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H W Stokes

Publications and source records attributed to H W Stokes.

52 records · Page 3Linked to original sources
PubMed1992

Identification of a chloroplast-encoded secA gene homologue in a chromophytic alga: possible role in chloroplast protein translocation.

SecA is one of seven Sec proteins that comprise the prokaryotic protein translocation apparatus. A chloroplast-encoded secA gene has been identified from the unicellular chromophytic alga Pavlova lutherii. The gene predicts a protein that is related to the SecA proteins of Escherichia coli and Bacillus subtilis. The presence of secA, as well as the previously described secY and hsp70 genes, on the chloroplast genome of P. lutherii suggests that this eukaryotic organism utilises protein translocation mechanisms similar to those of bacterial cells.

Adenosine Triphosphatases↗
PubMed1992

The integron In1 in plasmid R46 includes two copies of the oxa2 gene cassette.

The sequence of the insert region of the integron In1 found in the IncN plasmid R46 was completed. The insert region is 2929 bases long and includes four gene cassettes, two of which are identical copies of the oxa2 gene cassette flanking an aadA1 cassette. The fourth cassette encodes an open reading frame orfD. From comparison of these data with published maps and sequences it is argued that the integrons found in the IncN plasmids pCU1 and R1767 and in the transposon Tn2410 are closely related to In1 from R46. Both site-specific gene insertion and recA-dependent recombination are likely to have contributed to the evolution of these integrons.

Amino Acid Sequence↗
PubMed1992

Induced expression of a Drosophila hsp70 promoter-fusion transgene is reduced after repeated heat shocks.

Levels of transcripts produced by a heat shock protein 70 (hsp70)-antisense white transgene in Drosophila were measured after single and multiple heat shocks to determine whether the hsp70 promoter could produce sustained high levels of transgene transcripts. A single heat shock resulted in typical highly inducible levels of RNA, but the amount of antisense RNA was substantially reduced after multiple heat shocks. Endogenous hsp70 mRNA levels were also less abundant after multiple heat shocks as compared to a single heat shock. The hsp70 promoter is unsuitable for use in fusion gene constructs for long term expression studies where repeated heat shocks are required.

Animals↗
PubMed1991

Sequence analysis of the inducible chloramphenicol resistance determinant in the Tn1696 integron suggests regulation by translational attenuation.

The sequence of the Tn1696 determinant for inducible nonenzymatic chloramphenicol resistance has been determined. The cml region, the fourth insert of the Tn1696 integron, is 1547 bases and includes a 59-base element at the 3' end, as is typical of integron inserts. One gene, designated cmlA and predicting a polypeptide of 44.2 kDa, is encoded in the insert. However, the cmlA region shows one feature not previously found in an integron insert. A promoter is located within the cmlA insert, and translational attenuation signals related to those of the inducible cat and ermC genes found in gram-positive organisms are also present. The regulatory region includes a leader peptide of nine amino acids, a ribosome stall sequence related to those preceding cat genes, and two alternative pairs of stem-loop structures which either sequester or disclose the ribosome binding site and start codon preceding the cmlA gene.

Amino Acid Sequence↗
PubMed1991

Site-specific insertion of genes into integrons: role of the 59-base element and determination of the recombination cross-over point.

From examination of published DNA sequences of genes found inserted at a specific site in integrons, all genes are shown to be associated, at their 3' ends, with a short imperfect inverted repeat sequence, a 59-base element or relative of this element. The similarity of the arrangement of gene inserts in the integron and in the Tn7 transposon family is described. A refined consensus for the 59-base element is reported. Members of this family are highly diverged and the relationship of a group of longer elements to the 59-base elements is demonstrated. The ability of 59-base elements of different length and sequence to act as sites for recombination catalysed by the integron-encoded DNA integrase is demonstrated, confirming that elements of this family have a common function. The ability of elements located between gene pairs to act as recombination sites has also been demonstrated. The recombination cross-over point has been localized to the GTT triplet which is conserved in the core sites, GTTRRRY, found at the 3' end of 59-base elements. Recombination at the core site found in inverse orientation at the 5' end of the 59-base elements was not detected, and the sequences responsible for orientation of the recombination event appear to reside within the 59-base element. A model for site-specific insertion of genes into integrons and Tn7-like transposons is proposed. Circular units consisting of a gene associated with a 59-base element are inserted into an ancestral element which contains neither a gene nor a 59-base element.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1991

A new trimethoprim resistance gene, dhfrX, in the In7 integron of plasmid pDGO100.

A new trimethoprim resistance determinant, designated dhfrX, was identified in the In7 integron of pDGO100. The sequence of the dhfrX dihydrofolate reductase is up to 28% identical to the sequences of several known dihydrofolate reductase proteins. The dhfrX gene is adjacent to the second 3'-conserved segment of the In7 integron, but the first 77 bases of this segment are not present.

Amino Acid Sequence↗
PubMed1990

The structure of a partial duplication in the integron of plasmid pDGO100.

A family of novel potentially mobile DNA elements called integrons, has recently been described (H. W. Stokes and R. M. Hall, 1989, Mol. Microbiol. 3, 1669-1683). The integrons present in the plasmids pDGO100 and pSa are unusual in that they include a duplication of the sulI gene which is located in one of the two conserved segments that make up these elements. In order to define the nature of the duplication in pDGO100, we have sequenced the sulI gene region located between the aadB and the dhfr genes of pDGO100. This region includes the first 1355 bases of the 2026-base 3'-conserved segment present in the integrons of Tn21, R46 and R388, and the sequence identity in pDGO100 ceases 24 bases beyond the end of the sulI gene. This position corresponds to the center of a 59-base element, a remnant of which is located at the end of sulI. This finding suggests that the 59-base element may have been involved in the event which gave rise to the partial duplication.

