybiphenyl, is encoded by the bphC gene of Pseudomonas pseudoalcaligenes KF707 (K. Furukawa and T. Miyazaki, J. Bacteriol. 166:392-398, 1986). We determined the nucleotide sequence of a DNA fragment of 2,040 base pairs which included the bphC gene. The fragment included one open reading frame of 912 base pairs to accommodate the enzyme. The predicted processed amino acid sequence of the enzyme subunit consisted of 302 residues, and its 12 NH2-terminal residues were in perfect agreement with those determined for the enzyme. Approximately 10 base pairs upstream from the initiation codon for 2,3-dihydroxybiphenyl dioxygenase, there was a base sequence complementary to the 3' end of the 16S rRNA from Pseudomonas aeruginosa. There was no promoterlike sequence in the region upstream of the bphC gene, but another long open reading frame was present. A putative bphD gene encoding a metacleavage compound-hydrolyzing enzyme was suggested in the region downstream of the bphC gene.     

Nucleotide sequence of Escherichia coli purF and deduced amino acid sequence of glutamine phosphoribosylpyrophosphate amidotransferase

The Escherichia coli gene purF, coding for 5-phosphoribosylamine:glutamine pyrophosphate phosphoribosyltransferase (amidophosphoribosyltransferase) was subcloned from a ColE1-purF plasmid into pBR322. Amidophosphoribosyltransferase levels were elevated more than 5-fold in the ColE1-purF plasmid-bearing strain compared to the wild type control, and a further 10- to 13-fold elevation was observed in several pBR322 derivatives. The nucleotide sequence of a 2478-base pair PvuI-HinfI fragment encoding purF was determined. The purF45 structural gene codes for a 56,395 Mr protein chain having 504 amino acid residues. Methionine-1 is removed by processing in vivo leaving cysteine as the NH2-terminal residue. The deduced amino acid sequence was confirmed by comparisons with the NH2-terminal amino acid sequence determined by automated Edman degradation (Tso, J. Y., Hermodson, M. A., and Zalkin, H. (1982) J. Biol. Chem. 257, 3532-3536) and amino acid analyses of CNBr peptides including a 4-residue peptide from the CO2H terminus of the enzyme. Nucleotide sequences characteristic of bacterial promoter-operator regions were identified in the 5' flanking region. The coding region appears to be preceded by a 277-297 nucleotide mRNA leader. A deletion removing the putative promoter-operator region results in defective purF expression.     

Pyridoxal 5'-phosphate-dependent histidine decarboxylase. Nucleotide sequence of the hdc gene and the corresponding amino acid sequence

The nucleotide sequence of a 1.3-kilobase NaeI fragment from Morganella morganii AM-15 that contains the gene for histidine decarboxylase has been determined. The gene was initially identified among total chromosomal digests using a mixed sequence oligonucleotide probe corresponding to amino acids 11-16 of histidine decarboxylase and then cloned on a 5.5-kilobase PstI fragment. The structural gene contains 1131 nucleotides and encodes 377 amino acids with the sequence: (sequence: in text). The independently determined NH2-terminal sequence of this enzyme (Tanase, S., Guirard, B. M., and Snell, E. E. (1985) J. Biol. Chem. 260, 6738-6746) and the amino acid sequences of two tryptic peptides reported in the accompanying paper (Hayashi, H., Tanase, S., and Snell, E. E. (1986) J. Biol. Chem. 261, 11003-11009) are localized in the sequence presented here; the lysine that binds pyridoxal phosphate is situated at residue 232, whereas the serine that binds the adduct formed between pyridoxal phosphate and the inhibitor alpha-fluoromethylhistidine is positioned at residue 322.     

Molecular cloning and nucleotide sequence of a cDNA clone coding for the cell attachment domain in human fibronectin

A cDNA clone coding for the cell attachment domain in human fibronectin has been isolated using synthetic oligonucleotides. Three sets of mixed tetradecamer oligonucleotides were synthesized based on amino acid sequences in the 108-amino acid cell attachment domain (Pierschbacher, M. D., Ruoslahti, E., Sundelin, J., Lind, P., and Peterson, P. A. (1982) J. Biol. Chem. 257, 9593-9597). One of these sets was made complementary to amino acids located near the COOH terminus of the cell attachment domain and synthesized as a mixture of 24 sequences. This oligonucleotide mixture was used to prime cDNA synthesis with mRNA prepared from a human fibrosarcoma as a template. A cDNA library was constructed with the oligonucleotide-primed sequences in the vector pBR322. Colonies that hybridized with the primer were isolated from the library and further identified by hybridization with oligonucleotides deduced from an amino acid sequence located 45 amino acid residues NH2-terminal of the primer sequence. One clone which hybridized to both probes was characterized in detail. The insert was 380 base pairs long and its nucleotide sequence agreed completely with the corresponding amino acid sequence of human plasma fibronectin, showing that the sequences for this region are identical in plasma fibronectin and fibronectin from a cell line. This clone should be useful for studies on the expression of fibronectins and may also allow for the production of the biologically active cell attachment domain of fibronectin in bacteria.     

