| Abstract: | 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. |
