Search PubMed⌕ Search

Biomedical subjects

B A Roe

Publications and source records attributed to B A Roe.

At least 91 records · Page 5Linked to original sources
PubMed1995

The esg locus of Myxococcus xanthus encodes the E1 alpha and E1 beta subunits of a branched-chain keto acid dehydrogenase.

The esg locus of Myxococcus xanthus appears to control the production of a signal that must be transmitted between cells for the completion of multicellular development. DNA sequence analysis suggested that the esg locus encodes the E1 decarboxylase (composed of E1 alpha and E1 beta subunits) of a branched-chain keto acid dehydrogenase (BCKAD) that is involved in branched-chain amino acid (BCAA) metabolism. The properties of an esg::Tn5 insertion mutant supported this conclusion. These properties include: (i) the growth yield of the mutant was reduced with increasing concentrations of the BCAAs in the medium while the growth yield of wild-type cells increased, (ii) mutant extracts were deficient in BCKAD activity, and (iii) growth of the mutant in media with short branched-chain fatty acids related to the expected products of the BCKAD helped to correct the mutant defects in growth, pigmentation and development. The esg BCKAD appears to be involved in the synthesis of long branched-chain fatty acids since the mutant contained reduced levels of this class of compounds. Our results are consistent with a model in which the esg-encoded enzyme is involved in the synthesis of branched-chain fatty acids during vegetative growth, and these compounds are used later in cell-cell signalling during development.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1995

The primary structure of Clostridium septicum alpha-toxin exhibits similarity with that of Aeromonas hydrophila aerolysin.

The gene for Clostridium septicum alpha-toxin was cloned and expressed in Escherichia coli from C. septicum BX96. The toxin was determined to be 443 amino acids in length, with a 31-residue signal peptide that was removed from the toxin during secretion. No extended hydrophobic regions were observed in the mature toxin sequence. Expression of alpha-toxin in E. coli BL21 resulted in the production of ATpro, which was identical to native toxin from C. septicum with respect to activity and activation. The proteolytic activation site for alpha-toxin was determined to be on the carboxy-terminal side of arginine 398, which lies within the sequence KKRRGKR-398SVD. Previous work showing similarities in activation and mechanism between alpha-toxin and Aeromonas hydrophila aerolysin was extended to the primary structures of both toxins. The DNA-derived primary sequence of alpha-toxin exhibited 27% identity and 72% similarity over a 387-residue region with the primary structure of the A. hydrophila aerolysin toxin, a level of similarity heretofore unobserved between toxins produced by a gram-positive organism and a gram-negative organism.

Aeromonas hydrophila↗
PubMed1994

Sequence of the rec-2 locus of Haemophilus influenzae: homologies to comE-ORF3 of Bacillus subtilis and msbA of Escherichia coli.

The nucleotide sequence of a 4243-bp PstI fragment containing the rec-2 gene of Haemophilus influenzae was determined. The amino acid (aa) sequences of four putative proteins were deduced from the corresponding open reading frames (ORFs). The 2400-bp ORF2 accounted for rec-2, based on the sequences of DNA fragments that contained rec-2::mini-Tn10 mutations. The rec-2 gene encoded a putative 800-aa protein with a M(r) of 90,561. Sequence analysis suggested that the rec-2 product contained nine highly probable integral membrane-spanning segments. Database searches showed that rec-2 was homologous to the comE-ORF3 gene of Bacillus subtilis. This hypothesis is consistent with the known involvement of both of these genes in the passage of transforming DNA through the competent-cell envelope. Although the sequences of the other three ORFs were incomplete, sufficient data were available to allow inferences about their homologies to other genes. ORF4, which overlapped ORF1, was homologous to the Escherichia coli dnaK suppressor gene, dksA, and therefore was named dsh-1 (dnaK suppressor homolog). Mutations in dsh-1 and its putative promoter region caused a mild sensitivity to UV light, but did not affect DNA recombination. ORF3, located downstream from rec-2, was homologous to msbA, an essential gene of E. coli with extensive similarity to the ATP-dependent translocators. ORF3 was named msh-1 (msbA homolog). Mutations in msh-1 had no effects on genetic transformation. The close juxtaposition of rec-2 and msh-1 implied that the expression of msh-1 could be linked to the translation of the rec-2 ORF.

