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PubMed1981

Nucleotide sequence coding for the respiratory NADH dehydrogenase of Escherichia coli. UUG initiation codon.

The nucleotide sequence of the structural gene coding for the respiratory NADH dehydrogenase of Escherichia coli has been determined by the chain-termination method. The reading frame for the protein starts with the unusual initiation codon UUG and predicts an amino acid sequence of 434 residues (Mr = 47 304). The reading frame was confirmed by protein chemical studies including determination of the N-terminal sequence of the protein. The product made in vivo was found to have threonine as its N-terminal residue, indicating that the initiating N-formylmethionine had been removed by post-translational processing.

Amino Acid Sequence↗
PubMed2002

Compensation for nucleotide bias in a genome by representation as a discrete channel with noise.

MOTIVATION: Calculation of the information content of motifs in genomes highly biased in nucleotide composition is likely to lead to overestimates of the amount of useful information in the motif. Calculating relative information can compensate for biases, however the resulting information content is the amount seen by an observer and not by a macromolecule binding to the motif. The latter is needed to calculate the discriminatory power of the motif and to compare motifs between species. RESULTS: By treating a biased genome as a discrete channel with noise, in accordance with Shannon Information Theory, we were able to remove both 'Distortion' and 'Noise' from the motif and recover a more instructive biological 'signal.' A Java application, LogoPaint, was developed to remove nucleotide bias distortion and triplet frequency noise from motifs, calculate information content and present the motif as a logo. We demonstrate how this technique can 'unmask' motifs in the translation initiation regions of bacteria that are obscured by strong sequence biases. AVAILABILITY: LogoPaint is available to all users from the authors as an executable JAR file. Source code is available by arrangement.

Algorithms↗
PubMed1999

Complete sequence of the mitochondrial DNA of the red alga Porphyra purpurea. Cyanobacterial introns and shared ancestry of red and green algae.

The mitochondrial DNA (mtDNA) of Porphyra purpurea, a circular-mapping genome of 36,753 bp, has been completely sequenced. A total of 57 densely packed genes has been identified, including the basic set typically found in animals and fungi, as well as seven genes characteristic of protist and plant mtDNAs and specifying ribosomal proteins and subunits of succinate:ubiquinone oxidoreductase. The mitochondrial large subunit rRNA gene contains two group II introns that are extraordinarily similar to those found in the cyanobacterium Calothrix sp, suggesting a recent lateral intron transfer between a bacterial and a mitochondrial genome. Notable features of P. purpurea mtDNA include the presence of two 291-bp inverted repeats that likely mediate homologous recombination, resulting in genome rearrangement, and of numerous sequence polymorphisms in the coding and intergenic regions. Comparative analysis of red algal mitochondrial genomes from five different, evolutionarily distant orders reveals that rhodophyte mtDNAs are unusually uniform in size and gene order. Finally, phylogenetic analyses provide strong evidence that red algae share a common ancestry with green algae and plants.

Amino Acid Sequence↗
PubMed1983

The nucleotide sequence of the rho gene of E. coli K-12.

We have determined the nucleotide sequence of the rho gene which encodes the E. coli K-12 transcription termination factor. The structural gene was located on a cloned 3.6 kilobase BglII-HindIII restriction fragment by the introduction of the insertion element gamma delta and analysis of the recombinant plasmids by restriction analysis and in maxicells. The coding region consists of 1260 nucleotides directing the synthesis of a polypeptide 419 amino acids in length with a calculated molecular weight of 46,094. The deduced amino acid composition, amino-terminal protein sequence and calculated molecular weight are consistent with the data from the analysis of purified rho protein (16). We have shown that the rho genes from E. coli K-12, B and C strains are located on PvuII-HindIII fragments of the same size by hybridization to the rho (K-12) coding sequences.

Amino Acid Sequence↗
PubMed2005

Mitochondrial genomes of two demosponges provide insights into an early stage of animal evolution.

Mitochondrial DNA (mtDNA) of multicellular animals (Metazoa) is typically a small ( approximately 16 kbp), circular-mapping molecule that encodes 37 tightly packed genes. The structures of mtDNA-encoded transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs) are usually highly unorthodox, and proteins are translated with multiple deviations from the standard genetic code. In contrast, mtDNA of the choanoflagellate Monosiga brevicollis, the closest unicellular relative of animals, is four times larger, contains 1.5 times as many genes, and lacks mentioned peculiarities of animal mtDNA. To investigate the evolutionary transition that led to the specific organization of metazoan mtDNA, we determined complete mitochondrial sequences from the demosponges Geodia neptuni and Tethya actinia, two representatives of the most basal animal phylum, the Porifera. We found that poriferan mtDNAs resemble those of other animals in their compact organization, lack of introns, and a well-conserved animal-like gene order. Yet, they contain several extra genes, encode bacterial-like rRNAs and tRNAs, and use a minimally derived genetic code. Our findings suggest that the evolution of the typical metazoan mtDNA has been a multistep process in which the compact genome organization and the reduced gene content were established prior to the reduction of tRNA and rRNA structures and the introduction of multiple changes of the translation code.