Amino Acid Sequence↗
PubMed1989

A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions: integrons.

A family of novel mobile DNA elements is described, examples of which are found at several independent locations and encode a variety of antibiotic resistance genes. The complete elements consist of two conserved segments separated by a segment of variable length and sequence which includes inserted antibiotic resistance genes. The conserved segment located 3' to the inserted resistance genes was sequenced from Tn21 and R46, and the sequences are identical over a region of 2026 bases, which includes the sulphonamide resistance gene sull, and two further open reading frames of unknown function. The complete sequences of both the 3' and 5' conserved regions of the DNA element have been determined. A 59-base sequence element, found at the junctions of inserted DNA sequences and the conserved 3' segment, is also present at this location in the R46 sequence. A copy of one half of this 59-base element is found at the end of the sull gene, suggesting that sull, though part of the conserved region, was also originally inserted into an ancestral element by site-specific integration. Inverted or direct terminal repeats or short target site duplications, both of which are characteristics of class I and class II transposons, are not found at the outer boundaries of the elements described here. Furthermore, the conserved regions do not encode any proteins related to known transposition proteins, except the DNA integrase encoded by the 5' conserved region which is implicated in the gene insertion process. Mobilization of this element has not been observed experimentally; mobility is implied from the identification of the element in at least four independent locations, in Tn21, R46 (IncN), R388 (IncW) and Tn1696. The definitive features of these novel elements are (i) that they include site-specific integration functions (the integrase and the insertion site); (ii) that they are able to acquire various gene units and act as an expression cassette by supplying the promoter for the inserted genes. As a consequence of acquiring different inserted genes, the element exists in a variety of forms which differ in the number and nature of the inserted genes. This family of elements appears formally distinct from other known mobile DNA elements and we propose the name DNA integration elements, or integrons.

Amino Acid Sequence↗
PubMed1985

Sequence of the ebgA gene of Escherichia coli: comparison with the lacZ gene.

We have sequenced the ebgA (evolved beta-galactosidase) gene of Escherichia coli K12. The sequence shows 50% nucleotide identity with the E. coli lacZ gene, demonstrating that the two genes are related by descent from a common ancestral gene. Comparison of the two sequences suggests that the ebgA gene has recently been under selection. A significant excess of identical, rather than synonymous, codons used to encode identical amino acids at the same positions in the aligned sequences implies that some form of selection is operating directly at the DNA level. This selection is independent of, and in addition to, selection based on codon usage or on function of the gene products.

Amino Acid Sequence↗
PubMed1985

Sequence of the ebgR gene of Escherichia coli: evidence that the EBG and LAC operons are descended from a common ancestor.

The sequence of ebgR, the gene that encodes the EBG repressor, was determined. There is 44% DNA sequence identity between ebgR and lacI, the gene that encodes the LAC repressor. There is also 25% identity between the amino acid sequence of lacI and the deduced amino acid sequence of ebgR. The sequence of 596 bp distal to ebgA, the structural gene for EBG beta-galactosidase, was also determined. Within that region there were two sequences, 74 and 100 bp long, that showed 46% and 50% identity, respectively, to sequences in the first 600 bp of lacY, the structural gene for the lactose permease. The organization and direction of transcription of the repressor and structural genes of the two operons are identical. Taken together with the homology between ebgA and lacZ (as demonstrated in the companion article in this issue), this provides strong evidence that the EBG and LAC operons are descended from a common ancestor. The map position of these two operons supports the notion that these operons diverged following a genome duplication event in an ancestor of Escherichia coli.

Amino Acid Sequence↗
PubMed1984

Topological repression of gene activity by a transposable element.

The ebgA (evolved beta-galactosidase) gene of Escherichia coli was isolated as part of a 9.6-kilobase (kb) sequence cloned into plasmid pBR322. The position of the ebgA gene within that 9.6-kilobase sequence was identified by insertional inactivation by means of the transposon gamma-delta. In addition to the gamma-delta insertions that inactivate ebgA by disrupting the coding sequence, seven additional gamma-delta insertions reduce expression of the gene by a factor of greater than 200 by insertions elsewhere into the replicon. One of these insertions is into the pBR322 sequence itself. This action at a distance to reduce expression requires that gamma-delta is cis with respect to the ebgA gene. The effect is independent of the orientation or position of gamma-delta within the replicon, but it does depend both upon the orientation of the ebgA-bearing sequence within the replicon and upon the total size of the replicon. Transcription readthrough (promoter occlusion) does not explain this phenomenon, and we suggest that the presence of gamma-delta may alter the local supercoiling in the region of the ebgA promoter in such a way as to inhibit transcription. This repression by a transposable element appears to represent a novel mechanism for altering gene expression.

DNA Restriction Enzymes↗
PubMed1983

Comparison of plasmids in strains of Zymomonas mobilis.

Four strains of Zymomonas mobilis were examined for their resistance to antimicrobial agents and found to have similar resistance profiles. Plasmid DNA was extracted and purified by CsCl dye-buoyant density centrifugation; molecular weights were determined by agarose gel electrophoresis and electron microscopy. All four strains harbored a large plasmid (46 X 10(6) Da) and a smaller plasmid (16-21 X 10(6) Da) whose molecular weight was strain dependent. Two strains, Ag11 and ATCC 10988, had smaller plasmids of unique molecular weight. Homology existed between the plasmids in the four strains as shown by cross-reaction in DNA-DNA blot hybridizations. Only one plasmid appeared unique to the host from which it was isolated.

Anti-Bacterial Agents↗