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At least 631 records · Page 35Linked to original sources
PubMed2006

A bacterial arginine-agmatine exchange transporter involved in extreme acid resistance.

The arginine-dependent extreme acid resistance response of Escherichia coli operates by decarboxylating arginine. AdiC, a membrane antiporter, catalyzes arginine influx coupled to efflux of the decarboxylation product agmatine, effectively exporting a proton in each turnover. Using the adiC coding sequence under control of a tetracycline promoter in an E. coli vector, we expressed and purified the transport-protein with a yield of approximately 10 mg/liter bacterial culture. Glutaraldehyde cross-linking experiments indicate that the protein is a homodimer in detergent micelles and lipid membranes. Purified AdiC reconstituted into liposomes exchanges arginine and agmatine in a strictly coupled, electrogenic fashion. Kinetic analysis yields K(m) approximately 80 microm for Arg, in the same range as its dissociation constant determined by isothermal titration calorimetry.

Agmatine↗
PubMed2004

A reassessment of the response of the bacterial ribosome to the frameshift stimulatory signal of the human immunodeficiency virus type 1.

HIV-1 uses a programmed -1 ribosomal frameshift to produce the precursor of its enzymes. This frameshift occurs at a specific slippery sequence followed by a stimulatory signal, which was recently shown to be a two-stem helix, for which a three-purine bulge separates the upper and lower stems. In the present study, we investigated the response of the bacterial ribosome to this signal, using a translation system specialized for the expression of a firefly luciferase reporter. The HIV-1 frameshift region was inserted at the beginning of the coding sequence of the luciferase gene, such that its expression requires a -1 frameshift. Mutations that disrupt the upper or the lower stem of the frameshift stimulatory signal or replace the purine bulge with pyrimidines decreased the frameshift efficiency, whereas compensatory mutations that re-form both stems restored the frame-shift efficiency to near wild-type level. These mutations had the same effect in a eukaryotic translation system, which shows that the bacterial ribosome responds like the eukaryote ribosome to the HIV-1 frameshift stimulatory signal. Also, we observed, in contrast to a previous report, that a stop codon immediately 3' to the slippery sequence does not decrease the frameshift efficiency, ruling out a proposal that the frameshift involves the deacylated-tRNA and the peptidyl-tRNA in the E and P sites of the ribosome, rather than the peptidyl-tRNA and the aminoacyl-tRNA in the P and A sites, as commonly assumed. Finally, mutations in 16S ribosomal RNA that facilitate the accommodation of the incoming aminoacyl-tRNA in the A site decreased the frameshift efficiency, which supports a previous suggestion that the frameshift occurs when the aminoacyl-tRNA occupies the A/T entry site.

Codon, Terminator↗
PubMed1980

Nucleotide sequence of the thrA gene of Escherichia coli.

The thrA gene of Escherichia coli codes for a single polypeptide chain having two enzymatic activities required for the biosynthesis of threonine, aspartokinase I and homoserine dehydrogenase I. This gene was cloned in a bacterial plasmid and its complete nucleotide sequence was established. It contains 2460 base pairs that encode for a polypeptide chain of 820 amino acids. The previously determined partial amino acid sequence of this protein is in good agreement with that predicted from the nucleotide sequence. The gene contains an internal sequence that resembles the structure of bacterial ribosome-binding sites, with an AUG preceded by four triplets, each of which can be converted to a nonsense codon by a single mutation. This suggests that the single polypeptide chain was formed by the fusion of two genes and that initiation of translation may occur inside the gene to give a protein fragment having only the homoserine dehydrogenase activity.

Aspartokinase Homoserine Dehydrogenase↗
PubMed1994

An efficient Shine-Dalgarno sequence but not translation is necessary for lacZ mRNA stability in Escherichia coli.

The 5' ends of many bacterial transcripts are important in determining mRNA stability. A series of Shine-Dalgarno (SD) sequence changes showed that the complementarity of the SD sequence to the anti-SD sequence of 16S rRNA correlates with lacZ mRNA stability in Escherichia coli. Several initiation codon changes showed that an efficient initiation codon is not necessary to maintain lacZ mRNA stability. A stop codon in the 10th codon of lacZ increased mRNA stability. Therefore, ribosomal binding via the SD sequence but not translation of the coding region is necessary to maintain lacZ mRNA stability.

Codon↗
PubMed1988

Polarity effects in the hisG gene of salmonella require a site within the coding sequence.

