Molecular cloning of a gene encoding a 45,000-dalton polypeptide associated with bile acid 7-dehydroxylation in Eubacterium sp. strain VPI 12708. 1988

W B White, and J P Coleman, and P B Hylemon
Department of Microbiology and Immunology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298.

Eubacterium sp. strain VPI 12708 is an intestinal anaerobic bacterium which possesses an inducible bile acid 7-dehydroxylation activity. Two cholic acid-induced polypeptides with apparent molecular weights of 27,000 and 45,000, respectively, coeluted with bile acid 7-dehydroxylation activity upon anaerobic high-performance gel filtration chromatography of crude cellular protein extracts. The 45,000-dalton polypeptide was purified to greater than 95% homogeneity by high-performance liquid chromatography gel filtration and high-performance liquid-DEAE chromatography. The first 28 amino acid residues of the N terminus of this polypeptide were determined by gas-phase sequencing, and a corresponding mixed oligonucleotide (20-mer) was synthesized. Southern blot analysis of EcoRI total digests of chromosomal DNA showed a 2.6-kilobase fragment which hybridized to the 32P-labeled 20-mer. This fragment was enriched for by size fractionation of an EcoRI total digest of genomic DNA and ligated into bacteriophage lambda gt11. Recombinant phage containing the putative gene encoding the 45,000-dalton polypeptide were detected with the 32P-labeled 20-mer by plaque hybridization techniques. The insert was 2.6 kilobases in length and may contain the entire coding sequence for the 45,000-dalton polypeptide. The 2.6-kilobase insert was subcloned into pUC8 and transformed into Escherichia coli DH5 alpha. However, the 45,000-dalton polypeptide was not detected in cell extracts of this organism when specific antibody was used. Preliminary nucleic acid sequence data correlated exactly with the amino acid sequence. A cholic acid-induced mRNA species of greater than 6 kilobases in size was identified by Northern (RNA) blot analysis of total RNA, suggesting that the gene coding for this polypeptide is part of a larger operon.

UI MeSH Term Description Entries
D007118 Immunoassay A technique using antibodies for identifying or quantifying a substance. Usually the substance being studied serves as antigen both in antibody production and in measurement of antibody by the test substance. Immunochromatographic Assay,Assay, Immunochromatographic,Assays, Immunochromatographic,Immunoassays,Immunochromatographic Assays
D007525 Isoelectric Focusing Electrophoresis in which a pH gradient is established in a gel medium and proteins migrate until they reach the site (or focus) at which the pH is equal to their isoelectric point. Electrofocusing,Focusing, Isoelectric
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D009693 Nucleic Acid Hybridization Widely used technique which exploits the ability of complementary sequences in single-stranded DNAs or RNAs to pair with each other to form a double helix. Hybridization can take place between two complimentary DNA sequences, between a single-stranded DNA and a complementary RNA, or between two RNA sequences. The technique is used to detect and isolate specific sequences, measure homology, or define other characteristics of one or both strands. (Kendrew, Encyclopedia of Molecular Biology, 1994, p503) Genomic Hybridization,Acid Hybridization, Nucleic,Acid Hybridizations, Nucleic,Genomic Hybridizations,Hybridization, Genomic,Hybridization, Nucleic Acid,Hybridizations, Genomic,Hybridizations, Nucleic Acid,Nucleic Acid Hybridizations
D010088 Oxidoreductases The class of all enzymes catalyzing oxidoreduction reactions. The substrate that is oxidized is regarded as a hydrogen donor. The systematic name is based on donor:acceptor oxidoreductase. The recommended name will be dehydrogenase, wherever this is possible; as an alternative, reductase can be used. Oxidase is only used in cases where O2 is the acceptor. (Enzyme Nomenclature, 1992, p9) Dehydrogenases,Oxidases,Oxidoreductase,Reductases,Dehydrogenase,Oxidase,Reductase
D010455 Peptides Members of the class of compounds composed of AMINO ACIDS joined together by peptide bonds between adjacent amino acids into linear, branched or cyclical structures. OLIGOPEPTIDES are composed of approximately 2-12 amino acids. Polypeptides are composed of approximately 13 or more amino acids. PROTEINS are considered to be larger versions of peptides that can form into complex structures such as ENZYMES and RECEPTORS. Peptide,Polypeptide,Polypeptides
D002850 Chromatography, Gel Chromatography on non-ionic gels without regard to the mechanism of solute discrimination. Chromatography, Exclusion,Chromatography, Gel Permeation,Chromatography, Molecular Sieve,Gel Filtration,Gel Filtration Chromatography,Chromatography, Size Exclusion,Exclusion Chromatography,Gel Chromatography,Gel Permeation Chromatography,Molecular Sieve Chromatography,Chromatography, Gel Filtration,Exclusion Chromatography, Size,Filtration Chromatography, Gel,Filtration, Gel,Sieve Chromatography, Molecular,Size Exclusion Chromatography
D003001 Cloning, Molecular The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells. Molecular Cloning
D004262 DNA Restriction Enzymes Enzymes that are part of the restriction-modification systems. They catalyze the endonucleolytic cleavage of DNA sequences which lack the species-specific methylation pattern in the host cell's DNA. Cleavage yields random or specific double-stranded fragments with terminal 5'-phosphates. The function of restriction enzymes is to destroy any foreign DNA that invades the host cell. Most have been studied in bacterial systems, but a few have been found in eukaryotic organisms. They are also used as tools for the systematic dissection and mapping of chromosomes, in the determination of base sequences of DNAs, and have made it possible to splice and recombine genes from one organism into the genome of another. EC 3.21.1. Restriction Endonucleases,DNA Restriction Enzyme,Restriction Endonuclease,Endonuclease, Restriction,Endonucleases, Restriction,Enzymes, DNA Restriction,Restriction Enzyme, DNA,Restriction Enzymes, DNA
D004591 Electrophoresis, Polyacrylamide Gel Electrophoresis in which a polyacrylamide gel is used as the diffusion medium. Polyacrylamide Gel Electrophoresis,SDS-PAGE,Sodium Dodecyl Sulfate-PAGE,Gel Electrophoresis, Polyacrylamide,SDS PAGE,Sodium Dodecyl Sulfate PAGE,Sodium Dodecyl Sulfate-PAGEs

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