Molecular cloning of cDNA for rat mitochondrial 3-hydroxyacyl-CoA dehydrogenase.
Messenger RNA for 3-hydroxyacyl-CoA dehydrogenase, a mitochondrial matrix enzyme of fatty acid beta-oxidation, was purified from livers of di(2-ethylhexyl)phthalate-treated rats by immunoadsorption of hepatic free polysomes to fixed cells of Staphylococcus aureus and enrichment for poly(A)-rich RNA by oligo(dT)-cellulose chromatography. Plasmid cDNA was constructed from this poly(A)-rich RNA by a modification of the method of Okayama and Berg and was transformed into the Escherichia coli DH1 strain. Plasmids containing cDNA sequences coding for 3-hydroxyacyl-CoA dehydrogenase were screened by differential colony hybridization, and were identified by hybrid-arrested translation and hybrid-selected translation. Plasmid pHADH-1, which contains a 1400-base-pair insert, hybridized to rat 3-hydroxyacyl-CoA dehydrogenase mRNA with a length of 1700 bases. Determination of the dehydrogenase mRNA by in vitro translation and dot-blot analysis with the cDNA probe showed that the induction of the enzyme in rat liver by di(2-ethylhexyl)phthalate could be attributed to an increase in the mRNA concentration.
Amaya
Y
Y
Takiguchi
M
M
Hashimoto
T
T
Mori
M
M
D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant; D004051:Diethylhexyl Phthalate / Q000494:pharmacology; D007120:Immunochemistry; D066298:In Vitro Techniques; D008297:Male; D008930:Mitochondria, Liver / Q000201:enzymology*; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D011061:Poly A / Q000032:analysis; D014176:Protein Biosynthesis; D012313:RNA / Q000032:analysis; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D004274:DNA, Recombinant; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D004247:DNA; D004051:Diethylhexyl Phthalate; D015094:3-Hydroxyacyl CoA Dehydrogenases; D004262:DNA Restriction Enzymes