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At least 865 records · Page 48Linked to original sources
PubMed2000

A post-translational modification in the GGQ motif of RF2 from Escherichia coli stimulates termination of translation.

A post-translational modification affecting the translation termination rate was identified in the universally conserved GGQ sequence of release factor 2 (RF2) from Escherichia coli, which is thought to mimic the CCA end of the tRNA molecule. It was shown by mass spectrometry and Edman degradation that glutamine in position 252 is N:(5)-methylated. Overexpression of RF2 yields protein lacking the methylation. RF2 from E.coli K12 is unique in having Thr246 near the GGQ motif, where all other sequenced bacterial class 1 RFs have alanine or serine. Sequencing the prfB gene from E.coli B and MRE600 strains showed that residue 246 is coded as alanine, in contrast to K12 RF2. Thr246 decreases RF2-dependent termination efficiency compared with Ala246, especially for short peptidyl-tRNAs. Methylation of Gln252 increases the termination efficiency of RF2, irrespective of the identity of the amino acid in position 246. We propose that the previously observed lethal effect of overproducing E.coli K12 RF2 arises through accumulating the defects due to lack of Gln252 methylation and Thr246 in place of alanine.

Alanine↗
PubMed1994

Endpoint bias in large Tn10-catalyzed inversions in Salmonella typhimurium.

A genetic strategy identified Salmonella typhimurium strains carrying large (> 40 kb) Tn 10-catalyzed inversions; the inverted segments were characterized by XbaI digestion and pulsed field gel electrophoresis. Two size classes of large inversions were found. More than half of the inversions extended 40-80 kb either clockwise or counterclockwise of the original Tn10 site. The remaining inversions extended up to 1620 kb (33% of the genome), but the distal endpoints of these inversions were not randomly scattered throughout the chromosome. Rather, each Tn10 repeatedly yielded similar (though not identical) inversions. The biased endpoint selection may reflect the limited search for target DNA sequences by the Tn10 transposase, and the spatial proximity of the donor and target regions in the folded S. typhimurium nucleoid. Using this interpretation, the data suggest that DNA sequences 40-80 kb clockwise and counterclockwise of the insertion site are in spatial proximity with the insertion, perhaps reflecting the organization of DNA into approximately 120-kb nucleoid domains. In addition, the data predict the spatial proximity of several distant DNA regions, including DNA sequences equidistant from the origin of DNA replication.

Base Sequence↗
PubMed1974

Ultraviolet mutagenesis and its repair in an Escherichia coli strain containing a nonsense codon.

Ultraviolet mutagenesis and its repair were studied mainly in WU36-10-89, a uvr(-) strain of Escherichia coli containing a UAG mutation in a gene for leucine biosynthesis. Following ultraviolet (UV) irradiation revertants appearing with or without direct photoreactivation (PR) were classified according to the presence and type of suppressor they contained. We find UV mutation production to be quite specific. An analysis of revertants produced by UV indicates they are formed mainly from GC --> AT and that the miscoding is due to a cytosine residue at the site of mutation in a cytosine-thymine (CT) dimer. We propose that the dimer serves as template during some aspects of repair replication and at the time of replication the C in the dimer directs the insertion of A in the complementary strand. We also note that C --> A and T -->G changes caused by a CT dimer occur much less frequently.

Base Sequence↗
PubMed1990

The molecular evolution of bacterial alkaline phosphatase: correlating variation among enteric bacteria to experimental manipulations of the protein.

The phylogenetic distribution of the gene coding for bacterial alkaline phosphatase (phoA) was examined in nine species of enteric bacteria closely related to Escherichia coli. The nucleotide and protein sequences from the E. fergusonii and Serratia marcescens genes are presented. The spatial distribution of replaced amino acid residues in the aligned sequences is shown to be highly nonrandom and can be correlated with specific regions within the tertiary structure of the protein. There is an avoidance of replacements within the beta sheet of the protein, and there is an excess of replacements elsewhere, particularly in solvent-exposed residues. In addition, all positions across alpha helices do not accept replacements with equal frequency; there is a bias toward acceptance of replacements in the carboxyl ends of helices. To examine this further, mutations within the E. coli phoA gene were created using site-directed mutagenesis. The patterns seen from the sequence comparisons were verified in the laboratory-created mutants. The average activity of mutations within or near the beta sheet was approximately one-third of that within or near alpha helices, and multiple mutations within the carboxyl ends of alpha helices always possessed greater activity than did multiple mutations within the corresponding amino ends. The results indicate that identifiable regions within the protein are under different selective pressures and are therefore evolving at different rates.

