Cloning and sequencing of the HU-2 gene of Escherichia coli

Summary The Escherichia coli HU-2 gene was cloned using a DNA fragment from the HU-1 gene as a probe. The amino acid sequence of the HU-2 protein deduced from the nucleotide sequence is in good agreement with the published sequence. The nucleotide sequence has a possible promoter and a typical ribosomal binding site upstream of the translation initiation codon (AUG) and a possible rhoindependent terminater site downstream of the termination codon (UAA) of the gene.     

An analysis of nucleotide and amino acid variability in the barcode region of cytochrome c oxidase I (cox1) in fishes

The 655 bp cytochrome c oxidase subunit I barcode region of single specimens of 388 species of fishes (four Holocephali, 61 Elasmobranchii and 323 Actinopterygii) was examined. All but two (Urolophus cruciatus and Urolophus sufflavus) showed different cox1 nucleotide sequences (99.5% species discrimination); the two that could not be resolved are suspected to hybridize. Most of the power of cox1 nucleotide sequence analysis for species identification comes from the degenerate nature of the genetic code and the highly variable nature of the third codon position of amino acids. Variation at the third codon position is bimodally distributed, and the more variable mode is dominated by amino acids with four or six codons, while the less variable mode is dominated by amino acids with two codons. The ratio of nonsynonymous to synomymous changes is much less than one, indicating that this gene is subject to strong purifying selection. Consequently, cox1 amino acid sequence diversity is much less than nucleotide sequence diversity and has very poor species resolution power. Fourteen of the 16 amino acid residues recognized as having important functions in the region of cox1 sequenced were completely conserved over all 388 species (and the bovine cox1 sequence), with one fish species varying at one of these sites, and three fish at another site. No significant differences in amino acid conservation were observed between residues in helices, strands and turns. Patterns of nucleotide and amino acid variability were very similar between elasmobranchs and actinopterygians.     

Nucleotide sequence of the gene for Escherichia coli ribosomal protein S15 (rpsO)

Summary The nucleotide sequence of the ribosomal protein gene rpsO (S15) and its flanking region were determined. The amino acid sequence of S15 protein deduced from the nucleotide sequence is in good agreement with the published amino acid sequence with one exception. The nucleotide sequence shows two probable promoter sites about 100 nucleotides upstream from the initiation codon (AUG) of rpsO. Inspection of the sequence also revealed structural homology between the distal part of rpsO and the reported S15 binding region in 16S rRNA.     

Structure and expression of the gene encoding ribosomal protein S1 from the cyanobacterium Synechococcus sp. strain PCC 6301: striking sequence similarity to the chloroplast ribosomal protein CS1

We isolated a 38 kDa ssDNA-binding protein from the unicellular cyanobacterium Synechococcus sp. strain PCC 6301 and determined its N-terminal amino acid sequence. A genomic clone encoding the 38 kDa protein was isolated by using a degenerate oligonucleotide probe based on the amino acid sequence. The nucleotide sequence and predicted amino acid sequence revealed that the 38 kDa protein is 306 amino acids long and homologous to the nuclear-encoded 370 amino acid chloroplast ribosomal protein CS1 of spinach (48% identity), therefore identifying it as ribosomal protein (r-protein) S1. Cyanobacterial and chloroplast S1 proteins differ in size from Escherichia coli r-protein S1 (557 amino acids). This provides an additional evidence that cyanobacteria are closely related to chloroplasts. The Synechococcus gene rps1 encoding S1 is located 1.1 kb downstream from psbB, which encodes the photosystem 11 P680 chlorophyll a apoprotein. An open reading frame encoding a potential protein of 168 amino acids is present between psbB and rps1 and its deduced amino acid sequence is similar to that of E. coli hypothetical 17.2 kDa protein. Northern blot analysis showed that rps1 is transcribed as a monocistronic mRNA.     

The comparative amino acid sequences, substrate specificities and gene or cDNA nucleotide sequences of some prokaryote and eukaryote amidinotransferases: implications for evolution

The amino acid sequences of the amidinotransferases and the nucleotide sequences of their genes or cDNA from four Streptomyces species (seven genes) and from the kidneys of rat, pig, human and human pancreas were compared. The overall amino acid and nucleotide sequences of the prokaryotes and eukaryotes were very similar and further, three regions were identified that were highly identical. Evidence is presented that there is virtually zero chance that the overall and high identity regions of the amino acid sequence similarities and the overall nucleotide sequence similarities between Streptomyces and mammals represent random match. Both rat and lamprey amidinotransferases were able to use inosamine phosphate, the amidine group acceptor of Streptomyces. We have concluded that the structure and function of the amidinotransferases and their genes has been highly conserved through evolution from prokaryotes to eukaryotes. The evolution has occurred with: (1) a high degree of retention of nucleotide and amino acid sequences; (2) a high degree of retention of the primitive Streptomyces guanine+cytosine (G+C) third codon position composition in certain high identity regions of the eukaryote cDNA; (3) a decrease in the specificities for the amidine group acceptors; and (4) most of the mutations silent in the regions suggested to code for active sites in the enzymes.     

