[Cloning of double-stranded DNA--a transcript of rat transferrin mRNA]

Two-stage synthesis of double-stranded DNA was performed using purified rat transferrin mRNA as a template, reverse transcriptase and DNA polymerase I. Double-stranded transcripts of transferrin mRNA were cloned as recombinant plasmid derivatives of pBR322. The insert length in these plasmids varied from 150 to 1500 bp. Clones carrying transferrin mRNA sequences were identified using colony hybridization and Southern blot hybridization with 32P-cDNA probe. Nick-translated DNAs from transformed clones hybridized with a single component of rat liver polysomal RNA that corresponded to transferrin mRNA in its molecular weight (0.86 MD). In hybridization selection cell-free translation test cloned plasmid DNAs hybridized specifically with rat liver poly(A) +RNA that programmed the cell-free synthesis of a polypeptide identical to pretransferrin in antigenic properties and molecular weight.