Alkaline Phosphatase↗
PubMed1983

The pyruvate dehydrogenase complex of Escherichia coli K12. Nucleotide sequence encoding the pyruvate dehydrogenase component.

The nucleotide sequence of a 3780-base-pair segment of DNA containing the aceE gene encoding the pyruvate dehydrogenase component (E1) of the pyruvate dehydrogenase complex of Escherichia coli, has been determined by the dideoxy chain-termination method. The aceE structural gene comprises 2655 base pairs (885 codons, excluding the initiation codon AUG), it is preceded by a good ribosome binding site and several potential RNA polymerase binding sites. Its polarity and location in the restriction map of the corresponding segment of DNA are consistent with it being the proximal gene in the ace operon, as defined in previous genetic and post-infection labelling studies. The relative molecular mass (99474), composition (885 amino acids), amino-terminal residue and carboxy-terminal sequence predicted from the nucleotide sequence are in excellent agreement with published information obtained from studies with the purified pyruvate dehydrogenase component (E1). The nucleotide sequence also contains a second gene (gene A) situated upstream of the aceE gene. It appears to be an independent gene containing 708 base pairs (236 codons) and encoding a weakly expressed product (protein A; Mr = 27049) of unknown function.

Amino Acids↗
PubMed1983

The pyruvate dehydrogenase complex of Escherichia coli K12. Nucleotide sequence encoding the dihydrolipoamide acetyltransferase component.

The nucleotide sequence of the aceF gene, which encodes the dihydrolipoamide acetyltransferase component (E2) of the pyruvate dehydrogenase complex of Escherichia coli K12, has been determined using the dideoxy chain-termination method. The aceF gene comprises 1887 base pairs (629 codons excluding the initiation codon AUG); it is preceded by a short intercistronic segment of 14 base pairs containing a good ribosomal binding site, and it is followed closely by a potential rho-independent terminator. The results extend by 1980 base pairs the previously sequenced segment of 3780 base pairs containing the structural gene (aceE) of the pyruvate dehydrogenase component (E1) and they confirm that aceE and aceF are the proximal and distal genes of the ace operon. The amino terminus, carboxy-terminal sequence and amino acid composition of the acetyltransferase subunit predicted from the nucleotide sequence are in excellent agreement with previous studies with the purified protein. The predicted molecular weight (Mr = 65959) confirms experimental values derived from sedimentation equilibrium analysis and indicates that the higher values (78000-89000) that have been reported are due to unusual features of the protein that lead to anomalous mobilities during sodium dodecyl sulphate/polyacrylamide gel electrophoresis and in gel filtration. The primary structure fully supports conclusions, based on limited tryptic proteolysis, that the acetyltransferase subunit possesses two heterologous domains: the lipoyl domain and the subunit binding and catalytic domain. The lipoyl domain corresponds to the amino-terminal segment of the protein. It is acidic and contains three remarkably homologous repeating units of approximately 100 amino acids, each possessing a potential lipoyl binding site and a region that is characteristically rich in alanine and proline residues. The subunit binding and catalytic domain occupies most of the residual polypeptide in the carboxy-terminal segment.

Acetyltransferases↗
PubMed1984

The fimA gene encoding the type-1 fimbrial subunit of Escherichia coli. Nucleotide sequence and primary structure of the protein.

The nucleotide sequence was determined of a region of 1450 base pairs encompassing the fimA gene for the subunit of type 1 fimbriae of Escherichia coli as well as flanking regions containing potential regulator sequences. The 'translated' protein contains a 23-residue signal peptide; the processed fimbrial subunit consists of 158 amino acid residues yielding a relative molecular mass of 15706. The elucidated sequence shows significant homology with those of other E. coli fimbrial proteins.

Amino Acid Sequence↗
PubMed1971

Transcription of nonrepeated DNA in mouse brain.

Under normal conditions of DNA renaturation, about 60 percent of mouse DNA fragments renature at a rate consistent with their being present only once per sperm. These nonrepeated sequences (also called single-copy or unique) may be used in RNA-DNA hybridization experiments to provide quantitative estimates of RNA diversity. About 10 percent of the mouse single-copy sequences are transcribed in mouse brain tissue. Estimates of about 3 percent were obtained for mouse liver and kidney RNA's. If only one of the complementary DNA strands is transcribed, this hybridization value implies that the equivalent of at least 300,000 different sequences of 1000 nucleotides are expressed in mouse brain tissue. It is suggested that the large amount of DNA in mammals is functionally important, and that a substantial proportion of the genome is expressed in the brain.