A single site in the middle of the coding sequence of the hisG gene of Salmonella is required for most of the polar effect of mutations in this gene. Nonsense and insertion mutations mapping upstream of this point in the hisG gene all have strong polar effects on expression of downstream genes in the operon; mutations mapping promotor distal to this site have little or no polar effect. Two previously known hisG mutations, mapping in the region of the polarity site, abolish the polarity effect of insertion mutations mapping upstream of this region. New polarity site mutations have been selected which have lost the polar effect of upstream nonsense mutations. All mutations abolishing the function of the site are small deletions; three are identical, 28-bp deletions which have arisen independently. A fourth mutation is a deletion of 16 base pairs internal to the larger deletion. Several point mutations within this 16-bp region have no effect on the function of the polarity site. We believe that a small number of polarity sites of this type are responsible for polarity in all genes. The site in the hisG gene is more easily detected than most because it appears to be the only such site in the hisG gene and because it maps in the center of the coding sequence.

Base Sequence↗
PubMed1992

Mapping of binding sites for monoclonal antibodies to chick tropoelastin by recombinant DNA techniques.

A fusion molecule consisting of the entire coding sequence of mature chicken tropoelastin preceded by 14 amino acids of the signal peptide and 9 amino acids of vector origin has been expressed in a recombinant bacterial system and purified. The molecule has been used as immunogen for the production of hybridomas. Monoclonal antibodies which bound specifically the immunogen were also reactive with tropoelastin purified from chick aorta and stained elastic fibers in aorta sections by immunofluorescence. The region of tropoelastin containing the antigenic determinant recognized by each antibody has been identified by a recombinant DNA expression strategy based on the use of cDNA clones spanning different portions of the coding sequence. It could be shown that several antibodies were directed against unique epitopes; among these, a group of antibodies bound specifically to the sequence (PGVGV)n. Other antibodies were found to recognize antigenic determinants present more than once in the molecule. The monoclonal antibodies thus characterized will be useful reagents in studying the function of the different domains of tropoelastin.

Animals↗
PubMed2001

Cloning, characterization, and possible origin of the Prevotella loescheii dnaK homolog.

Heat shock proteins play an important role in bacterial survival and response to environmental stress. We cloned the Prevotella loescheii HSP70 homolog (dnaK) and characterized the coding sequence, regulatory regions, and evolutionary relationships to other bacteria. Predicted proteins encoded by the P. loescheii dnaK homolog (open reading frame ORF-1) and two downstream coding regions, ORF-2 and ORF-3, are highly homologous to the proteins encoded by ORF-4 (dnaK), ORF-5, and ORF-6 from the dnaK region of Porphyromonas gingivalis. The dnaK promoter resembles other HSP (heat shock protein) promoters. Alignment of the predicted protein encoded by ORF-2 showed significant homology to the Bacteroides fragilis tnpA gene from the transposon Tn4555, whereas the ORF-3 protein showed homology to B. fragilis transposase (Tn5220) and integrase (Tn4555) proteins. This suggests a transposition-like event may be responsible for transfer of these genes between Porphyromonas and Prevotella.

Amino Acid Sequence↗
PubMed1995

An antibody VH domain with a lox-Cre site integrated into its coding region: bacterial recombination within a single polypeptide chain.

Bacterial lox-Cre recombination within a single antibody VH domain was achieved through integration of a loxP site into its coding sequence. The 5' half of the VH gene, in which the H2 loop was replaced by a mutant loxP site, was fused to geneIII in an 'acceptor' fd-phage vector containing also a wild type loxP site. With a 'donor' plasmid vector harbouring the 3' half of the VH gene flanked by the same, differing loxP sites it recombined into a full-length VH with the loxP site-H2 loop. This VH was purified from bacterial periplasm, where it folded into a typical immunoglobulin domain. The system allows the generation of large VH repertoires using lox-Cre recombination.

Amino Acid Sequence↗
PubMed1987

The genes of the Paracoccus denitrificans bc1 complex. Nucleotide sequence and homologies between bacterial and mitochondrial subunits.

The genes for the three subunits of the cytochrome bc1 complex from the bacterium Paracoccus denitrificans were identified by screening a gene library constructed in pBR 322 for expression using a cytochrome c1-specific antibody. These three genes coding for the FeS subunit, cytochrome b, and cytochrome c1 were located on contiguous sites on the genome in a presumed operon arrangement. The DNA-deduced amino acid sequence shows that all three subunits are homologous to corresponding polypeptides of the mitochondrial cytochrome bc1 complex. Cytochrome c1 of Paracoccus is much larger than its mitochondrial counterpart due to an extra 150 amino acids of unique, highly acidic composition; in addition, it is most likely synthesized as a precursor polypeptide.

Amino Acid Sequence↗
PubMed1974

Translational reinitiation: reinitiation of lac repressor fragments at three internal sites early in the lac i gene of Escherichia coli.

