Isolation of rat liver 5S RNA genes

Abstract

A method for the isolation of structural genes, whose transcripts do not contain terminal poly(A) sequences, is presented. Poly(A) tails of predicted length were synthesized at the 3'-OH ends of RNA molecules employing Escherichia coli ATP : RNA adenyltransferase (EC 2.7.7.19). The gene isolation was performed in two steps: (a) enrichment of DNA fragments carrying the genes of interest, (b) subsequent cloning and amplification of these fragments. The enrichment of given gene sequences requires separate purifications of each of two complementary DNA chains, followed by their annealing. Rat liver 5S RNA genes were isolated by this method. DNA (+)chains were obtained by hybridizing EcoRI-digested and denatured rat liver DNA fragments with the 5S RNA-poly(A). The complex was purified on an oligo(dT)-cellulose column. DNA (-)chains were purified by hybridizing DNA fragments with the 5S cDNA which was coupled to oligo(dT)-cellulose. In the isolated and annealed double-stranded DNA preparation the enrichment of 5S DNA was 350--400-fold, when compared with the total genomic DNA. These purified DNA sequences were inserted into the EcoRI site of pBR325. Out of 1200 recombinant-plasmid colonies, analyzed in the colony-hybridization test, 4 hybridized with [125I]5S RNA.