PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “bacterial coding sequence”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 757 records · Page 42Linked to original sources
PubMed1996

A polyferredoxin with eight [4Fe-4S] clusters as a subunit of molybdenum formylmethanofuran dehydrogenase from Methanosarcina barkeri.

Formylmethanofuran dehydrogenase (Fmd) from Methanosarcina barkeri is a molybdenum iron-sulfur protein involved in methanogenesis. The enzyme contains approximately 30 mol non-heme iron/mol and 30 mol acid-labile sulfur/mol. We report here the cloning and sequencing of the encoding genes, and that these genes form a transcription unit fmdEFACDB. Evidence is provided that the subunit FmdB harbours the molybdenum-containing active site and may bind one [4Fe-4S] cluster. fmdF encodes a protein with four tandemly repeated bacterial-ferredoxin-like domains and is predicted to be a polyferredoxin that could contain as many as 32 iron atoms in eight [4Fe-4S] clusters. The other genes code for proteins without sequence motifs characteristic for iron-sulfur proteins. These findings suggest that most of the iron-sulfur clusters present in the purified formylmethanofuran dehydrogenase are associated with the subunit FmdF. The finding that FmdF forms a tight complex with the other subunits of formylmethanofuran dehydrogenase indicates a function of the polyferredoxin in the reaction catalyzed by the enzyme. fmdE encodes a protein not present in the purified enzyme. All six genes of the fmd operon were expressed in Escherichia coli and yielded proteins of expected molecular masses. A malE-fmdF gene fusion was constructed and expressed in E. coli, making the apoprotein of the polyferredoxin available in preparative amounts.

Aldehyde Oxidoreductases↗
PubMed1986

Expression of reovirus p14 in bacteria and identification in the cytoplasm of infected mouse L cells.

Reovirus genome segment S1 is transcribed by the virion-associated polymerase to form a single mRNA species that codes for two polypeptides: the 49-kDa cell-attachment protein, sigma 1, starting from the first A-U-G in the S1 transcript, and a 14-kDa nonstructural, basic protein initiated from the second A-U-G in a different reading frame (Ernst and Shatkin, 1985; Jacobs et al., 1985; Shatkin, 1985). To confirm that p14 is made in reovirus-infected cells, determine its intracellular location, and generate sufficient amounts of the polypeptide to begin an analysis of its presumptive role in the virus life cycle, the p14 coding sequence of an S1 cDNA clone was subcloned into the EcoRI site downstream of the lambda PL promoter in the bacterial expression vector, pEV-vrf1. The vector was modified to align the ribosome binding site with the p14 initiator codon, and transcription was placed under control of lambda cIts in a compatible plasmid. Transformed Escherichia coli RRI incubated at 42 degrees produced a new polypeptide of approximately 14 kDa as determined by SDS-PAGE. This polypeptide reacted specifically with rabbit antisera made against synthetic peptides corresponding to exposed regions of authentic p14 as predicted from the S1 cDNA sequence. Antipeptide sera also precipitated a approximately 14-kDa polypeptide in lysates of reovirus-infected mouse L cells, demonstrating the synthesis of p14 in vivo. Immunofluorescence experiments indicate that p14 accumulates in the cytoplasm of infected L cells.

Animals↗
PubMed1987

Proteolytic activity of the cowpea mosaic virus encoded 24K protein synthesized in Escherichia coli.

The function of the 24-kilodalton (24K) protein encoded by cowpea mosaic virus (CPMV) has been studied by constructing a bacterial expression plasmid that contained a cloned chimeric segment consisting of partial DNA copies of CPMV M-RNA (including sequences coding for both capsid proteins) and B-RNA (including sequences coding for the 24K protein). Viral sequences were transcribed from the phage T7 promoter phi 10 of plasmid pT7-6 using T7-RNA polymerase expressed from plasmid pGP1-2 present in the same cells. Upon inducing the synthesis of T7-RNA polymerase several new polypeptides that contained CPMV-specific sequences were expressed, as demonstrated by immunoprecipitation and immunoblotting. Furthermore a proteolytic activity was detected in induced cells which cleaved the viral protein sequences specifically at two glutamine-glycine sites. One of the cleavage products represented capsid protein VP23. The proteolytic activity was absent when an 87-bp deletion was introduced in the coding region for the 24K protein, indicating that this protein represented the protease involved in the proteolytic processing at those specific sites.

