Molecular cloning of cDNA sequences for avian malic enzyme. Nutritional and hormonal regulation of malic enzyme mRNA levels in avian liver cells in vivo and in culture.
A double-stranded cDNA library constructed from the total poly(A+) RNA of goose uropygial gland was screened for recombinants containing sequences complementary to malic enzyme mRNA. Replicate arrays of 1400 colonies were hybridized independently with 32P-labeled cDNAs copied from two populations of hepatic RNA derived from tissues which differed by about 35-fold with respect to the relative synthesis of malic enzyme. Forty-eight of the colonies which gave differential signals were further screened by hybrid-selected translation. DNA from one of these contained an insert of 970 base pairs and selected an mRNA which directed the synthesis of malic enzyme in a cell-free system. The malic enzyme sequences were subcloned into the single-stranded bacteriophage M13mp8. The subclones were used to prepare 32P-labeled single-stranded hybridization probe. Northern analysis indicated that malic enzyme mRNA from both goose and chicken is about 2100 bases in length. Hepatic malic enzyme mRNA concentration is stimulated 30- to 50-fold or more when neonatal chicks or goslings, respectively, are fed for 24 h. When added to chick embryo hepatocytes in culture, triiodothyronine stimulated malic enzyme mRNA accumulation by more than 100-fold. Glucagon inhibited the thyroid hormone-stimulated accumulation of malic enzyme mRNA by 99%. In all instances, malic enzyme mRNA concentration was closely correlated with the relative rate of malic enzyme synthesis. These results suggest that nutritional and hormonal regulation of malic enzyme synthesis occurs at the pretranslational level.
D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D002645:Chickens; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis*; D004032:Diet; D005777:Geese; D005934:Glucagon / Q000494:pharmacology; D008099:Liver / Q000201:enzymology*; D008291:Malate Dehydrogenase / Q000235:genetics*; D011200:Poultry / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism*; D014284:Triiodothyronine / Q000494:pharmacology
D012333:RNA, Messenger; D014284:Triiodothyronine; D004247:DNA; D005934:Glucagon; D008291:Malate Dehydrogenase