MOLECULAR-CLONING OF CDNA FOR RAT MITOCHONDRIAL 3-OXOACYL-COA THIOLASE

Messenger RNA of rat 3-oxoacyl-CoA thiolase (acetyl-CoA acyltransferase), a mitochondrial matrix enzyme involved in fatty acid .beta.-oxidation, was enriched by immunoprecipitation of rat liver free polysomes and recombinant plasmids were prepared from the enriched mRNA by a modification of the vector-primer method of Okayama and Berg. The transformants were initially screened for 3-oxoacyl-CoA thiolase cDNA sequences by differential colony hybridization with [32P]cDNAs, synthesized from the immunopurified and unpurified mRNAs. The cDNA clones for 3-oxoacyl-CoA thiolase were identified by hybrid-arrested translation and hybrid-selected translation. One of the clones, designaed pT1-1, contained a 700-base insert and hybridized to a mRNA species of 1.6 .times. 103 bases in rat liver. The transformants were rescreened using the cDNA insert of pT1-1 as a hybridization probe and a clone (pT1-19) with a 1.5 .times. 103-base insert was obtained. Activity and concentration of 3-oxoacyl-CoA thiolase mRNA were quantified by in vitro translation and dot-blot analysis using the cDNA insert as a hybridization probe. The level of translatable and hybridizable mRNA in rat liver was increased about 5.1-fold and 4.6-fold, respectively, after administration of di-(2-ethylhexyl)phthalate, a potent inducer of the enzyme. The 3-oxoacyl-CoA thiolase mRNA levels thus determined correlated closely with levels of the activity and amount of this enzyme.