Amino Acid Sequence↗
PubMed1999

Characterization and binding of intracellular antibody fragments to the hepatitis C virus core protein.

The monoclonal antibody C7-50 binds to the HCV core protein with high sensitivity and specificity. The coding sequences of the variable domains of the antibody were determined following cDNA cloning of the Fab and sFv fragments. Subsequently, intracellular expression and binding of these antibody fragments to the HCV core protein as a potential antiviral approach were studied. There was high specificity and sensitivity of binding of bacterially expressed, recombinant C7-50 Fab to HCV core as measured by EIA and immunoblot. For expression in mammalian cells, the C7-50 antibody was subcloned in the sFv format by the introduction of a (Gly(4)Ser)(3) linker spaced between light and heavy chains. Northern and Western blot analysis as well as confocal microscopy established the targeted expression of the C7-50 sFv antibody fragment in the endoplasmic reticulum of transfected cells. The colocalization and intracellular binding of the antibody fragment to HCV core protein was confirmed by immunoprecipitation and subsequent immunoblot analysis. This study demonstrates that gene delivery of cDNA coding sequences inducing intracellular expression of C7-50 antibody fragments leads to binding of the antibody fragment to the HCV core protein within the secretory compartment of transfected cells. Intracellular immunization represents a promising antiviral approach to interfere with the life cycle of HCV.

Amino Acid Sequence↗
PubMed1988

Synthesis and expression in Escherichia coli of the gene encoding monocyte-derived neutrophil-activating factor: biological equivalence between natural and recombinant neutrophil-activating factor.

The neutrophil-activating factor (NAF) purified from the conditioned medium of lipopolysaccharide-stimulated human monocytes was sequenced and found to consist of 72 amino acids: SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRA ENS. Purified preparations of natural NAF contained, in addition to this main form, minor amounts of three amino-terminal variants with 77 (+AVLPR), 70, and 69 residues. A gene coding for the 72-amino acid NAF was synthesized, cloned, and expressed in Escherichia coli. Western (immunologic) blot analysis of crude bacterial extracts, with an antiserum raised against natural NAF, revealed a single band that comigrated with natural NAF. Recombinant NAF purified to homogeneity had identical amino- and carboxyl-terminal sequences to the 72-amino acid natural NAF. Recombinant NAF was tested on human neutrophils and had the same activity and potency as natural NAF in inducing chemotaxis, rapidly increasing cytosolic free Ca2+, activating the respiratory burst, and releasing specific and azurophilic granular contents.

Amino Acid Sequence↗
PubMed2001

Human calcitonin receptor-like receptor for adrenomedullin: genomic structure, eight single-nucleotide polymorphisms, and haplotype analysis.

Adrenomedullin (ADM), a peptide characterized by persistent hypotensive activity, is thought to be involved when the control mechanism of blood pressure is deranged, because its plasma concentration is upregulated in hypertensive patients. The receptor for ADM, a molecular complex consisting of calcitonin-receptor-like receptor (CRLR) and receptor-activity-modifying protein 2 (RAMP2), is activated through a unique intracellular transport mechanism. By analyzing the nucleotide sequences of bacterial artificial chromosome (BAC) clones, we have established that the gene encoding CRLR is spread over a genomic distance of 103,145 bases; it contains 15 exons interrupted by 14 introns, including 1 that spans more than 60 kilobases. Exons 1-3 constitute the 5' noncoding region; exons 4 through 15 are coding elements, of which exons 8 to 14 encode seven transmembrane domains. Eight novel single-nucleotide polymorphisms (SNPs) and their allelic frequencies in the Japanese population were found by direct sequencing of 32 alleles; two SNPs were in the 5' flanking region, one in exon 2, and the other five around intron-exon junctions. Eight haplotypes were constructed using these alleles in our Japanese population sample. The data establish a basis for investigations to detect molecular variants in the ADM receptor that might alter control of blood pressure and confer on individuals a predisposition to essential hypertension.

Alleles↗
PubMed1987

Expression of porcine pancreatic phospholipase A2. Generation of active enzyme by sequence-specific cleavage of a hybrid protein from Escherichia coli.

The cDNA coding for the porcine pancreatic prophospholipase A2 (proPLA) has been cloned and expressed in E. coli. Expression of proPLA could only be obtained in the form of intracellular aggregates after fusing the 15 kDa proPLA to a large (greater than or equal to 45 kDa) bacterial peptide. The fusion protein was readily purified from cell lysates, and specifically cleaved. Cleavage of the fusion protein was achieved with either hydroxylamine (at Asn/Gly sequences in the denatured protein), or trypsin (between the pro- and the mature PLA in the renatured fusion protein). The former method releases a proPLA-like enzyme, while the latter directly yields PLA. Renaturation of the fusion protein was made possible by the use of a recently reported new S-sulphonation method. The released (pro)PLA was purified (yields of 2-3 mg/ltr of culture medium), and showed identical properties compared to native (pro)PLA.

