Cloning the double-stranded RNA genes of reovirus: sequence of the cloned S2 gene

Abstract

The genes of the Dearing strain of reovirus serotype 3, which consist of double-stranded RNA, have been cloned into pBR322 by tailing both strands of each gene with poly(A), transcribing them with reverse transcriptase, self-hybridizing the cognate plus and minus cDNA strands, incubating them with Escherichia coli DNA polymerase I to ensure that they are complete, and cloning the double-stranded cDNA molecules by standard procedures. The sequence of the cloned S2 gene has been determined. The sequence at the termini are exactly the same as those at the ends of the native double-stranded RNA gene. The gene is 1,329 nucleotides long and possesses a single long open reading frame that starts at the first initiation codon (residue 19) and extends for 331 codons, sufficient to encode a protein of the same size as the known S2 gene product, protein sigma 2, a major reovirus core component (Mr, 38,000). A second open reading frame of 85 codons, in a different phase, starts close to where the first ends. The protein translated from this reading frame is unknown.