Ordered transcription of RNA tumor virus genomes

Abstract

Recent improvements in the reaction conditions have allowed a detailed characterization of the DNA products synthesized in vitro by the reverse transcriptase of RNA tumor viruses from the 70 S genome. We have investigated the size distribution of DNA products synthesized in vitro under different reaction conditions using both Rous sarcoma virus (RSV) and Moloney murine leukemia virus (M-MuLV). Analysis of the reaction products on polyacrylamide gels shows that the majority of DNA synthesized, especially at high concentrations of dNTPs, comprised a set of DNA chains of discrete length the longest of which cover a major fraction of the genome. The effect of varying the time of incubation was to increase the fraction of the total DNA in longer chains. However, small DNA chains less than 200 nucleotides long were still abundant after 500 minutes. By varying which of the four dNTPs was at low concentration we revealed two classes of DNA fragments of discrete length, those present regardless of which dNTP was at low concentration (structural stops) and those which were made only when a specific dNTP was at a low concentration (sequence stops). A strong structural stop which gave a fragment 135 nucleotides long (M-MuLV) or 100 nucleotides long (RSV) comprised more than half of the population of DNA molecules even at the highest dNTP concentrations. The DNA products of the endogenous and exogenous (reconstructed) reactions were identical. The DNA chains of discrete length were initiated with a tRNA (tRNA^(Trp) RSV, tRNA^(Pro) M-MuLV). The DNA fragments of both RSV and M-MuLV have a common initiation sequence. We have sequenced the first 18 nucleotides of M-MuLV. The initial sequences of RSV (AATGAAGC) and M-MuLV (AATGAAAGA) are remarkably similar suggesting a common evolutionary ancestry. We analyzed the pyrimidine tracts of DNA chains of increasing length by electrophoresis followed by homochromatography. The pyrimidine tracts of the shorter pieces form a subset of the pyrimidine tracts of the longer pieces. These data demonstrate that the in vitro products of discrete length were initiated with a tRNA primer at a unique site along the 35 S genome and grew by linear extension.