Complete nucleotide sequence of cDNA and deduced amino acid sequence of rat liver arginase

Arginase (EC 3.5.3.1) catalyzes the last step of urea synthesis in the liver of ureotelic animals. The nucleotide sequence of rat liver arginase cDNA, which was isolated previously (Kawamoto, S., Amaya, Y., Oda, T., Kuzumi, T., Saheki, T., Kimura, S., and Mori, M. (1986) Biochem. Biophys. Res. Commun. 136, 955-961) was determined. An open reading frame was identified and was found to encode a polypeptide of 323 amino acid residues with a predicted molecular weight of 34,925. The cDNA included 26 base pairs of 5'-untranslated sequence and 403 base pairs of 3'-untranslated sequence, including 12 base pairs of poly(A) tract. The NH2-terminal amino acid sequence, and the sequences of two internal peptide fragments, determined by amino acid sequencing, were identical to the sequences predicted from the cDNA. Comparison of the deduced amino acid sequence of the rat liver arginase with that of the yeast enzyme revealed a 40% homology.     

The nucleotide sequence of the chicken thymidine kinase gene and the relationship of its predicted polypeptide to that of the vaccinia virus thymidine kinase.

The entire DNA nucleotide sequence of a 3.0 kilobase pair Hind III fragment containing the chicken cytoplasmic thymidine kinase gene was determined. Oligonucleotide linker insertion mutations distributed throughout this gene and having known effects upon gene activity ( Kwoh , T.J., Zipser , D., and Wigler , M. 1983. J. Mol. Appl. Genet. 2, 191-200), were used to access regions of the Hind III fragment for sequencing reactions. The complete nucleotide sequence, together with the positions of the linker insertion mutations within the sequence, allows us to propose a structure for the chicken thymidine kinase gene. The protein coding sequence of the gene is divided into seven small segments (each less than 160 base pairs) by six small introns (each less than 230 base pairs). The proposed 244 amino acid polypeptide encoded by this gene bears strong homology to the vaccinia virus thymidine kinase. No homology with the thymidine kinases of the herpes simplex viruses was found.     

Nucleotide sequence analysis of the Pseudomonas putida PpG7 salicylate hydroxylase gene (nahG) and its 3'-flanking region

Gene nahG of naphthalene/salicylate catabolic plasmid NAH7 encodes a protein of molecular weight 45,000, salicylate hydroxylase. This enzyme catalyzes the formation of catechol from salicylate, a key intermediate in naphthalene catabolism. DNA sequence analysis of the 3.1-kilobase HindIII fragment containing the nahG locus reveals an open reading frame (ORF) of 1305 base pairs that corresponds to a protein of 434 amino acid residues. The predicted amino acid sequence of salicylate hydroxylase is in agreement with the molecular weight, NH2-terminal amino acid sequence, and total amino acid composition of the purified salicylate hydroxylase [You, I.-S., Murray, R. I., Jollie, D., & Gunsalus, I. C. (1990) Biochem. Biophys. Res. Commun. 169, 1049-1054]. The amino acid sequence between positions 8 and 37 of salicylate hydroxylase shows homology with known ADP binding sites of other FAD-containing oxidoreductases, thus confirming its biochemical function. The sequence of the Pseudomonas putida salicylate hydroxylase was compared with those of other similar flavoproteins. A small DNA segment (831 base pairs) disrupts the continuity of the known gene order nahG and nahH, the latter encoding catechol 2,3-dioxygenase. The complete nucleotide sequence of the intergenic region spanning genes nahG and nahH has been determined and its biological role proposed.     

Human microsomal xenobiotic epoxide hydrolase. Complementary DNA sequence, complementary DNA-directed expression in COS-1 cells, and chromosomal localization

A lambda gt11 expression library constructed from human liver mRNA was screened with an antibody against human microsomal xenobiotic epoxide hydrolase. The clone pheh32 contains an insert of 1742 base pairs with an open reading frame coding for a protein of 455 amino acids with a calculated Mr of 52,956. The nucleotide sequence is 77% similar to the previously reported rat xenobiotic epoxide hydrolase cDNA sequence. The deduced amino acid sequence of the human epoxide hydrolase is 80% similar to the previously reported rabbit and 84% similar to the deduced rat protein sequence. The NH2-terminal amino acids deduced from the human xenobiotic epoxide hydrolase cDNA are identical to the published 19 NH2-terminal amino acids of the purified human xenobiotic epoxide hydrolase protein. Northern blot analysis revealed a single mRNA band of 1.8 kilobases. Southern blot analysis indicated that there is only one gene copy/haploid genome. The human xenobiotic epoxide hydrolase gene was assigned to the long arm of human chromosome 1. Several restriction fragment length polymorphisms were observed with the human epoxide hydrolase cDNA. pheh32 was expressed as enzymatically active protein in cultured monkey kidney cells (COS-1).     