ATP-Binding Cassette Transporters↗
PubMed1994

DNA sequence of direct repeats of the sulI gene of plasmid pSa.

The restriction enzyme and genetic map of the antibiotic-resistance region of plasmid pSa is related to Tn21 integrons by the insertion of 5.4 kb containing a chloramphenicol resistance gene (catII) and a 1.1-kb direct repeat. We report here the nucleotide sequences of both copies of the repeat with adjoining sequences. They were identical for 1065 bp and contained the entire coding sequence of the sulfanilamide resistance gene, sulI. Since only the first copy of the repeat confers sulfonamide resistance, this leads to the conclusion that no promoter was available for the second copy. The sequence of the pSa sulI gene was identical to several published sulI sequences from other plasmids. The first junction point of the catII-containing insert was identical to the sequence for pDG0100; the second junction occurred farther into the 3'-conserved segment of integrons than does that of pDG0100. A recent report of these junction sequences for pSa and pDG0100 differs from our sequences by one nucleotide. Two additional differences were an insert of 41 bases and a single base insertion between sulI and ORF341 in our sequence. Our sequenced regions have been assigned GenBank Accession Nos. UO4277 and UO4278 for the first and second sulI genes of pSa, respectively.

Amino Acid Sequence↗
PubMed1994

The complete nucleotide sequences of the SacBII Kan domain of the P1 pAD10-SacBII cloning vector and three cosmid cloning vectors: pTCF, svPHEP, and LAWRIST16.

The complete nucleotide sequence of the 16,009-bp SacBII Kan domain of the P1 pAD10-SacBII cloning vector and the sequences of three cosmid cloning vectors, pTCF (7941 bp), svPHEP (9201 bp), and LAWRIST16 (5194 bp) have been determined. A modified diatomaceous earth (Prep-A-Gene)-based procedure, which rapidly yields highly supercoiled double-stranded DNA from recombinant P1 and cosmid clones suitable for generating shotgun libraries, also has been developed. The isolated recombinant DNAs were physically sheared to generate 1- to 2-kb fragments that then were blunt-ended and subcloned into double-stranded pUC-based sequencing vectors. The double-stranded sequencing templates were isolated by an alkaline lysis method and subjected to Taq polymerase catalyzed fluorescent end-labeled primer cycle sequencing. After shotgun sequence assembly, contig gaps were closed and ambiguities were resolved via Sequenase catalyzed fluorescent dye-terminator sequencing.

Bacteriophage P1↗
PubMed1993

The hemoglobins of the bullfrog, Rana catesbeiana. The cDNA-derived amino acid sequences of the alpha chains of adult hemoglobins B and C: their roles in deoxygenation-induced aggregation.

The adult bullfrog (Rana catesbeiana) has two major tetrameric hemoglobins, B and C, which share a common beta chain but have different alpha chains. Components B and C associate upon deoxygenation to form a complex of the form BC2, a trimer of tetramers that depends on contacts between the alpha B and alpha C chains. Nucleotide sequences of cDNA transcripts for these chains have been determined. Transcripts were identified by analysis of the amino acid compositions of the tryptic peptides of the components and by partial amino acid sequencing. These results, together with the amino acid sequence of the beta chain (Tam, L.-T., Gray, G. P., and Riggs, A. F. (1986) J. Biol. Chem. 261, 8290-8294), permit an analysis of the structures of the alpha 2 beta 2 tetramers of hemoglobins B and C. Molecular modeling suggests possible residues at the alpha B-alpha C interfaces in the BC2 trimer and additional alpha C-alpha C contacts that would form a closed ring of six alpha chain subunits that would further stabilize the BC2 trimer. Phylogenetic analysis of the alpha B sequence suggests that it may be a "tadpole" chain, the temporal expression of which has shifted from larva to adult.

