At least 104 nucleotides are transposed from the 5' terminus of the avian sarcoma virus genome to the 5' termini of smaller viral mRNAs.

Cells producing avian sarcoma virus (ASV) contain at least three virus-specific mRNAs, two of which are encoded within the 3' half of the viral genome. Each of these viral RNAs can hybridize with single-stranded DNA(cDNA5') that is complementary to a sequence of 101 nucleotides found at the 5' terminus of the ASV genome, but not within the 3' half of the genome. We proposed previously (Weiss, Varmus and Bishop, 1977) that this nucleotide sequence may be transposed to the 5' termini of viral mRNAs during the genesis of these RNAs. We now substantiate this proposal by reporting the isolation and chemical characterization of the nucleotide sequences complementary to cDNA5' in the genome and mRNAs of the Prague B strain of ASV. We isolated the three identified classes of ASVmRNA (38, 28 and 21S) by molecular hybridization; each class of RNA contained a "capped" oligonucleotide identical to that found at the 5' terminus of the ASV genome. When hybridized with cDNA5', each class of RNA gave rise to RNAase-resistant duplex hybrids that probably encompassed the full extent of cDNA5'. The molar yields of duplex conformed approximately to the number of virus-specific RNA molecules in the initial samples; hence most if not all of the molecules of virus-specific RNA could give rise to the duplexes. The duplexes prepared from the various RNAs all contained the capped oligonucleotide found at the 5' terminus of the viral genome and had identical "fingerprints" when analyzed by two-dimensional fractionation following hydrolysis with RNAase T1. In contrast, RNA representing the 3' half of the ASV genome did not form hybrids with cDNA5'. We conclude that a sequence of more than 100 nucleotides is transposed from the 5' end of the ASV genome to the 5' termini of smaller viral RNAs during the genesis of these RNAs. Transposition of nucleotide sequences during the production of mRNA has now been described for three families of animal viruses and may be a common feature of mRNA biogenesis in eucaryotic cells. The mechanism of transposition, however, and the function of the transposed sequences are not known.     

Homologous terminal sequences of the genome double-stranded RNAs of bluetongue virus.

The 3'-terminal sequences of the 10 double-stranded RNA genome segments of bluetongue virus (serotypes 10 and 11) were determined. The double-stranded RNAs were 3' labeled with [5'-32P]pCp and resolved into 10 segments by electrophoresis. After denaturation, the two complementary strands of segments 4 through 10 were resolved into fast- and slow-migrating species by polyacrylamide gel electrophoresis, and their 3' end sequences were determined. Complete RNase T1 digestion of the individual 3'-labeled double-stranded RNA segments yielded two labeled oligonucleotides, one of which migrated faster than the other on 20% polyacrylamide-7 M urea gels. Sequence analyses of the two oligonucleotides of segments 4 through 10 confirmed the corresponding RNA sequence data. For RNA segments 1 through 3 the oligonucleotide analyses gave comparable results. The 3'-terminal sequences of the fast-migrating RNA species were HOCAAUUU. . . ; those of the slow-migrating RNA species were HOCAUUCACA. . . . Similar results were obtained for double-stranded RNA from bluetongue virus serotypes 10 and 11. Beyond the common termini, the sequences for each segment varied considerably.     

Nucleotide sequence of cucumber mosaic virus RNA. 1. Presence of a sequence complementary to part of the viral satellite RNA and homologies with other viral RNAs

The nucleotide sequence of the 3389 residues of RNA 1 (Mr 1.15 X 10(6) of the Q strain of cucumber mosaic virus (CMV) was determined, completing the primary structure of the CMV genome (8617 nucleotides). CMV RNA 1 was sequenced by the dideoxy-chain-termination method using M13 clones carrying RNA 1 sequences as well as synthetic oligonucleotide primers on RNA 1 as a template. At the 5' end of the RNA there are 97 noncoding residues between the cap structure and the first AUG (98-100), which is the start of a single long open-reading frame. This reading frame encodes a translation product of 991 amino acid residues (Mr 110791) and stops 319 nucleotide residues from the 3' end of RNA 1. In addition to the conserved 3' region present in all CMV RNAs (307 residues in RNA 1), RNAs 1 and 2 have highly homologous 5' leader sequences, a 12-nucleotide segment of which is also conserved in the corresponding RNAs of brome mosaic virus (BMV). CMV satellite RNA can form stable base pairs with a region of CMV RNAs 1 and 2 including this 12-nucleotide sequence, implying a regulatory function. This conserved sequence is part of a hairpin structure in RNAs 1 and 2 of CMV and BMV and in CMV satellite RNA. The entire translation products of RNA 1 of CMV and BMV could be aligned with significant homology. Less prominent homologies were found with alfalfa mosaic virus RNA 1 translation product and with tobacco mosaic virus Mr-126000 protein.     

Nucleotide sequences at the terminal of La Crosse virus RNAs.

The 5' and 3'-terminal sequences of the three RNA molecules which make up the genomes of La Crosse virus are reported. Eleven nucleotides at both the 5' and 3' termini of all three RNAs are conserved and complementary. In addition more extensive unique sequence complementarity is present in at least two of the three RNAs.     

An apparent deletion of an oligonucleotide detected by RNA fingerprint in the nondiabetogenic B variant of encephalomyocarditis virus is caused by a point mutation.

The diabetogenic D variant of encephalomyocarditis virus (EMC-D) was previously shown to be different from the nondiabetogenic B variant of encephalomyocarditis virus (EMC-B) by a single spot in an oligonucleotide fingerprint after RNase T1 digestion of their genomic RNAs. An oligoribonucleotide was missing from EMC-B but was present in EMC-D. The oligoribonucleotide specific to EMC-D was isolated from a two-dimensional polyacrylamide gel and sequenced as 5'-ACAAUCUCACUUUUCCAACAACAG-3'. Molecular hybridizations of EMC-D and EMC-B genomic RNAs with a DNA primer complementary to the EMC-D-specific oligoribonucleotide revealed that the absence of a corresponding spot in EMC-B was due to a point mutation rather than a deletion. By sequencing a cloned cDNA of EMC-B corresponding to the EMC-D-specific oligoribonucleotide, the point mutation was identified as a G for EMC-B and an A for EMC-D transversion at base 9 of the oligonucleotide. Comparative sequence analysis of eight randomly picked RNA segments around the EMC-D-specific oligoribonucleotide revealed that there were no base changes between EMC-D and EMC-B. It is concluded that the diabetogenic EMC-D viral genome differs from the nondiabetogenic EMC-B viral genome by at least a point mutation.