Animals↗
PubMed1992

Nucleotide sequence and transcriptional regulation of the yeast recombinational repair gene RAD51.

The RAD51 gene of Saccharomyces cerevisiae is required both for recombination and for the repair of DNA damage caused by X rays. Here we report the sequence and transcriptional regulation of this gene. The RAD51 protein shares significant homology (approximately 50%) over a 70-amino-acid with the RAD57 protein (J.A. Kans and R.K. Mortimer, Gene 105:139-140, 1991), the product of another yeast recombinational repair gene, and also moderate (approximately 27%), but potentially significant, homology with the bacterial RecA protein. The homologies cover a region that encodes a putative nucleotide binding site of the RAD51 protein. Sequences upstream of the coding region for RAD51 protein share homology with the damage response sequence element of RAD54, an upstream activating sequence required for damage regulation of the RAD54 transcript, and also contain two sites for restriction enzyme MluI; the presence of MluI restriction sites has been associated with cell cycle regulation. A 1.6-kb transcript corresponding to RAD51 was observed, and levels of this transcript increased rapidly after exposure to relatively low doses of X-rays. Additionally, RAD51 transcript levels were found to that of a group of genes involved primarily in DNA synthesis and replication which are thought to be coordinately cell cycle regulated. Cells arrested in early G1 were still capable of increasing levels of RAD51 transcript after irradiation, indicating that increased RAD51 transcript levels after X-ray exposure are not solely due to an X-ray-induced cessation of the cell cycle at a period when the level of RAD51 expression is normally high.

Amino Acid Sequence↗
PubMed1993

The high-molecular-weight human mucin is the primary salivary carrier of ABH, Le(a), and Le(b) blood group antigens.

Because many bacteria interact with the carbohydrate portions of receptor molecules, factors controlling glycosylation probably influence the ability of salivary components to mediate bacterial adherence/clearance. Important sources of diversity in glycosylation are the ABO, secretor, and Lewis genes, which code for glycosyltransferases that add specific sugar sequences to the termini of carbohydrate chains of glycolipids and glycoproteins. We identified, by Western blotting, salivary glycoproteins carrying the ABH and Le(a) or Le(b) antigens. Samples of whole, unstimulated saliva were obtained from 19 subjects whose blood group was determined by agglutination of red blood cells with specific antisera. After centrifugation, the samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose. Glycoproteins carrying blood group antigens were identified by staining the blot with monoclonal antisera specific for the A, B, H, Le(a), or Le(b) antigens. The most intensely staining component from all the samples migrated at the same position as the high-molecular-weight mucin. Saliva samples from the nonsecretors contained only the Le(a) antigen. Samples from the secretors contained one or more of the ABH antigens and, variably, the Le(b) antigen. In all cases, the salivary blood group antigens corresponded to those found on the red blood cells of the same subject. The functional consequences of the expression of blood group antigens on the high-molecular-weight mucin are not known, but their presence could modulate the adherence of certain oral microorganisms that interact preferentially with this molecule.

ABO Blood-Group System↗
PubMed1994

A yeast system for expression of human cystathionine beta-synthase: structural and functional conservation of the human and yeast genes.

Human cystathionine beta-synthase (CBS; EC 4.2.1.22) deficiency results in a recessive genetic disorder whose clinical and biochemical manifestations vary greatly among affected individuals. In an effort to identify and analyze mutations in the human CBS gene, we have developed a yeast expression system for human CBS. We have cloned and sequenced a human cDNA that codes for CBS and have expressed the human CBS protein in yeast cells lacking endogenous CBS. The human enzyme produced in yeast is functional both in vitro and in vivo. We have also cloned and sequenced the yeast gene, CYS4, that codes for CBS. The predicted human and yeast CBS proteins are 38% identical and 72% similar to each other, as well as sharing significant similarity with bacterial cysteine synthase. These results demonstrate the evolutionary conservation of CBS and establish the utility of a yeast expression system for studying human CBS.

Animals↗
PubMed1993

A role for Bacteroides fragilis neuraminidase in bacterial growth in two model systems.