Amino Acid Sequence↗
PubMed1992

Duck liver 'malic' enzyme. Expression in Escherichia coli and characterization of the wild-type enzyme and site-directed mutants.

A cDNA for duck liver 'malic' enzyme (EC 1.1.1.40) was subcloned into pUC-8, and the active enzyme was expressed in Escherichia coli TG-2 cells as a fusion protein including a 15-residue N-terminal leader from beta-galactosidase coded by the lacZ' gene. C99S and R70Q mutants of the enzyme were generated by the M13 mismatch technique. The recombinant enzymes were purified to near homogeneity by a simple two-step procedure and characterized relative to the enzyme isolated from duck liver. The natural duck enzyme has a subunit molecular mass of approx. 65 kDa, and the following kinetic parameters for oxidative decarboxylation of L-malate at pH 7.0: Km NADP+ (4.6 microM); Km L-malate (73 microM); kcat (160 s-1); Ka (2.4 microM) and Ka' (270 microM), dissociation constants of Mn2+ at 'tight' (activating) and 'weak' metal sites; and substrate inhibition (51% of kcat. at 8 mM-L-malate). Properties of the E. coli-derived recombinant wild-type enzyme are indistinguishable from those of the natural duck enzyme. Kinetic parameters of the R70Q mutant are relatively unaltered, indicating that Arg-70 is not required for the reaction. The C99S mutant has unchanged Km for NADP+ and parameters for the 'weak' sites (i.e. inhibition by L-malate, Ka'); however, kcat. decreased 3-fold and Km for L-malate and Ka each increased 4-fold, resulting in a catalytic efficiency [kcat./(Km NADP+ x Km L-malate x Ka)] equal to 3.7% of the natural duck enzyme. These results suggest that the positioning of Cys-99 in the sequence is important for proper binding of L-malate and bivalent metal ions.

Amino Acid Sequence↗
PubMed1992

Cloning and DNA sequence of the Mycobacterium fortuitum var fortuitum plasmid pAL5000.

The complete nucleotide sequence of the Mycobacterium fortuitum var fortuitum plasmid pAL5000 has been determined. Computer analysis of this 4821-bp plasmid for protein coding regions, based on mycobacterial codon usage preferences, reveals the presence of two putative protein coding regions immediately downstream from typical mycobacterial promoter and ribosome binding sites. Both open reading frames, ORF1 and ORF2, produced proteins with the predicted respective sizes previously shown in minicell expression experiments [A. Labidi et al. (1985) FEMS Microbiol. Lett. 30, 221-225]. ORF1 encodes a putative 20-kDa basic protein with characteristics of a DNA binding protein involved in plasmid DNA replication. ORF2 encodes a 67-kDa protein with an amino-terminal sequence suggestive of a transported protein and a possible transmembrane anchor near its carboxyl-terminal. The current sequence and its analysis are more consistent with the minicell expression experiments than the previously published sequence of the pAL5000 plasmid [J. Rauzier et al. (1988) Gene 71, 315-321].

Amino Acid Sequence↗
PubMed1991

DNA sequence of a Salmonella-specific DNA fragment and the use of oligonucleotide probes for Salmonella detection.