Three early amber mutations in the lac i gene have been shown to arise from the codons corresponding to residues 7, 12, and 17 of the lac repressor polypeptide chain. All three mutations allow translational reinitiation at the same two sites, resulting in the synthesis of two lac repressor fragments. The amino-terminal sequences of these fragments show that the first site is the triplet coding for valine residue 23, while the second is the first internal in-phase AUG codon corresponding to residue 42. Translational reinitiation appears to be a common event in E. coli, since there are at least three such sites in the first 70 in-phase codons of the i-gene messenger RNA, and all amber mutants found in this region show translational reinitiation. Only one of these sites involves an AUG codon; the other two involve an in vivo ambiguity of the genetic code, in that the same codon can be translated into two different amino acids depending on whether it is recognized during initiation or elongation of protein biosynthesis. The two non-AUG codons are the codons corresponding to leucine residue 62 and valine residue 23 of the lac repressor.

Amino Acid Sequence↗
PubMed1985

N-Methyl transferase of Streptomyces erythraeus that confers resistance to the macrolide-lincosamide-streptogramin B antibiotics: amino acid sequence and its homology to cognate R-factor enzymes from pathogenic bacilli and cocci.

The nucleotide sequence of a structural gene ermE for ribosomal RNA (rRNA) N6-amino adenine N-methyl transferase (NMT) of Streptomyces erythraeus, cloned by Thompson et al. [Gene 20 (1982) 51-62], has been determined. The NMT amino acid (aa) sequence deduced from the nucleotide sequence contains extensive homology to aa sequences of cognate NMTs specified by: (1) plasmid pE194 from Staphylococcus aureus, 30% G + C, ermC; (2) plasmid pAM77 from Streptococcus sanguis, 43% G + C; as well as to (3) a chromosomal determinant from Bacillus licheniformis 759, 46% G + C, ermD, cloned in a recombinant plasmid pBD90. These findings suggest that all four NMT structural genes could have evolved from a common progenitor sequence despite the wide range of % G + C of the erm genes reflecting their current respective hosts. Comparison of the four NMT sequences with respect to localized hydrophobicity averaged over a moving window of 11 aa indicates that the common features of localized hydrophobicity that characterize the C-terminal portion of the ermE and ermD proteins are distinguishable from a contrasting pattern of hydrophobicity that characterizes the ermC and pAM77-coded proteins.

Amino Acid Sequence↗
PubMed2025

Cellular function of the GndA microprotein during heat shock.

Over the past 15 years, hundreds of previously undiscovered bacterial small open reading frames (sORFs) encoding microproteins of fewer than fifty amino acids have been identified. Biological functions have been ascribed to an increasing number of microproteins from intergenic regions and small RNAs, and many play integral roles in bacterial stress responses. However, despite numbering in the dozens in Escherichia coli, and hundreds in humans, same-strand frameshifted sORFs that internally overlap protein coding sequences remain understudied. To provide insight into nested genes, we characterized GndA, a frameshifted 36-amino acid microprotein nested within the 6-phosphogluconate dehydrogenase (6PGD) coding sequence. Using precise genome editing, we demonstrate independent contributions of GndA and 6PGD to cell growth at high temperature. GndA associates with membrane-associated complexes associated with electron transport and ATP generation, and supports ATP homeostasis during heat shock. Functional characterization of GndA thus adds to the catalog of bacterial microproteins that function in stress responses, while providing clear genetic evidence for the importance of an overlapping gene to cellular fitness.

6PGD↗
PubMed2003

CCLS96.1, a member of a multicopy gene family, may encode a non-coding RNA preferentially transcribed in reproductive organs of Silene latifolia.

Dioecy in the model dioecious plant Silene latifolia is determined genetically by its heteromorphic sex chromosomes. A bacterial artificial chromosome (BAC) clone, #19B12, was isolated by screening a BAC library from S. latifolia using polymerase chain reaction (PCR) with a set of sequence tagged site (STS) primers, ScD05, which are specific to the Y chromosome. A portion of #19B12 was subcloned to construct plasmid #25-1, with an insert of 7.8 kb. This 7.8-kb fragment encodes ScD05 homolog and an anther-specific gene, CCLS96.1. Northern blot analysis of CCLS96.1 indicated a faint band of 1.8 kb in male and female flower buds. 5' and 3' rapid amplification of cDNA ends (RACE) indicated that transcripts of CCLS96.1 are very varied in size. Moreover, semi-quantitative reverse transcription-PCR (RT-PCR) showed that CCLS96.1 was also expressed in both male and female leaves. RACE produced at least ten species of transcripts, with 79-97% similarity among them. However, no significant ORFs could be predicted from their nucleotide sequences, since each has numerous stop codons throughout all three reading frames. Genomic Southern hybridization showed that the S. latifolia genome contains numerous CCLS96.1 homologs. These results suggest that the transcripts of CCLS96.1 play some role as multiple non-coding RNAs in S. latifolia.

