Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the enteric bacterium, Escherichia coli, acyl coenzyme A synthetase (fatty acid:CoA ligase (AMP-forming) EC 6.2.1.3) activates exogenous long-chain fatty acids concomitant with their transport across the inner membrane into metabolically active CoA thioesters. These compounds serve as substrates for acyl-CoA dehydrogenase in the first step in the process of beta-oxidation. The acyl-CoA synthetase structural gene, fadD, has been identified on clone 6D1 of the Kohara E. coli gene library and by a process of subcloning and complementation analyses shown to be contained on a 2.2-kilobase NcoI-ClaI fragment of genomic DNA. The polypeptide encoded within this DNA fragment was identified following T7 RNA polymerase-dependent induction and estimated to be M(r) = 62,000 using SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of acyl-CoA synthetase was determined by automated sequencing to be Met-Lys-Lys-Val-Trp-Leu-Asn-Arg-Tyr-Pro. Sequence analysis of the 2.2-kilobase NcoI-ClaI fragment revealed a single open reading frame encoding these amino acids as the first 10 residues of a protein with a molecular weight of 62,028. The initiation codon for methionine was TTG. Primer extension of total in vivo mRNA from two fadD-specific oligonucleotides defined the transcriptional start at an adenine residue 60 base pairs upstream from the predicted translational start site. Two FadR operator sites of the fadD gene were identified at positions -13 to -29 (OD1) and positions -99 to -115 (OD2) by DNase I footprinting. Comparisons of the predicted amino acid sequence of the E. coli acyl-CoA synthetase to the deduced amino acid sequences of the rat and yeast acyl-CoA synthetases and the firefly luciferase demonstrated that these enzymes shared a significant degree of similarity. Based on the similar reaction mechanisms of these four enzymes, this similarity may define a region required for the same function.
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PMID:Cloning, sequencing, and expression of the fadD gene of Escherichia coli encoding acyl coenzyme A synthetase. 146 45

Rat liver mRNA encoding the precursor of medium-chain acyl-CoA dehydrogenase was purified to near homogeneity by polysome immunoadsorption using a polyclonal, monospecific antibody. A single-stranded, 32P-labeled cDNA probe was synthesized using the enriched mRNA as template and was used to screen directly 15,000 colonies from a total rat liver cDNA library constructed in pBR322. One clone [600 base pairs (bp)] was positively identified by hybrid-selected translation combined with mitochondrial processing of translated products. Using the isolated rat cDNA as probe, 43,000 colonies from a human liver cDNA library were screened. Three overlapping clones (1100 bp, 500 bp, and 400 bp) were isolated and positively identified by hybrid-selected translation. The largest human cDNA clone was subcloned into the transcription vector pGEM-2, which contains a bacteriophage T7 RNA polymerase promoter. In vitro transcription of this recombinant, followed by in vitro translation, showed that the cDNA clone coded for approximately 80% of the medium-chain acyl-CoA dehydrogenase protein. The sizes of rat and human mRNAs encoding the precursor of medium-chain acyl-CoA dehydrogenase were 2.2 and 2.4 kilobases long, respectively, as determined by blot hybridization analysis of electrophoretically fractionated poly(A)+ RNA. Southern blot analysis of DNAs from human-rodent somatic cell hybrids with an isolated human cDNA assigned the gene coding for this enzyme to the short arm of chromosome 1, band p31. The chromosomal assignment was confirmed by in situ hybridization of the probe to human metaphase cells. Direct screening of cDNA libraries using a highly enriched mRNA to generate a probe, as demonstrated in this study, may provide the most rapid and convenient approach to cDNA cloning of low-abundance mRNAs.
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PMID:Molecular cloning of cDNAs encoding rat and human medium-chain acyl-CoA dehydrogenase and assignment of the gene to human chromosome 1. 346 13