Molecular cloning of complementary DNA sequences of citrus tristeza virus RNA

Complementary DNA (cDNA) of citrus tristeza virus (CTV) RNA, synthesized using calf thymus DNA random primers, was converted to a double-stranded form and inserted into the PstI site of the Escherichia coli pBR322 plasmid by the G-C tailing method. Bacterial clones harbouring virus-specific sequences were detected by colony hybridization with a 32P-labelled viral RNA probe. Hybridization patterns of denatured virus RNA revealed the presence of three types of specific clones: those hybridizing with a distinct narrow band corresponding to the full-length virus RNA, those hybridizing with a broader band of virus RNA sequences, and those hybridizing with several distinct virus-related RNA bands. Similar patterns were obtained when these clones were hybridized to purified double-stranded RNA from CTV-infected plants. None of these cDNA clones hybridized with similarly treated preparations extracted from healthy plants. The origin of variation among the CTV clones is discussed.