Mapping RNase T -Resistant Oligonucleotides of Avian Tumor

The large RNase T,-resistant oligonucleotides of the nondefective (nd) Rous sarcoma viruses (RSV): Prague RSV of subgroup B (PR-B), PR-C and B77 of subgroup C; of their transformation-defective (td) deletion mutants: td PR-B, td PR-C, and td B77; and of replication-defective (rd) RSV( -) were completely or partially mapped on the 30 to 40S viral RNAs. The location of a given oligonucleotide relative to the poly(A) terminus of the viral RNAs was directly deduced from the smallest size of the poly(A)-tagged RNA fragment from which it could be isolated. Identification of distinct oligonucleotides was based on their location in the electrophoretic/chromatographic fingerprint pattern and on analysis of their RNase A-resistant fragments. The following results were obtained. (i) The number of large oligonucleotides per poly(A)-tagged fragment increased with increasing size of the fragment. This implies that the genetic map is linear and that a given RNase T1-resistant oligonucleotide has, relative to the poly(A) end, the same location on all 30 to 40S RNA subunits of a given 60 to 70S viral RNA complex. (ii) Three sarcoma-specific oligonucleotides were identified in the RNAs of PR-B, PR-C and B77 by comparison with the RNAs of the corresponding td viruses. The sarcoma-specific oligonucleotides of these three sarcoma viruses had very similar compositions. Based on the map positions of these oligonucleotides, sarcoma-specific sequences of nd viral RNAs were estimated to map between 6.6 and 20% away from the poly(A) end. (iii) As far as analyzed, the oligonucleotide maps of the RNAs of nd and td PR-B were the same with the exception of the sarcoma-specific sequences, as were the maps of nd and td B77 RNAs. (iv) Comparisons of the map locations of oligonucleotides from the three nd sarcoma virus strains analyzed suggested that homologous oligonucleotides are found in certain homologous map positions of the respective RNAs. This implies that these three virus strains probably have similar gene orders. (v) The complexity in daltons of the RNA of PR-C was 3.22 x 106 and that of B77 was 3.02 x 106. (vi) The RNase T,-resistant oligonucleotides of poly(A)-tagged RNA fragments ranging up to 15S from each nd/td virus pair studied here were very similar. This implies that they share a common heteropolymeric sequence at their poly(A) ends. (vii) Poly(A)-tagged 12S RNA fragments of all avian tumor viruses studied so far including that of RSV(-) shared one oligonucleotide, termed spot C, which mapped very near the poly(A) end. It may be part of a short terminal heteropolymeric sequence common to all avian tumor virus RNAs investigated. The following maps emerged for the poly(A) terminal sequences of the nd/td virus RNAs which were analyzed: they start with poly(A) (molecular weight = 60,000), continue with a heteropolymeric sequence shared partially or completely by nd and td viruses (molecular weight = 140,000), which is followed by sarcoma-specific sequences (molecular weight 300,000 to 450,000) in the case of nd sarcoma virus RNAs. Several mechanisms are discussed to explain the generation of deletion mutants from nd viruses.