Cloning, Molecular↗
PubMed1995

A secY homologous gene in the crenarchaeon Sulfolobus acidocaldarius.

The nucleotide sequence of an open reading frame, located upstream of the gene for adenylate kinase, was determined in the thermoacidophile crenarchaeon Sulfolobus acidocaldarius. Data bank searches identified the sequence as a secY homologous gene. The DNA derived protein sequence of total 463 amino acids contains 10 hydrophobic domains. A sequence alignment with other prokaryotic and eukaryotic secY sequences reveals significant homology, but the secY primary sequence of S. acidocaldarius shows only a low degree of similarity with the secY counterparts of the euryarchaea Methanococcus vannielii and Haloarcula marismortui. A transcription analysis indicates, that the secY gene is cotranscribed with the gene coding for adenylate kinase.

Adenylate Kinase↗
PubMed2004

High-level expression and purification of untagged and histidine-tagged HIV-1 reverse transcriptase.

We have devised simplified protocols to purify large quantities of histidine-tagged (His-tagged) and untagged heterodimeric forms of human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT). Here, we report the optimization of overexpression and purification of heterodimeric RT expressed in Escherichia coli. The coding sequences of p66 and p51 subunits of RT were amplified using PCR from HXB2 HIV-1 and cloned into a bacterial expression system. The resulting expression plasmids for the RT subunits, pET-RT66 and pET-RT51, were under a strong T7/lac promoter that is induced by isopropyl-beta-d-thiogalactopyranoside. Purification of heterodimeric forms of RT was facilitated by high-level expression of these subunits that represented approximately 30-40% of total cell protein. For purification of the His-tagged heterodimeric RT, cell pellet from cells expressing the untagged p66 subunit was mixed in excess with a cell pellet expressing tagged p51. For untagged heterodimeric RT, the pellet from cells expressing p51 was mixed in excess with pellet expressing p66. Subunit dimerization occurred during cell lysis. During the subsequent chromatography steps, stable p66/p51 heterodimer was purified to homogeneity. The heterodimeric nature of the final preparations of RT was confirmed by analytical gel filtration, mass spectrometry, and denaturing gel electrophoresis. Further, the sensitivity of these enzyme preparations to AZTTP indicated that the histidine tag had no effect on nucleoside inhibitor binding, nucleotide binding or insertion, or DNA binding. The application of these expression/purification methodologies represents a useful method to purify large quantities of heterodimeric RT for structural investigations and provides an efficient protocol to produce subunit-specific amino acid alterations necessary for unambiguous structure/function investigations.

Anti-HIV Agents↗
PubMed1973

Mechanism of protein chain termination: further characterization of a mutant defective in a new protein synthesis factor.

Mutant N4316 is conditionally lethal at 43 degrees . At 36 degrees it suppresses the termination codons UGA and UAA, but not the UAG codon or a missense mutant of T4 bacteriophage. In vitro, a factor rescues protein synthesis from a temperature-dependent arrest when N4316 extracts are used with RNA from bacteriophage f2. Analyses of the substrate in the arrested synthesis and of the product of the rescue reaction indicate that the factor works at the level of coat protein termination, and that it also affects the synthesis of noncoat protein products. The rescue factor is different from the release factors RF-1, RF-2, and RF-3. Model systems previously used to study release fail to score for at least one vital function in protein chain termination.

Bacterial Proteins↗
PubMed1973

Nucleotide sequence divergence in the -chain-structural genes of tryptophan synthetase from Escherichia coli, Salmonella typhimurium, and Aerobacter aerogenes.

Two different estimates were obtained for the extent of nucleotide sequence divergence in the structural genes of the tryptophan synthetase alpha-chains of Escherichia coli, Salmonella typhimurium, and Aerobacter aerogenes. One estimate was based on comparisons of the amino acid sequences of the respective alpha chains. The other was derived from measurements of the thermal stability of RNA-DNA hybrids formed with phage DNA carrying the alpha-chain structural gene of E. coli and labeled messenger RNA from the three bacterial species. Comparison of the two estimates suggests that during the course of evolution synonymous codon changes have accumulated in the alpha-chain-structural genes.