Structural characterization of protein secretion genes of the bacterial phytopathogen Xanthomonas campestris pathovar campestris: relatedness to secretion systems of other gram-negative bacteria

Summary The nucleotide sequence was determined of a 5.3 kb region of the Xanthomonas campestris pathovar campestris genome carrying a gene cluster encoding protein secretion and pathogenicity functions. A putative promoter sequence and five open reading frames (ORF) which may be part of an operon were revealed. The five predicted primary translation products comprise 531, 390, 147, 169 and 138 amino acids with M_r values of 58854, 42299, 15548, 18214 and 15108 respectively. A sixth, partial ORF is also present. Between ORF1 and ORF2 is a sequence of unknown function showing 7 by duplications. The deduced amino acid sequence of ORF1 is related to the Klebsiella pneumoniae PulE protein, to the Bacillus subtilis ComG ORF1 and to the Agrobacterium tumefaciens VirB ORF11 products. In addition, the deduced amino acid sequence of ORF2 showed homology to the Pu1F and to the ComG ORF2 products. The proteins encoded by ORF3, 4 and 5 showed amino acid homology to PulG, H and I products respectively. The proteins encoded by ORF2, 3, 4 and 5 showed significant hydrophobic domains which may represent membrane-spanning regions. By contrast the protein encoded by ORF1 was largely hydrophilic and had two putative nucleoside triphosphate binding sites.The nucleotide sequence data in this paper have been deposited in the EMBL, Genbank and DDBJ nucleotide sequence databases under the accession number X59079     

Spontaneous and reversible high-frequency frameshifts originating a phase transition in the carotenoid biosynthesis pathway of the phototrophic bacterium Rhodobacter sphaeroides 2.4.1

Summary The nucleotide sequence of the melB gene coding for the Na⁺(Li⁺)/melibiose symporter of Salmonella typhimurium LT2 was determined, and its amino acid sequence was deduced. It consists of 1428 bp, corresponding to a protein of 476 amino acid residues (calculated molecular weight 52800). The amino acid sequence is homologous to that of the melibiose permease of Escherichia coli K12, with 85% identical residues. All, except one, of the amino acid residues that have been reported to be important for cation or substrate recognition in the melibiose permease of E. coli are conserved in the melibiose permease of S. typhimurium. In addition, part of the sequence resembles the lactose permease of Streptococcus thermophilus, the animal glucose transporter (GLUT1), the plasmid-coded raffinose permease (RafB), and the NADH-ubiquinone oxidoreductase chain 4 (Nuo4) of Aspergillus amstelodami.The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBank/EMBL Data Bank with accession number X62101     

Similarity in Nucleotide Sequence of the Gene Encoding Nontoxic Component of Botulinum Toxin Produced by Toxigenic Clostridium butyricum Strain BL6340 and Clostridium botulinum Type E Strain Mashike

The complete nucleotide and deduced amino acid sequence of the nontoxic component of botulinum type E progenitor toxin is determined in recombinant plasmid pU9BUH containing about 6.0 kb HindIII fragment obtained from chromosomal DNA of Clostridium butyricum strain BL6340. The open reading frame (ORF) of this nontoxic component gene is composed of 3,486 nucleotide bases (1,162 amino acid residues). The molecular weight calculated from deduced amino acid residues is estimated 13,6810.1. The present study revealed that 33 nucleotide bases of 3,486 are different in the nontoxic component gene between C.butyricum strain BL6340 and C. botulinum type E strain Mashike. This corresponds to the difference of 17 amino acid residues in these nontoxic component.     

A dinucleotide mutation in dihydrodipicolinate synthase of Nicotiana sylvestris leads to lysine overproduction

