Isolation and characterization of milk protein nuclear RNAs in rat mammary gland

Abstract

Methods have been developed to isolate high-molecular-weight pre-mRNAs from lactating mammary gland, a tissue high in RNase levels. These methods involved isolation of nuclei at −20°C in 50% glycerol, and nucleic acid extraction using a guanidine thiocyanate-CsCl protocol. Specific RNAs were detected using α-, β-, and γ-casein and whey acidic protein nick-translated cDNA and genomic DNA probes by hybridization in situ to pre-mRNAs fractionated on agarose gels containing 10 m m methylmercuric hydroxide. Using these techniques it was possible to isolate poly(A)-containing gene-sized primary transcripts in the case of the two smaller genes, β-casein and whey acidic protein. A very complex pattern of pre-mRNAs was observed for the β-casein transcripts, including detection of a species which may represent an excised intron. Probes for the α- and γ-casein genes revealed much lower abundance and complexity of RNA precursors. These methods have proven useful in the initial analysis of RNA processing of these hormonally regulated milk protein gene transcripts.