Nucleotide sequence of the Bacillus stearothermophilus alpha-amylase gene.

The nucleotide sequence of the Bacillus stearothermophilus alpha-amylase gene and its flanking regions was determined. An open reading frame was found, comprising a total of 1,647 base pairs (549 amino acids) and starting from a GUG codon as methionine. It was shown by NH2-terminal amino acid sequence analysis that the extracellular amylase consisted of 515 amino acid residues, which corresponded to a molecular weight of 58,779. Thus the NH2-terminal portion of the gene encodes 34 amino acid residues as a signal peptide. The amino acid sequence deduced from the alpha-amylase gene was fairly homologous (61%) with that of another thermostable amylase from Bacillus amyloliquefaciens.     

Cloning of the Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase gene in Escherichia coli. Nucleotide sequence determination and properties of the plasmid-encoded enzyme

The Bacillus subtilis gene encoding glutamine phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase) was cloned in pBR322. This gene is designated purF by analogy with the corresponding gene in Escherichia coli. B. subtilis purF was expressed in E. coli from a plasmid promoter. The plasmid-encoded enzyme was functional in vivo and complemented an E. coli purF mutant strain. The nucleotide sequence of a 1651-base pair B. subtilis DNA fragment was determined, thus localizing the 1428-base pair structural gene. A primary translation product of 476 amino acid residues was deduced from the DNA sequence. Comparison with the previously determined NH2-terminal amino acid sequence indicates that 11 residues are proteolytically removed from the NH2 terminus, leaving a protein chain of 465 residues having an NH2-terminal active site cysteine residue. Plasmid-encoded B. subtilis amidophosphoribosyltransferase was purified from E. coli cells and compared to the enzymes from B. subtilis and E. coli. The plasmid-encoded enzyme was similar in properties to amidophosphoribosyltransferase obtained from B. subtilis. Enzyme specific activity, immunological reactivity, in vitro lability to O2, Fe-S content, and NH2-terminal processing were virtually identical with amidophosphoribosyltransferase purified from B. subtilis. Thus E. coli correctly processed the NH2 terminus and assembled [4Fe-4S] centers in B. subtilis amidophosphoribosyltransferase although it does not perform these maturation steps on its own enzyme. Amino acid sequence comparison indicates that the B. subtilis and E. coli enzymes are homologous. Catalytic and regulatory domains were tentatively identified based on comparison with E. coli amidophosphoribosyltransferase and other phosphoribosyltransferase (Argos, P., Hanei, M., Wilson, J., and Kelley, W. (1983) J. Biol. Chem. 258, 6450-6457).     

Molecular cloning, nucleotide sequence and expression of the gene encoding prepro-polygalacturonaseII of Aspergillus niger

PolygalacturonaseII of Aspergillus niger was fragmented using CNBr and the NH2-terminal fragment and another fragment were partially sequenced. The polygalacturonaseII (pgaII) gene was then isolated by using an oligonucleotide mixture based on the internal amino acid sequence as a probe. The nucleotide sequence of the pgaII structural gene was determined. It was found that polygalacturonaseII is synthesized as a precursor having an NH2-terminal prepro-sequence of 27 amino acids. The cloned gene was used to construct polygalacturonaseII over-producing A. niger strains. PolygalacturonaseII was isolated from one such strain and was determined to be correctly processed and to be fully active.     

Isolation and amino acid sequence of insulins and C-peptides of European bison (Bison bonasus) and fox (Alopex lagopus)

Insulins and C-peptides were extracted and purified from bison and fox pancreatic glands. The insulins were reduced and pyridylethylated, and the derived A- and B-chains separated by HPLC. Amino acid sequence determinations of the pyridylethylated A- and B-chains proved bisontine insulin to be identical to bovine insulin and fox insulin to be identical to dog and porcine insulin. Bisontine C-peptide proved to be identical to bovine C-peptide. The isolated fox C-peptide comprises 23 amino acid residues and probably represents a major tryptic fragment of a larger C-peptide. The fox C-peptide fragment is identical to the dog C-peptide (9-31) except for residue 3 (residue 11 in the dog C-peptide), which is aspartic acid as compared with glutamic acid in the dog C-peptide.     

