Stepwise biosynthesis in vitro of globin genes from globin mRNA by DNA polymerase of avian myeloblastosis virus.

Abstract

Two approaches were explored for the synthesis of double-stranded DNA from single-stranded DNA template complementary to rabbit 9S globin mRNA (cDNA). cDNA was elongated with dCMP or dTMP homopolymeric tracts using terminal deoxynucleotidyltransferase (EC 2.7.7.31; nucleosidetriphosphate:DNA deoxynucleotidylexotransferase). cDNA-dC, in the presence of an oligo(dG)1O primer, was an efficient template with either DNA polymerase of Escherichia coli (EC 2.7.7.7; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase) or RNA-directed DNA polymerase of avian myeloblastosis virus. cDNA-dT [with an oligo(dA)1O primer] functioned as template only with E. coli polymerase. cDNA, without homopolymeric tails, was also efficiently copied in the absence of oligonucleotide primer, by DNA polymerase of avian myeloblastosis virus or of E. coli. The product of the reaction consisted of long hairpin molecules which could be converted into DNA duplex (melting temperature, 93.degree.) by digestion with single-strand nuclease S1. A loop structure on the 3'' end of cDNA allowed DNA synthesis to take place by a self-priming mechanism. Some of the double-stranded DNA synthesized corresponded to the entire sequence of the 9S mRNA template. The synthesis of full-length double-stranded DNA from mouse globin mRNA and immunoglobulin light chain mRNA is also discussed.