Philip Goelet

Tobacco mosaic virus induces the synthesis of a family of 3′ coterminal messenger RNAs and their complements

At least eight 3′ coterminal RNA species, which may act as messenger RNAs, are synthesized during infection of tobacco ( Nicotiana tabacum ) by tobacco mosaic virus. These RNAs vary in length from 700 nucleotides to the genomic RNA of 6400 nucleotides and include the mRNAs for the coat protein and the 30,000 M r protein. RNAs that contain the viral origin of assembly are packaged and may be recovered from preparations of the virus. Complementary strand RNAs corresponding to each of the messengers are also synthesized during infection. It is proposed that the coterminal RNAs are synthesized during tobacco mosaic virus infection by a mechanism involving incomplete transcription of the viral RNA into negative strands that are subsequently transcribed to produce messenger RNAs.

Methods for cDNA cloning and sequencing tobacco mosaic virus RNA

DNA complementary to tobacco mosaic virus (TMV) RNA (cDNA) was prepared by priming reverse transcription with synthetic oligonucleotides. The cDNAs terminated prematurely at many specific sites and no transcripts longer than about 2000 nucleotides were obtained. However, the entire 6395 nucleotide long TMV RNA could be copied into cDNA by specific priming with a series of 13 to 17 residue long oligonucleotides or by non-specific priming with short, 4 to 7 residue, oligonucleotides. A number of different priming methods were used to convert the cDNA into double-stranded DNA (dsDNA). The double-stranded cDNA was recovered by shotgun cloning into M13 and analysed by sequencing. The frequency at which cDNA clones were recovered has been used to compare various cDNA cloning strategies.