Open AccessMolecular cloning of cDNA for rat mitochondrial 3‐hydroxyacyl‐CoA dehydrogenase
Author(s) -
AMAYA Yoshihiro,
TAKIGUCHI Masaki,
HASHIMOTO Takashi,
MORI Masataka
Publication year - 1986
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1986.tb09541.x
Subject(s) - complementary dna , microbiology and biotechnology , biology , messenger rna , biochemistry , rna , dehydrogenase , polysome , escherichia coli , enzyme , gene , ribosome
Messenger RNA for 3‐hydroxyacyl‐CoA dehydrogenase, a mitochondrial matrix enzyme of fatty acid β‐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.