Construction and selection of recombinant plasmids containing full-length complementary DNAs corresponding to rat...

We have used a synthetic deoxydecanucleotide to generate an insulin-specific cDNA probe suitable for selecting transformants that contain nearly full-length cDNAs corresponding to the mRNAs coding for rat insulins I and II. Double-stranded cDNA was synthesized from x-ray-induced rat insulinoma poly(A)-RNA, inserted in pBR322 plasmid DNA by the homopolymeric tailing technique, and cloned in Escherichia coli chi 1776. Colony hybridization with oligonucleotide-primed cDNA yielded 16 positive clones of which 7 corresponded to rat insulin I mRNA and 9 to rat insulin II mRNA. Restriction endonuclease maps of representative clones of each group indicated that these contained the complete coding sequences, as was confirmed by nucleotide sequence analysis of the 5' region of the cloned DNA for rat insulin II. Nucleotide sequence analysis also established the amino acid sequence of the prepeptide of rat preproinsulin II. Comparison of the amino acid sequence of the prepeptides of rat preproinsulin I and II shows that three conservative amino acid substitutions have occurred in this region of the molecule.