In vitro transcription of DNA from the 70S RNA of Rous sarcoma virus: identification and characterization of various size classes of DNA transcripts

Abstract

DNA transcripts, ranging from 1,500 to 4,500 nucleotides in length, can be identified in the DNA product synthesized in vitro by the Rous sarcoma virus-associated RNA-directed DNA polymerase. Although these DNA transcripts are considerably larger than the size classes of the DNA product previously reported, they only represent a minor proportion (less than 5%) of the total DNA synthesized in standard reaction mixtures containing rate-limiting concentration of one of the four deoxynucleoside triphosphates. However, the proportion of these larger transcripts relative to the total DNA product increases substantially when the enzymatic synthesis of DNA is performed in the presence of equimolar concentrations of deoxynucleoside triphosphate precursors. Both rate-zonal sedimentation in alkaline sucrose and nucleic acid hybridization techniques have confirmed the length and genetic complexity of these larger DNA transcripts. The identification of large DNA chains in the DNA product synthesized in vitro by the avian oncornavirus RNA-directed DNA polymerase provides an explanation for the paradox that exists between the limited number of primer sites per 70S RNA genome, the small size of the bulk of the DNA product, and the extent of the Rous sarcoma virus genome represented by the DNA product.