Molecular cloning of the complementary DNA copies of the common and cowpea strains of tobacco mosaic virus RNA.
Complementary DNA (cDNA) copies of the common (OM) and Cowpea (Cc) strains of tobacco mosaic virus (TMV) RNA polyadenylated in vitro have been synthesized with AMV reverse transcriptase and oligo (dT)10 as primer. The cDNA was converted to a double-stranded form by E. coli DNA polymerase I followed by S1 nuclease digestion. The double-stranded cDNA copies were cloned in pBR322 at the PstI site using the oligo(dC)-oligo(dG) tailing method. With the common strain, several clones containing inserts covering different parts of genomic RNA were selected using partially reconstituted RNA and partially stripped virus RNA as hybridization probes. Restriction mapping of three clones and their overlaps showed that cloned sequences covered about 4000 nucleotides of the common strain RNA from the 3' end. With the Cc strain, clones containing the 3' portion were selected by Southern hybridization using coat protein mRNA isolated from short particles as a probe. One cloned recombinant plasmid was found by restriction analysis and R-loop mapping to carry about a 1700 nucleotide sequence of Cc strain RNA from the 3' end. A restriction map of the OM strain was very similar to the map of the vulgare strain, as predicted from its nucleotide sequence, but completely different from that of Cc strain.
Meshi
T
T
Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Tokyo 113, Japan.
Takamatsu
N
N
Ohno
T
T
Okada
Y
Y