Cloning of cDNA sequences for murine malic enzyme and the identification of aberrantly large malic enzyme mRNA in MOD-1 null mice.

Abstract

Polysomes containing cytosolic malic enzyme mRNA and malic enzyme nascent chains were complexed with specific antibodies and purified by chromatography on protein A-Sepharose. When poly(A+) mRNA derived from the immunoselected polysomes was translated in vitro, full length malic enzyme (subunit Mr = 58,000) accounted for a significant fraction (approximately 20%) of the polypeptides synthesized. Double-stranded cDNA, synthesized using partially purified malic enzyme mRNA as a template, was inserted into pBR 322 and cloned. Twenty-five candidate malic enzyme cDNA clones were identified by differential hybridization. Four clones were studied further and each of these was shown to have malic enzyme cDNA sequences by hybrid-selected translation and specific immunoprecipitation. Plasmid pME1, which contains a 1400-base pair insert, hybridized to two mouse liver malic enzyme mRNAs with lengths of 2300 and 3500 bases. Similar analyses were performed on liver mRNAs isolated from MOD-1 mutant mice which lack cytosolic malic enzyme activity. These Northern blots disclosed a pair of aberrantly large malic enzyme mRNAs with lengths of 2800 and 4000 bases. Furthermore, anti-malic enzyme antibodies exclusively precipitated a polypeptide translation product with a Mr of 77,000 when MOD-1 mRNA was used to direct in vitro protein synthesis. Thus, it is possible that MOD-1 malic enzyme mRNA contains an additional polypeptide coding sequence. The translation of such a sequence might disrupt enzyme function and/or markedly decrease enzyme stability. The malic enzyme cDNA probe was also employed to demonstrate that the induction of malic enzyme in the livers of previously starved mice that were fed a high carbohydrate, fat-free diet was controlled pretranslationally by a parallel modulation of the malic enzyme mRNA concentration.