Localization of the 26-S RNA sequence on the viral genome type 42-S RNA isolated from SFV-infected cells

Abstract

32P-labeled virus-specific 42- and 26-S RNA has been prepared from BHK 21 cells infected with the togavirus Semliki Forest virus. Analyses of the oligonucleotides generated from these RNA species after digestion with RNAse T 1 by two-dimensional polyacrylamide gel electrophoresis (T 1-fingerprints) show that both RNA species contain poly(A) sequences and that the oligonucleotides of the 26-S RNA represent a subset of those present in 42-S RNA. The following findings indicate that the different nucleotide sequences are present on the 42-S RNA in the order 5′-terminus-42-S RNA-specific sequences-26-S RNA sequences-poly(A)-A OH. (1) Identical oligonucleotide patterns are found in the T 1-fingerprints of the 26-S RNA accumulating in infected cells and of poly(A)-containing fragments of the 42-S RNA which sediment around 26 S on sucrose density gradients. (2) The poly(A) fragments were isolated from T 1-fingerprints of 42-and 26-S RNA, digested with pancreatic RNAse, and the reaction products analyzed by electrophoresis on DEAE paper. The results indicate that both fragments contain a heteropolymeric sequence consisting of U 6C 2(AC) 1 (AU) 1(AAU) 1. (3) GMP has been detected in all oligonucleotides derived from T 1-fingerprints which have been analyzed so far, except in the poly(A)-containing oligonucleotides, indicating that the latter contain the 3′-termini of the molecules from which they were derived. (4) [ 3H]Adenosine-labeled 42-S RNA was digested with RNAse T 1 and pancreatic RNAse, the poly(A) fragments were isolated, and an [ 3H]adenosine to [ 3H]AMP radioactivity ratio of 1 to 97 was determined for these fragments. Possible implications of the organization of the different sequences on the 42-S RNA on its translation and the replication of SFV are discussed.