Isolation and Characterization of mRNA from Mammary Gland of Lactating Cow

Abstract

MRNA was isolated from mammary glands of lactating cow by affinity chromatography on poly(U)-Sepharose. The mRNA was heterogeneous on 3% agarose gel electrophoresis in the presence of 6m urea. The molecular weight of the main peak was estimated to be 3.3 x 10⁵. The mRNA was translated in a cell-free protein synthesizing system derived from wheat germ extract, and the translation products were analysed by the indirect immunoprecipitation method using specific antisera for casein components. About 50% of the total protein directed by this mRNA was casein. The relative amounts of α_s1-, β-,and k-casein in the translation products were nearly the same as those in bovine milk. The immunoprecipitates were analysed on sodium dodecyl sulfatepolyacrylamide gradient gel (15~20%) electrophoresis, and their mobilities were compared with those of dephosphorylated and non-glycosylated casein as standard, α_s1- and k Casein synthesized in vitro migrated more slowly than standard caseins, while synthesized β-casein migrated slightly faster than the standard β-casein.