Cloning of epoxide hydratase complementary DNA.
Tightly membrane-bound polysomes were isolated from livers of rats administered trans-stilbene oxide. Epoxide hydratase mRNA was enriched from these polysomes using immunochemical techniques and oligo(dT)-cellulose chromatography. This resulted in an increase in message concentration over that found in noninduced membrane-bound cDNA, synthesized from enriched mRNA, was inserted into the ampicillin resistance gene of pBR322 using oligo(dG)-oligo(dC) tailing. Clones containing sequences complementary to epoxide hydratase mRNA were selected by differential colony hybridization using [32P]cDNA synthesized from immunoenriched mRNA and [32P]cDNA synthesized from nonenriched mRNA. Plasmids from four clones, which only annealed with the enriched probe, were isolated and shown to specifically hybridize with epoxide hydratase mRNA by hybrid selection-translation. A composite restriction endonuclease map of the plasmid inserts was constructed which spanned 1310 base pairs and represented approximately 80% of the message sequence. The 3'-5' orientation of this map relative to the epoxide hydratase mRNA was also determined.
D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant / Q000378:metabolism*; D004851:Epoxide Hydrolases / Q000235:genetics*; D008099:Liver / Q000201:enzymology; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D011132:Polyribosomes / Q000201:enzymology; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics
D004274:DNA, Recombinant; D012333:RNA, Messenger; D004262:DNA Restriction Enzymes; D004851:Epoxide Hydrolases