By applying a mutagenesis/selection procedure to obtain resistance to a lysine analog, S-(2-aminoethyl)l -cysteine (AEC), a lysine overproducing mutant in Nicotiana sylvestris was isolated. Amino acid analyses performed throughout plant development and of different organs of the N. sylvestris RAEC-1 mutant, revealed a developmental-dependent accumulation of free lysine. Lysine biosynthesis in the RAEC-1 mutant was enhanced due to a lysine feedback-desensitized dihydrodipicolinate synthase (DHDPS). Several molecular approaches were undertaken to identify the nucleotide change in the dhdps-r1 gene, the mutated gene coding for the lysine-desensitized enzyme. The enzyme was purified from wild-type plants for amino end microsequencing and 10 amino acids were identified. Using dicotyledon dhdps probes, a genomic fragment was cloned from an enriched library of DNA from the homozygote RAEC-1 mutant plant. A dhdps cDNA, putatively full-length, was isolated from a tobacco cDNA library. Nucleotide sequence analyses confirmed the presence of the previously identified amino end preceded by a chloroplast transit peptide sequence. Nucleotide sequence comparisons, enzymatic and immunological analyses revealed that the tobacco cDNA corresponds to a normal type of DHDPS, lysine feedback-regulated, and the genomic fragment to the mutated DHDPS, insensitive to lysine inhibition. Functional complementation of a DHDPS-deficient Escherichia coli strain was used as an expression system. Reconstruction between the cDNA and genomic fragment led to the production of a cDNA producing an insensitive form of DHDPS. Amino acid sequence comparisons pointed out, at position 104 from the first amino acid of the mature protein, the substitution of Asn to lleu which corresponds to a dinucleotide mutation. This change is unique to the dhdps-r1 gene when compared with the wild-type sequence. The identification of the nucleotide and amino acid change of the lysine-desensitized DHDPS from RAEC-1 plant opens new perspectives for the improvement of the nutritional value of crops and possibly to develop a new plant selectable marker.     

Molecular Cloning and Expression of Proteinaceous α-Amylase Inhibitor Gene from Streptomyces nitrosporeus in Escherichia coli

The proteinaceous α-amylase inhibitor, T-76, gene was cloned by screening a Streptomyces nitrosporeus genomic library using a deoxyinosine-containing probe corresponding to the amino acid sequence of the inhibitor. The nucleotide sequence of the insert of a positive clone had an open reading frame of 330 bp that encoded a polypeptide of 110 amino acid residues with a calculated molecular mass of 11,306 daltons. The polypeptide begins with proximal basic amino acids and a region rich in hydrophobic amino acids that possibly act as a signal peptide for secretion, which is followed by a sequence consistent with the amino-terminal amino acid sequence of the T-76 inhibitor. Escherichia coli cells harboring the plasmid derivatives for expression produced the inhibitor in their periplasmic space. The amino-terminal sequence of the inhibitor produced by an E. coli transformant was identical to that of the T-76 inhibitor secreted by S. nitrosporeus. The amino acid sequence of the inhibitor deduced from nucleotide sequence showed significant homology to other proteinaceous α-amylase inhibitors.     

Phylogeny of nitrogenase sequences inFrankia and other nitrogen-fixing microorganisms

Summary The complete nucleotide sequence of a nitrogenase (nifH) gene was determined from a second strain (HRN18a) ofFrankia, an aerobic soil bacterium. The open reading frame is 870 bp long and encodes a polypeptide of 290 amino acids. The amino acid and nucleotide sequences were compared with 21 other published sequences. The twoFrankia strains were 96% similar at the amino acid level and 93% similar at the nucleotide level. A number of methods were used to infer phylogenies of these nitrogen fixers, based onnifH amino acid and nucleotide sequences. The results obtained do not agree completely with other phylogenies for these bacteria and thus make probable occurrences of lateral transfer of thenif genes. The time of divergence of the twoFrankia strains could be estimated at about 100 million years. The vanadium-dependent (Type 2) nitrogenase present inAzotobacter spp. appears to be a recent derivation from the conventional molybdenum-dependent (Type 1) enzyme, whereas the iron-dependent (Type 3) alternative nitrogenase would have a much older origin.     

Sequence analysis and HLA-DR genotyping of a novel HLA-DRw14 allele

Analysis of a Japanese population by oligonucleotide genotyping revealed that one Japanese HLA-DRw14 allele had a DRB1 genotype different from that of the known HLA-DRw14-related alleles, DRB1 ^* 1401 (DRw14-Dw9) and DRB1 ^* 1402 (DRw14-Dw16). The second exon of the DRB1 gene of the novel DRw14 allele (designated DRB1-14c) was amplified enzymatically and sequenced after cloning intto a plasmid vector. The amino acid sequence of the first domain in the DR1 chain encoded in the DRB1-14c allele was more similar to that of the DRB1 ^* 1401 allele (three amino acid substitutions).than to that of the DRB1 ^* 1402 allele (six amino acid substitutions). No polymorphic amino acid residue that could explain the common serologic HLA-DRw14 specificity was identified among the sequences of the three DRw14-related alleles. Sequence-specific oligonucleotides (SSOs) were synthesized on the basis of the DRB1-14c nucleotide sequence and used for genotyping of the Japanese population. These SSOs served as useful probes for identifying the DRB1-14c allele in a wide range of donors.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M33693.     