Hybridization specificity of a 1.8-kb HindIII DNA fragment isolated from Salmonella typhimurium by a molecular cloning technique was confirmed by colony hybridization with 327 Salmonella isolates of various serotypes and 56 non-Salmonella isolates including Enterobacteriaceae closely related to Salmonella, such as Escherichia coli, Klebsiella, Citrobacter and Shigella. It was found that this 1.8-kb DNA fragment was highly specific for all the Salmonella isolates tested. The DNA sequence of this 1.8-kb fragment was then determined by the dideoxynucleotide chain termination method. According to this DNA sequence, six oligonucleotide fragments ranging from 17- to 26-mer were then chemically synthesized and tested for their hybridization specificities. Results show that three of the six oligonucleotide fragments are highly specific for all 327 Salmonella strains tested and can be used as probes for the specific detection of Salmonella in foods or other samples.

Base Sequence↗
PubMed1990

The 56-kilodalton major protein antigen of Rickettsia tsutsugamushi: molecular cloning and sequence analysis of the sta56 gene and precise identification of a strain-specific epitope.

Lasting immunity against Rickettsia tsutsugamushi, the causative agent of scrub typhus fever, has been demonstrated to be strain specific. Two protein antigens of 110 and 56 kilodaltons (kDa) have been shown to exhibit strain-specific epitopes. The 56-kDa scrub typhus antigen (Sta56) is an abundant outer membrane protein of R. tsutsugamushi and is an antigen often recognized by humans infected with this obligate intracellular bacterium. In this study the complete gene encoding Sta56 (strain Karp) was cloned into pBR322 on a 2.3-kilobase genomic HindIII DNA fragment and the complete 56-kDa polypeptide was expressed in Escherichia coli. DNA sequence analysis of the 2.3-kilobase HindIII fragment revealed an open reading frame large enough to encode a 56-kDa polypeptide. A putative signal sequence was identified at the deduced amino terminus of the Sta56 polypeptide, and pulse-chase analysis of maxicells labeled with [35S]methionine demonstrated that a higher-molecular-weight precursor matures into the 56-kDa polypeptide. Epitope scanning analysis with synthetic peptides derived from the deduced amino acid sequence identified an octapeptide (located from amino acid residues 117 to 124) that was reactive with a Karp strain-specific monoclonal antibody (K13F88A). Other epitopes recognized by different monoclonal antibodies, including another Karp strain-specific monoclone (K1E106), were localized to different regions of the protein based on their reactivities with lambda gt11 recombinants expressing various portions of the sta56 gene.

Amino Acid Sequence↗
PubMed1989

Presence of the hypermodified nucleotide N6-(delta 2-isopentenyl)-2-methylthioadenosine prevents codon misreading by Escherichia coli phenylalanyl-transfer RNA.

The overall structure of transfer RNA is optimized for its various functions by a series of unique post-transcriptional nucleotide modifications. Since many of these modifications are conserved from prokaryotes through higher eukaryotes, it has been proposed that most modified nucleotides serve to optimize the ability of the tRNA to accurately interact with other components of the protein synthesizing machinery. When a cloned synthetic Escherichia coli tRNAPhe gene was transfected into a bacterial host that carried a defective phenylalanine tRNA-synthetase gene, tRNAPhe was overexpressed by 11-fold. As a result of this overexpression, an undermodified tRNAPhe species was produced that lacked only N6-(delta 2-isopentenyl)-2-methylthioadenosine (ms2i6A), a hypermodified nucleotide found immediately 3' to the anticodon of all major E. coli tRNAs that read UNN codons. To investigate the role of ms2i6A in E. coli tRNA, we compared the aminoacylation kinetics and in vitro codon-reading properties of the ms2i6A-lacking and normal fully modified tRNAPhe species. The results of these experiments indicate that while ms2i6A is not required for normal aminoacylation of tRNAPhe, its presence stabilizes codon-anticodon interaction and thereby prevents misreading of the genetic code.

Adenosine↗
PubMed1989

Automated methods for single-stranded DNA isolation and dideoxynucleotide DNA sequencing reactions on a robotic workstation.

