Molecular cloning of cDNA for acetyl-coenzyme A carboxylase.
Poly(A)+ RNA from lactating rat mammary glands was size-fractionated to enrich the relative amount of acetyl-CoA carboxylase mRNA. The enriched mRNA was used to generate a lambda gt11 cDNA library. Initial screening with polyclonal antiserum to acetyl-CoA carboxylase produced three positive clones. Western blot analysis revealed that two clones, lambda DH3 and lambda KH18, synthesized 165,000-dalton proteins that were recognized by antibodies to acetyl-CoA carboxylase and beta-galactosidase, indicating that acetyl-CoA carboxylase/beta-galactosidase fusion proteins were produced. Competition experiments with purified acetyl-CoA carboxylase further demonstrated that the fusion proteins contained acetyl-CoA carboxylase protein segments. Antibodies which are specific to the fusion proteins were isolated. These antibodies cross-reacted only with acetyl-CoA carboxylase in a preparation of partially purified acetyl-CoA carboxylase. In addition, the antibodies immunoprecipitated enzyme activity from a crude liver homogenate. Northern blot analysis of total RNA revealed two RNA species: one 10 kilobases and the other 3.0 kilobases. The levels of these RNA species increased when starved animals were fed a fat-free diet, indicating that they are coordinately regulated.
D000103:Acetyl-CoA Carboxylase / Q000235:genetics*; D000818:Animals; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004041:Dietary Fats / Q000008:administration & dosage; D005260:Female; D007774:Lactation; D008025:Ligases / Q000235:genetics*; D008099:Liver / Q000201:enzymology; D008297:Male; D008321:Mammary Glands, Animal / Q000201:enzymology; D008970:Molecular Weight; D011061:Poly A / Q000378:metabolism; D011247:Pregnancy; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013217:Starvation / Q000378:metabolism
D004041:Dietary Fats; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D004247:DNA; D008025:Ligases; D000103:Acetyl-CoA Carboxylase