Nucleotide sequence of katG of Salmonella typhimurium LT2 and characterization of its product, hydroperoxidase I

Summary The nucleotide sequence of katG from Salmonella typhimurium was determined revealing an open reading frame of 2181 by that could encode a 727 amino acid protein. The predicted sequence of the encoded hydroperoxidase I (HPI) was found to be 90% similar to HPI from Escherichia coli and was one amino acid longer. The physical and enzymatic properties of HPI from both Salmonella typhimurium and Escherichia coli were found to be virtually identical despite the 10% divergence in sequence.     

Nucleotide Sequence of the Gene Encoding β-Isopropylmalate Dehydrogenase (leuB) from Bacteroides fragilis

The complete nucleotide sequence of the gene (leuB) coding for β-isopropylmaiate dehydrogenase of Bacteroides fragilis was determined. An open reading frame of 1,061 nucleotides was detected that could encode a polypeptide of 353 amino acid residues with a calculated molecular mass of 39,179 Da. The deduced amino acid sequence of the β-isopropylmalate dehydrogenase from B. fragilis showed substantial sequence similarity with the β-isopropylmalate dehydrogenases from other bacteria.     

The JAK2/STAT3 Signal Pathway Regulates the Expression of Genes Related to Skeletal Muscle Development and Energy Metabolism in Mice and Mouse Skeletal Muscle Cells

The gene encoding a β-galactosidase from Entevobacter cloacae GAO was cloned and expressed in Escherichia coli. The nucleotide sequence of the insert of a positive clone had an open reading frame of 3084 bp that encoded a polypeptide of 1028 amino acid residues with a calculated molecular mass of 116,677 daltons. The amino acid sequence of β-galactosidase deduced from the nucleotide sequence, especially the sequence around the putative active site and of the fourteen regions, showed significant homology to β-galactosidases of other microorganisms, E. coli, Klebsiella pneumoniae, Lactobacillus bulgaricus, and Clostridium acetobutylicum.     

Cloning and Sequence Analysis of a Protease-encoding Gene from the Marine Bacterium Alteromonas sp. Strain O-7

The gene (aprI) encoding alkaline serine protease (AprI; subtilase) from Alteromonas sp. strain O-7 was cloned and sequenced. The nucleotide sequence of aprI has been identified. The deduced amino acid sequence indicated that aprI codes for a precursor of 715 amino acids and the precursor is composed of four regions including a signal peptide, an N-terminal pro-region, a mature protease region and a C-terminal extension region of 215 amino acids as previously described for aprII [H. Tsujibo et al., Gene, 136, 247–251 (1993)]. The amino acid sequence of the mature AprI (AprI-M) showed high sequence homology with those of other class I subtilases. The C-terminal region was characterized by a repeat of 94 amino acids residues, which showed about 50% similarity with those of the C-terminal pro-region of several known proteases from Gram-negative bacteria.     

Nucleotide sequence of the virG locus of the Agrobacterium tumefaciens plasmid pTiC58

The nucleotide sequence of the virG locus of the nopaline type plasmid pTiC58 of Agrobacterium tumefaciens has been determined. It contains an open reading frame (ORF) of 759 nucleotides and has 77% homology to the virG sequences of octopine type plasmids. Differences between the sequences of the two types of Ti plasmids in the region of virG are located predominantly outside the ORF. The amino acid sequences inferred from the two virG genes show 80% homology to each other and each shows the same moderate homologies to amino acid sequences derived from genes in a family of two-component regulatory systems. Specific differences in nucleotide and amino acid sequences as well as a structure-function model for the gene product are discussed.     

Characterization of cDNA clones encoding the extrinsic 23 kDa polypeptide of the oxygen-evolving complex of photosystem II in pea

The 23 kDa polypeptide of the oxygen-evolving complex of photosystem II has been extracted from pea photosystem II particles by washing with 1 M NaCl and purified by anion-exchange chromatography. The N-terminal amino acid sequence has been determined and specific antisera have been raised in rabbits and used to screen a pea-leaf cDNA library in gt11. Determination of the nucleotide sequence of two clones provided the nucleotide sequence for the full 23 kDa polypeptide. The deduced amino acid sequence showed it to code for a mature protein of 186 amino acid residues with an N-terminal presequence of 73 amino acid residues showing a high degree of conservation with previously reported 23 kDa sequences from spinach and Chlamydomonas. Southern blots of genomic DNA from pea probed with the labelled cDNA gave rise to only one band suggesting that the protein is encoded by a single gene. Northern blots of RNA extracted from various organs indicated a message of approximately 1.1 kb, in good agreement with the size of the cDNA, in all chlorophyll-containing tissues. Western blots of protein extracted from the same organs indicated that the 23 kDa polypeptide was present in all major organs of the plant except the roots.Abbreviations bis-Tris bis (2-hydroxyethyl) imino-tris (hydroxymethyl)-methane - pfu plaque-forming units