Automated procedures have been developed for both the simultaneous isolation of 96 single-stranded M13 chimeric template DNAs in less than two hours, and for simultaneously pipetting 24 dideoxynucleotide sequencing reactions on a commercially available laboratory workstation. The DNA sequencing results obtained by either radiolabeled or fluorescent methods are consistent with the premise that automation of these portions of DNA sequencing projects will improve the reproducibility of the DNA isolation and the procedures for these normally labor-intensive steps provides an approach for rapid acquisition of large amounts of high quality, reproducible DNA sequence data.

Automation↗
PubMed1988

Aminoacylation of synthetic DNAs corresponding to Escherichia coli phenylalanine and lysine tRNAs.

Synthetic DNA oligomers (tDNAs) corresponding to Escherichia coli tRNA(Phe) or tRNA(Lys) have been synthesized with either deoxythymidine (dT) or deoxyuridine (dU) substituted in the positions occupied by ribouridine or its derivatives. The tDNAs inhibited the aminoacylation of their respective tRNAs with their cognate amino acids, but not the aminoacylation of tRNA(Leu) with Leu. In the presence of aminoacyl-tRNA synthetase, species of both a tDNA(Phe) synthesized with a 3' terminal riboadenosine and a tDNA(Lys) containing only deoxynucleotides could be aminoacylated with the appropriate amino acids, although the Michaelis constant Km and observed maximal rate Vmax values for aminoacylation were increased by three- to fourfold and decreased by two- to threefold, respectively. The aminoacylation of synthetic tDNAs demonstrates that the ribose backbone of a tRNA is not absolutely required for tRNA aminoacylation.

Base Sequence↗
PubMed1988

Nucleotide sequence of the gene coding for a 130-kDa mosquitocidal protein of Bacillus thuringiensis israelensis.

The nucleotide sequence of pVB131 containing the gene coding for a 130-kDa Bacillus thuringiensis israelensis (B.t.isr) mosquitocidal protein was determined. The pVB131 plasmid was constructed by Sekar and Carlton [Gene 33 (1985) 151-158]. Our sequencing revealed only one open reading frame large enough to code for a protein of 130 kDa. The translation start site was determined by sequencing the protein isolated from B.t.isr. The amino acid sequence of the protein was deduced from the nucleotide sequence, and its Mr was determined as 128,505. Immunological and biochemical analyses of B.t.isr mosquitocidal proteins indicated that the 130-kDa protein coded by pVB131 was indeed expressed in B.t.isr. Comparing the peptide sequence of the 130-kDa B.t.isr toxin with the sequences of other B.t. toxins having activities specific to lepidopteran species showed that several domains were highly homologous. This suggests that they are evolutionarily related to each other, and in the evolutionary process the sequences in the homologous domains that are important to the insecticidal activity have been conserved.

Amino Acid Sequence↗
PubMed1987

The chicken vimentin gene. Nucleotide sequence, regulatory elements, and comparison to the hamster gene.

Here we report the nucleotide sequence of the chicken vimentin gene and its deduced primary amino acid sequence. A comparison of this gene to other intermediate filament protein genes demonstrates that both exon size and position are strongly conserved features of this multigene family. In addition, the hamster and chicken vimentin genes exhibit strong identity at the level of nucleotide (74%) and amino acid (80%) sequence. Interestingly, 40% of total sequence diversity is localized to the N terminus or "head" region of these genes whereas other protein domains (rod and C terminus) are remarkably identical in both nucleotide (81%) and amino acid (89%) sequence. Even stronger amino acid identity (100%) is exhibited in certain subdomains which may define regions crucial for filament formation and function. Not surprisingly, vimentin is more homologous across animal species than it is to other intermediate filament protein members (e.g. desmin) within the same species. A comparison of 5'-flanking sequences of the hamster and chicken genes as well as other characterized promoter elements (SV40, HSV-TK) reveals homologous sequence elements which may define common and/or unique sites involved in the modulation of gene expression. The implications of these sequence elements for both tissue-specific and developmental expression of the vimentin gene are discussed.

Amino Acid Sequence↗