Amino Acid Sequence↗
PubMed1983

Evidence for use of rare codons in the dnaG gene and other regulatory genes of Escherichia coli.

Amino acid sequence and composition data of Escherichia coli dnaG primase protein and its tryptic peptides have confirmed that the dnaG gene contains an unusually high number of codons that are not frequently used in most E. coli genes. In 25 E. coli proteins analyzed the codons AUA, UCG, CCU, CCC, ACG, CAA, AAT, and AGG are infrequently used, occurring as 4% of the total codons in the reading frame and 11% and 10% in the nonreading frames. In dnaG they occur as 11% in the reading frame and 12% in the nonreading frames. The rpsU and rpoD genes, which flank the dnaG gene [Smiley, B. L., Lupski, J. R., Svec, P. S., McMacken, R. & Godson, G. N. (1982) Proc. Natl. Acad. Sci. USA 79, 4550-4554], however, have normal codon usage. Translational modulation using isoaccepting tRNA availability may therefore be part of the mechanism of keeping the dnaG gene expression low, while expression of the adjacent rpsU and rpoD genes on the same mRNA transcript is high.

Amino Acid Sequence↗
PubMed1995

Nonsense suppressor and antisuppressor mutations at the 1409-1491 base pair in the decoding region of Escherichia coli 16S rRNA.

Using a genetic selection for suppressors of a UGA nonsense mutation in trpA, we have isolated a G to A transition mutation at position 1491 in the decoding region of 16S rRNA. This suppressor displayed no codon specificity, suppressing UGA, UAG and UAA nonsense mutations and +1 and -1 frameshift mutations in lacZ. Subsequent examination of a series of mutations at G1491 and its base-pairing partner C1409 revealed various effects on nonsense suppression and frameshifting. Mutations that prevented Watson-Crick base pairing between these residues were observed to increase misreading and frameshifting. However, double mutations that retained pairing potential produced an antisuppressor or hyperaccurate phenotype. Previous studies of antibiotic resistance mutations and antibiotic and tRNA footprints have placed G1491 and C1409 near the site of codon-anticodon pairing. The results of this study demonstrate that the nature of the interaction of these two residues influences the fidelity of tRNA selection.

Base Sequence↗
PubMed1989

Cloning and characterization of beta-lactam biosynthetic genes.

Seven genes coding for two different enzymes of the penicillin/cephalosporin biosynthetic pathway have been cloned from fungal and bacterial sources. Using amino acid sequences derived from the purified enzymes, oligonucleotide probes were designed to hybridize to their cognate genes in a genomic library. The high degree of similarity (57%) between enzymes of bacterial and fungal origin suggests a horizontal transfer of a primordial beta-lactam pathway, probably from a bacterial cell to a fungal cell. Overproduction of the proteins in Escherichia coli has allowed further study of the mechanism of action of these important enzymes.

Amino Acid Sequence↗
PubMed1975

Purification and characterization of a new factor which restores protein synthesis in a conditionally lethal mutant of Escherichia coli.

The mutant Escherichia coli strain, N4316, has a temperature-sensitive defect in a protein factor required for translation in vitro of bacteriophage f2 RNA. We have purified the normal counterpart of this factor from a wild-type strain, using as an assay its ability to restore the activity of mutant extracts at non-permissive temperature. Our final preparation is free of known initiation, propagation, and release factors, proving that the factor is a new component required for translation. The new factor has a molecular weight of 95000 with preliminary data suggesting a subunit structure. 70% of this protein is found in the soluble-cell fraction, the rest being associated with 70-S ribosomes. Kinetic analyses indicate that the factor acts early in translation. Expression of the defect is highly dependent on the Mg2+ concentration, no temperature-sensitivity being apparent at 15 mM Mg2+. At lower Mg2+ concentrations, the defect is expressed only with natural mRNAs such as f2 RNA, and not with artifical polymers such as poly(U). This specificity suggests that the factor may function in events coded by special sequences in the natural messengers

Bacterial Proteins↗
PubMed1983

Nucleotide sequence of the lipoamide dehydrogenase gene of Escherichia coli K12.