Nucleotide sequence of the G6-amylase gene from alkalophilic Bacillus sp. H-167

The nucleotide sequence of the G6-amylase gene from alkalophilic Bacillus sp. H-167 was determined. The open reading frame of the gene consisted of 2865 base pairs, encoding 955 amino acids. The NH2-terminal amino acid sequence analysis of the G6-amylase indicated that the enzyme had a single peptide of 33 amino acid residues and the mature enzyme was composed of 922 amino acids, giving a molecular mass of 102,598. Identity of the NH2-terminal amino acid sequences among each component of the multiform G6-amylase suggested the proteolytic processing of the COOH-terminal side of the enzyme. The DNA sequence and the deduced amino acid sequence of the G6-amylase gene showed no homology with those of other bacterial alpha-amylases although the consensus amino acid sequences of the active center were well conserved.     

Deletion of a highly conserved tetrapeptide sequence of the proinsulin connecting peptide (C-peptide) inhibits proinsulin to insulin conversion by transfected pituitary corticotroph (AtT20) cells

The biological function of the connecting peptide (C-peptide) of proinsulin is unknown. Comparison of all known C-peptide sequences reveals the presence of a highly conserved peptide sequence, Glu/Asp-X-Glu/Asp (X being a hydrophobic amino acid), adjacent to the Arg-Arg doublet at the B chain/C-peptide junction. Furthermore, the next amino acid in the C-peptide sequence is also acidic in many animal species. To test the possible involvement of this hydrophilic domain in insulin biosynthesis, we constructed a mutant of the rat proinsulin II gene lacking the first four amino acids of the C-peptide and expressed either the normal (INS) on the mutated (INSDEL) genes in the AtT20 pituitary corticotroph cell line. In both cases immunoreactive insulin (IRI) was stored by the cells and released upon stimulation by cAMP. In the INS expressing cells, the majority of IRI, whether stored or released in response to a secretagogue, was mature insulin. By contrast, most of the stored and releasable IRI in the INSDEL expressing cells appeared to be (mutant) proinsulin or conversion intermediate with little detectable native insulin. Release of the mutant proinsulin and/or conversion intermediates was stimulated by cAMP. These results suggest that the mutant proinsulin was appropriately targeted to secretory granules and released predominantly via the regulated pathway, but that the C-peptide deletion prevented its conversion to native insulin.     

Nucleotide sequence of the gene encoding the hydrogenase from Desulfovibrio vulgaris (Hildenborough)

The nucleotide sequence of the 4.7-kb SalI/EcoRI insert of plasmid pHV 15 containing the hydrogenase gene from Desulfovibrio vulgaris (Hildenborough) has been determined with the dideoxy chain-termination method. The structural gene for hydrogenase encodes a protein product of molecular mass 45820 Da. The NH2-terminal sequence of the enzyme deduced from the nucleic acid sequence corresponds exactly to the amino acid sequence determined by Edman degradation. The nucleic acid sequence indicates that a N-formylmethionine residue precedes the NH2-terminal amino acid Ser-1. There is no evidence for a leader sequence. The NH2-terminal part of the hydrogenase shows homology to the bacterial [8Fe-8S] ferredoxins. The sequence Cys-Ile-Xaa-Cys-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Cys-Pro-Xaa-Xaa-Ala-(Ile) occurs twice both in the hydrogenase and in [8Fe-8S] ferredoxins, where the Cys residues have been shown to coordinate two [4Fe-4S] clusters [Adman, E. T., Sieker, L. C. and Jensen, L. H. (1973) J. Biol. Chem. 248, 3987-3996]. These results, therefore, suggest that two electron-transferring ferredoxin-like [4Fe-4S] clusters are located in the NH2-terminal segment of the hydrogenase molecule. There are ten more Cys residues but it is not clear which four of these could participate in the formation of the third cluster, which is thought to be the hydrogen binding centre. Another gene, encoding a protein of molecular mass 13493 Da, was found immediately downstream from the gene for the 46-kDa hydrogenase. The nucleic acid sequence suggests that the hydrogenase and the 13.5-kDa protein belong to a single operon and are coordinately expressed. Since dodecylsulfate gel electrophoresis of purified hydrogenase indicates the presence of a 13.5-kDa polypeptide in addition to the 46-kDa component, it is proposed that the hydrogenase from D. vulgaris (Hildenborough) is a two-subunit enzyme.     

Biosynthesis of bacterial glycogen. Primary structure of Escherichia coli ADP-glucose synthetase as deduced from the nucleotide sequence of the glg C gene

The nucleotide sequence of the glg C gene of Escherichia coli K12, coding for ADP-glucose synthetase, has been determined. The structural gene consists of 1293 base pairs, which specify a protein of 431 amino acids. The amino acid sequence deduced from the DNA sequence is consistent with the known NH2-terminal amino acid sequence and the amino acid composition of ADP-glucose synthetase. The translation start of the structural gene of glycogen synthase, glg A, starts immediately after termination of the glg C gene.