Two Bacteroides fragilis neuraminidase-deficient mutants were used to study the role of neuraminidase activity in growth of B. fragilis in tissue culture monolayers (CHO cells) and in the in vivo rat granuloma pouch. The nanH structural gene for neuraminidase was cloned from B. fragilis TM4000 and was used to create two isogenic strains with chromosomal disruptions at the nanH gene. B. fragilis VRC404 contains an insertion flanked by disrupted copies of the nanH gene, and B. fragilis VRC426 contains a deletion of a significant portion of nanH coding sequences. The insertion mutant VRC404 is capable of reverting to nanH+. It grew as well as the wild type in CHO monolayers. However, between 48 and 72 h after infection, the bacterial population was enriched with nanH+ bacterial cells (10 to 20%). In the rat pouch 48 h after infection, more than 90% of the population sampled had become nanH+. The deletion mutant VRC426 showed a severe growth defect in the rat pouch model. In addition, VRC426 was efficiently outgrown by the wild type in competition experiments, even when the mutant was present at 10 times the number of wild-type cells at the time of infection. A common characteristic of both model systems is a drastic decrease in the free glucose concentration 16 to 24 h postinfection. We suggest that neuraminidase activity may be required for B. fragilis to grow to maximal levels in the tissue culture and rat pouch systems by making other carbon sources available after glucose levels are reduced.

Animals↗
PubMed1986

Human complement factor H: isolation of cDNA clones and partial cDNA sequence of the 38-kDa tryptic fragment containing the binding site for C3b.

We isolated cDNA clones coding for the functionally important tryptic N-terminal 38-kDa fragment of human complement control protein factor H using polyclonal and monoclonal antibodies to screen a human liver cDNA library cloned in a bacterial expression vector, PEX-1. By testing the reactivity of antibodies specific for the recombinant proteins produced by individual clones with proteolytic fragments of serum H the exact position of these cDNA clones within H was mapped. One clone, H-19, coding for the 38-kDa fragment of H was sequenced and found to code for 289 amino acids derived from the 38-kDa N-terminal fragment as well as for the first 108 amino acids belonging to the complementary 142-kDa tryptic fragment. The derived protein sequence could be arranged in 6 highly homologous repeats of about 60 amino acids each, the homology between the repeats being determined by the characteristic position of cysteine, proline, glycine, tyrosine and tryptophane residues. The region coding for the epitope recognized by one of our monoclonal antibodies was localized by subcloning restriction fragments of H-19 into the expression plasmid and testing for the expression of this epitope.

Amino Acid Sequence↗
PubMed1987

Nucleotide sequence of a gene cluster involved in entry of E colicins and single-stranded DNA of infecting filamentous bacteriophages into Escherichia coli.

Mutations in fii or tolA of the fii-tolA-tolB gene cluster at 17 min on the Escherichia coli map render cells tolerant to high concentrations of the E colicins and do not allow the DNA of infecting single-stranded filamentous bacteriophages to enter the bacterial cytoplasm. The nucleotide sequence of a 1,854-base-pair DNA fragment carrying the fii region was determined. This sequence predicts three open reading frames sequentially coding for proteins of 134, 230, and 142 amino acids, followed by the potential start of the tolA gene. Oligonucleotide mutagenesis of each open reading frame and maxicell analysis demonstrated that all open reading frames are expressed in vivo. Sequence analysis of mutant fii genes identified the 230-amino acid protein as the fii gene product. Chromosomal insertion mutations were constructed in each of the two remaining open reading frames. The phenotype resulting from an insertion of the chloramphenicol gene into the gene coding for the 142-amino acid protein is identical to that of mutations in fii and tolA. This gene is located between fii and tolA, and we propose the designation of tolQRA for this cluster in which tolQ is the former fii gene and tolR is the new open reading frame. The protein products of this gene cluster play an important role in the transport of large molecules such as the E colicins and filamentous phage DNA into the bacterium.

Amino Acid Sequence↗
PubMed1981

A gene chimaera of SV40 and mouse beta-globin is transcribed and properly spliced.

A gene chimaera of the first exon from simian virus 40 (SV40) sequences coding for T antigen placed upstream from the third exon from mouse beta-globin, and separated by the resultant new chimaeric intron, was cloned into a bacterial plasmid. When transfected into monkey cells, the gene chimaera was transcribed, polyadenylated and spliced using the donor splice site from SV40 and the acceptor splice site from mouse beta-globin. This result suggests that a donor site from one gene can be spliced to an acceptor site from another gene.

Antigens, Neoplasm↗