The nucleotide sequence of a 1980-base-pair segment of DNA, containing the lpd gene encoding the lipoamide dehydrogenase component (E3) of the pyruvate dehydrogenase complex of Escherichia coli K12, has been determined by the dideoxy chain-termination method. The lpd structural gene comprises 1419 base pairs (473 codons, excluding the initiating AUG codon). It is preceded by a good promoter and an excellent ribosome binding site and it ends with a typical rho-independent terminator sequence. The results confirm that the lpd gene is an independent gene linked to, but not part of, the ace operon that encodes the E1 and E2 components of the pyruvate dehydrogenase complex. The location and transcriptional polarity of the lpd gene relative to the restriction map of the corresponding region of DNA, are completely consistent with previous genetic and post-infection labelling studies. The composition, Mr (50554 or 51274 if the FAD cofactor is included), amino-terminal sequence and carboxy-terminal sequence predicted from the nucleotide sequence are in excellent agreement with previous studies on the purified enzyme. The enzyme also exhibits a remarkable degree of sequence homology with peptides of the pig heart enzyme and with other pyridine nucleotide disulphide oxidoreductases whose sequences have been defined: human erythrocyte glutathione reductase and plasmid-encoded mercuric reductase.

Amino Acid Sequence↗
PubMed1986

Structural and functional analysis of a cloned delta endotoxin of Bacillus thuringiensis berliner 1715.

A plasmid-encoded crystal protein gene (bt2) has been cloned from Bacillus thuringiensis berliner 1715. In Escherichia coli, it directs the synthesis of the 130-kDa protein (Bt2) which is toxic to larvae of Pieris brassicae and Manduca sexta. Comparison of the deduced amino acid sequence of this Bt2 protein with the B. thuringiensis kurstaki HD1 Dipel, B. thuringiensis kurstaki HD73 and B. thuringiensis sotto crystal protein sequences suggests that homologous recombination between the different genes has occurred during evolution. Treatment of the Bt2 protein with trypsin or chymotrypsin yields a 60-kDa protease-resistant and fully toxic polypeptide. The minimal portion of the Bt2 protein required for toxicity has been determined by analysing the polypeptides produced by deletion derivatives of the bt2 gene. It coincides with the 60-kDa protease-resistant Bt2 fragment and it starts between amino acids 29 and 35 at the N-terminus and terminates between positions 599 and 607 at the C-terminus.

Amino Acid Sequence↗
PubMed1988

Pilin-gene phase variation of Moraxella bovis is caused by an inversion of the pilin genes.

Moraxella bovis Epp63 can express either of two different pilin proteins, called alpha and beta. We have previously cloned and sequenced the beta-pilin gene and now report that DNAs isolated from bacteria expressing alpha pilin have hybridization patterns consistently different from those of bacteria expressing beta pilin. The phase variation between alpha- and beta-pilin gene expression appears to be associated with an inversion of about 2 kilobases of DNA, whose endpoints occur within the coding region of the expressed pilin gene. Comparisons of the beta-pilin gene sequence with those of well-studied bacterial inversion systems revealed a stretch of 58% sequence similarity (21 of 36 base pairs) between the left inverted repeat of the Salmonella typhimurium flagellar hin control region and the amino-terminal portion of the beta-pilin gene.

Bacterial Outer Membrane Proteins↗
PubMed1997

Genetic analysis of chlorophyll biosynthesis.

During this decade, there have been major advancements in the understanding of genetic loci involved in synthesis of the family of Mg-tetrapyrroles known as chlorophylls and bacteriochlorophylls. Molecular genetic analysis of Mg-tetrapyrrole biosynthesis was initiated by the performance of detailed sequence and mutational analysis of the photosynthesis gene cluster from Rhodobacter capsulatus. These studies provided the first detailed understanding of genes involved in bacteriochlorophyll a biosynthesis. In the short time since these studies were initiated, most of the chlorophyll biosynthesis genes have been identified by virtue of their ability to complement bacteriochlorophyll a biosynthesis mutants as well as by sequence homology comparisons. This review is centered on a discussion of our current understanding of bacterial, algal, and plant genes that code for enzymes in the Mg-branch of the tetrapyrrole biosynthetic pathway that are responsible for synthesis of chlorophylls and bacteriochlorophylls.

Chlorophyll↗