Molecular cloning of a gene encoding a 45,000-dalton polypeptide associated with bile acid 7-dehydroxylation in Eubacterium sp. strain VPI 12708
Eubacterium sp. strain VPI 12708 is an intestinal anaerobic bacterium which possesses an inducible bile acid 7-dehydroxylation activity. Two choli 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 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 /sup 32/P-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 /sup 32/P-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 polypeptides. The 2.6-kilobase insert was subcloned into pUC8 and transformed into Escherichia coli DH5..cap 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.
- Research Organization:
- Virginia Commonwealth Univ., Richmond
- OSTI ID:
- 5409415
- Journal Information:
- J. Bacteriol.; (United States), Vol. 170:2
- Country of Publication:
- United States
- Language:
- English
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BACTERIA
MOLECULAR BIOLOGY
BILE ACIDS
BIOCHEMICAL REACTION KINETICS
POLYPEPTIDES
GENES
GENETIC MAPPING
AMINO ACID SEQUENCE
BACTERIOPHAGES
DNA SEQUENCING
DNA-CLONING
HYBRIDIZATION
PHOSPHORUS 32
RECOMBINANT DNA
TRACER TECHNIQUES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CARBOXYLIC ACIDS
CLONING
DAYS LIVING RADIOISOTOPES
DNA
HYDROXY COMPOUNDS
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGHT NUCLEI
MAPPING
MICROORGANISMS
MOLECULAR STRUCTURE
NUCLEI
NUCLEIC ACIDS
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
PARASITES
PEPTIDES
PHOSPHORUS ISOTOPES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
STEROIDS
STEROLS
STRUCTURAL CHEMICAL ANALYSIS
VIRUSES
550401* - Genetics- Tracer Techniques
550701 - Microbiology- Tracer Techniques