In vitro transcription of reconstituted 35S RNA·tRNA trp template·primer complexes by the avian oncornavirus DNA polymerase Effect of temperature on the size of the DNA transcripts

Abstract

The synthesis of long DNA transcripts from avian sarcoma virus 35S RNA·tRNA trp template·primer complexes in reconstituted reactions in vitro is temperature dependent. At 15° DNA transcripts accumulate that are 100 nucleotides in length (DNA 100), whereas at 30° DNA transcripts ranging to 3000 nucleotides in length (DNA >100) are synthesized from these template·primer complexes. By hybridization analysis we have demonstrated that DNA 100 synthesized at 15° contains nucleotide sequences complementary to the 5′ end of the viral RNA genome whereas longer DNA transcripts synthesized at 30° contain nucleotide sequences complementary to the 3′ terminus of the viral genome. Pulse-chase experiments indicate that the 5′-located DNA 100 transcripts are precursors of the larger DNA transcripts. These data are consistent with the elongation of 5′ terminally located DNA transcripts at the 3′ end of the viral genome.