Base Sequence↗
PubMed1991

The sequence of a 6.3 kb segment of yeast chromosome III reveals an open reading frame coding for a putative mismatch binding protein.

We report the sequence of a 6.3 kb segment of DNA mapping near the end of the right arm of chromosome III of Saccharomyces cerevisiae. The sequence reveals a major open reading frame coding for a putative protein of 1047 amino acids with a striking similarity to the bacterial proteins involved in recognition of mismatched DNA base pairs. This is particularly interesting as the existence of a yeast mismatch repair system similar to that of bacteria has been postulated for some years, but a yeast protein homologous to the bacterial mismatch binding protein had not been identified. The results of a comparison of the putative yeast mismatch binding protein with the bacterial mismatch binding proteins and with two cognate mammalian sequences, support the idea that a similar mismatch repair system may be present also in mammalian cells. The possibility that all of these proteins may have evolved from a common ancestral gene is also discussed.

Amino Acid Sequence↗
PubMedDate not supplied

A bioinformatics based approach to discover small RNA genes in the Escherichia coli genome.

The recent explosion in available bacterial genome sequences has initiated the need to improve an ability to annotate important sequence and structural elements in a fast, efficient and accurate manner. In particular, small non-coding RNAs (sRNAs) have been difficult to predict. The sRNAs play an important number of structural, catalytic and regulatory roles in the cell. Although a few groups have recently published prediction methods for annotating sRNAs in bacterial genome, much remains to be done in this field. Toward the goal of developing an efficient method for predicting unknown sRNA genes in the completed Escherichia coli genome, we adopted a bioinformatics approach to search for DNA regions that contain a sigma70 promoter within a short distance of a rho-independent terminator. Among a total of 227 candidate sRNA genes initially identified, 32 were previously described sRNAs, orphan tRNAs, and partial tRNA and rRNA operons. Fifty-one are mRNAs genes encoding annotated extremely small open reading frames (ORFs) following an acceptable ribosome binding site. One hundred forty-four are potentially novel non-translatable sRNA genes. Using total RNA isolated from E. coli MG1655 cells grown under four different conditions, we verified transcripts of some of the genes by Northern hybridization. Here we summarize our data and discuss the rules and advantages/disadvantages of using this approach in annotating sRNA genes on bacterial genomes.

Base Sequence↗
PubMed1987

[Design of a hybrid gene coding for the leader sequence of Bacillus amyloliquefaciens alpha-amylase and for human proinsulin].

The chemically synthesized structure gene of human proinsulin was cloned in E. coli on the secretory vector containing regulatory elements of the Bacillus amyloliquefaciens alpha-amylase gene. The proinsulin gene was inserted by the EcoRI site located immediately after the DNA area encoding the alpha-amylase signal peptide. The E. coli cells transformed by such a plasmid produced hybrid protein consisting of the alpha-amylase signal peptide, five amino acid residues after the gene mating and human proinsulin. For accurate mating of the alpha-amylase gene leader sequence and proinsulin gene directed mutagenesis was performed on the filiform phage M13 mp9 with synthetic oligonucleotide. The hybrid gene was transferred to the vector molecule capable of replicating in Bacillus subtilis. It was shown that in the cells of both E. coli and B. subtilis there is synthesized protein interacting by the radio-immunological data with antibodies to porcine insulin, a large portion of immunologically active protein being detected in the periplasmic space of E. coli cells and in the culture fluid of B. subtilis cells which was indicative of proinsulin secretion directed by the alpha-amilase regulatory elements.

Amino Acid Sequence↗
PubMed1996

Genomic cloning of a human heat shock protein 40 (Hsp40) gene (HSPF1) and its chromosomal localization to 19p13.2.

The Hsp40 (heat shock protein with molecular size of approximately 40 kDa) is one of the mammalian homologues of bacterial DnaJ heat shock protein. We have isolated and characterized a genomic DNA clone encompassing the entire coding sequences of the human Hsp40 cDNA. The Hsp40 gene (HGMW-approved symbol HSPF1) is composed of three exons divided by two introns. The 5' region of the gene is highly GC rich, and there are multiple basal elements for transcription factors including typical heat shock elements. The Hsp40 gene has been assigned to chromosome 19 band p13.2 by in situ hybridization.

Binding Sites↗