Amino Acid Sequence↗
PubMed1994

Molecular characterization of an aldehyde/alcohol dehydrogenase gene from Clostridium acetobutylicum ATCC 824.

A gene (aad) coding for an aldehyde/alcohol dehydrogenase (AAD) was identified immediately upstream of the previously cloned ctfA (J. W. Cary, D. J. Petersen, E. T. Papoutsakis, and G. N. Bennett, Appl. Environ. Microbiol. 56:1576-1583, 1990) of Clostridium acetobutylicum ATCC 824 and sequenced. The 2,619-bp aad codes for a 96,517-Da protein. Primer extension analysis identified two transcriptional start sites 83 and 243 bp upstream of the aad start codon. The N-terminal section of AAD shows homology to aldehyde dehydrogenases of bacterial, fungal, mammalian, and plant origin, while the C-terminal section shows homology to alcohol dehydrogenases of bacterial (which includes three clostridial alcohol dehydrogenases) and yeast origin. AAD exhibits considerable amino acid homology (56% identity) over its entire sequence to the trifunctional protein encoded by adhE from Escherichia coli. Expression of aad from a plasmid in C. acetobutylicum showed that AAD, which appears as a approximately 96-kDa band in denaturing protein gels, provides elevated activities of NADH-dependent butanol dehydrogenase, NAD-dependent acetaldehyde dehydrogenase and butyraldehyde dehydrogenase, and a small increase in NADH-dependent ethanol dehydrogenase. A 957-bp open reading frame that could potentially encode a 36,704-Da protein was identified upstream of aad.

Alcohol Dehydrogenase↗
PubMed1991

The genes coding for the antigen 85 complexes of Mycobacterium tuberculosis and Mycobacterium bovis BCG are members of a gene family: cloning, sequence determination, and genomic organization of the gene coding for antigen 85-C of M. tuberculosis.

A gene encoding the 33-kDa secreted protein of Mycobacterium tuberculosis (antigen 85-C) was isolated and sequenced. The corresponding DNA sequence contains a 1,020-bp coding region. The deduced amino acid sequence corresponds to a 340-residue protein consisting of a 46-amino-acid signal peptide and a 294-amino-acid mature protein. Comparison with previously described genes for the 30-kDa antigen (the alpha antigen of M. bovis BCG, also called antigen 85-B) and the 32-kDa antigens from M. bovis BCG and M. tuberculosis (antigens 85-A) indicates that the three genes share considerable sequence homology (70.8 to 77.5%) but may also code for distinctive epitopes. Strong differences among the three sequences are clearly visible upstream and downstream from the region coding for the mature proteins. The three genes have been detected in the genome of M. bovis BCG by Southern blot hybridization with three type-specific probes. Furthermore, hybridization of large DNA fragments (100 to 1,000 kbp) from M. tuberculosis separated by pulsed-field gel electrophoresis showed that the three genes coding for the antigen 85 complex are not clustered within the bacterial genome.

Amino Acid Sequence↗
PubMed1986

The nucleotide sequence of the Escherichia coli K12 dnaJ+ gene. A gene that encodes a heat shock protein.

The Escherichia coli dnaJ gene product is required for bacteriophage lambda DNA replication at all temperatures. It is also essential for bacterial viability in at least some conditions, since mutations in it result in temperature-sensitive bacterial growth. We have previously cloned the dnaJ gene and shown that its product migrates as a Mr 37,000 polypeptide under denaturing conditions. Here we present the primary DNA sequence of the dnaJ gene. It codes for a processed basic protein (63 basic and 51 acidic amino acids) composed of 375 amino acids totaling Mr 40,973. The predicted NH2-terminal amino acid sequence, overall amino acid composition, and isoelectric point agree well with those of the purified protein. We present evidence that the rate of expression of the dnaJ protein is increased by heat shock under the control of the htpR (rpoH) gene product.

Amino Acid Sequence↗
PubMed2005

Contact with host cells induces a DNA repair system in pathogenic Neisseriae.

DNA repair systems play a major role in maintaining the integrity of bacterial genomes. Neisseria meningitidis, a human pathogen capable of colonizing the human nasopharynx, possesses numerous DNA repair genes but lacks inducible DNA repair systems such as the SOS response, present in most bacteria species. We recently identified a set of genes upregulated by contact with host cells. An open reading frame having high homology with the small subunit of Escherichia coli exonuclease VII (xseB) belongs to this regulon. The increased sensitivity of a mutant in this coding sequence to UV irradiation, alkylating agent and nalidixic acid demonstrates the participation of this gene in meningococcal DNA repair. In addition, the upregulation of the transcription of this open reading frame upon interaction of N. meningitidis with host cells increased not only the bacterial ability to repair its DNA but also the rate of phase variation by frameshifting. Together these data demonstrate that N. meningitidis possesses an inducible DNA repair system that might be used by the bacteria to adapt to its niches when it is colonizing a new host.

Bacterial Proteins↗
PubMed1995

The hrp gene locus of Pseudomonas solanacearum, which controls the production of a type III secretion system, encodes eight proteins related to components of the bacterial flagellar biogenesis complex.

Five transcription units of the Pseudomonas solanacearum hrp gene cluster are required for the secretion of the HR-inducing PopA1 protein. The nucleotide sequences of two of these, units 1 and 3, have been reported. Here, we present the nucleotide sequence of the three other transcription units, units 2, 4 and 7, which are together predicted to code for 15 hrp genes. This brings the total number of Hrp proteins encoded by these five transcription units to 20, including HrpB, the positive regulatory protein, and HpaP, which is apparently not required for plant interactions. Among the 18 other proteins, eight belong to protein families regrouping proteins involved in type III secretion pathways in animal and plant bacterial pathogens and in flagellum biogenesis, while two are related solely to proteins involved in secretion systems. For the various proteins found to be related to P. solanacearum Hrp proteins, those in plant-pathogenic bacteria include proteins encoded by hrp genes. For Hrp-related proteins of animal pathogens, those encoded by the spa and mxi genes of Shigella flexneri and of Salmonella typhimurium and by the ysc genes of Yersinia are involved in type III secretion pathways. Proteins involved in flagellum biogenesis, which are related to Hrp proteins of P. solancearum, include proteins encoded by fli and flh genes of S. typhimurium, Bacillus subtilis and Escherichia coli and by mop genes of Erwinia carotovora. P. solanacearum Hrp proteins were also found to be related to proteins of Rhizobium fredii involved in nodulation specificity.

Amino Acid Sequence↗
PubMed1986

A gene in Paracoccus for subunit III of cytochrome oxidase.

The region of Paracoccus denitrificans chromosome where the genes coding for cytochrome oxidase (cytochrome aa3) subunits are located has been cloned. DNA sequencing revealed an open reading frame that codes for a protein homologous to the subunit III of the eukaryotic, mitochondrial enzyme. This subunit is absent from the isolated Paracoccus oxidase. It now seems that it is part of the native enzyme in the bacterial cytoplasmic membrane. This may explain the observed discrepancies in the function of the isolated enzyme.

Base Sequence↗
PubMed1996

Cloning and sequencing of the gene encoding alpha-acetolactate decarboxylase from Leuconostoc oenos.

The alsD gene encoding alpha-acetolactate decarboxylase was isolated from a genomic library of Leuconostoc oenos, using a screening procedure developed on microtiter plates. The nucleotide sequence of alsD encodes a putative protein of 239 amino acids showing significant similarity with other bacterial alpha-acetolactate decarboxylases. Upstream from alsD lies an open reading frame (alsS) which is highly similar to bacterial genes coding for catabolic alpha-acetolactate synthases. Northern (RNA) blotting analyses indicated the presence of a 2.4-kb dicistronic transcript of alsS and alsD. This suggests that the alsS and alsD genes are organized in a single operon.

Acetolactate Synthase↗
PubMed1998

Cloning and expression of a marine bacterial beta-galactoside alpha2,6-sialyltransferase gene from Photobacterium damsela JT0160.

Sialyltransferase 0160, a bacterial sialyltransferase which catalyzes the incorporation of NeuAc from CMP-NeuAc into the galactose residue of the carbohydrate chain at position 6, is produced by Photobacterium damsela JT0160. The gene coding for sialyltransferase 0160 (bst) was cloned, sequenced, and expressed in Escherichia coli. The sialyltransferase 0160 gene contains an open reading frame of 2,028 base pairs encoding a protein of 675 amino acid residues. The deduced amino acid sequence of sialyltransferase 0160 did not contain the sialylmotif and had no significant similarity to mammalian sialyltransferases. Crude extracts of cultured E. coli MV1184 cells carrying an expression plasmid for the sialyltransferase 0160 gene showed sialyltransferase activity, which was identified as beta-galactoside alpha2,6-sialyltransferase activity by enzymatic reaction product analysis. In addition, when mutant genes, lacking 3'-coding regions for COOH-terminal portions of the protein, which are thought to form alpha-helix structures, were expressed in E. coli MV1184, soluble-form enzymes were obtained. This implies that the COOH-terminal portion of sialyltransferase 0160 is required for membrane binding.

Amino Acid Sequence↗