Molecular Cloning of Complementary DNA Sequences of Citrus Tristeza Virus RNA

Abstract

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.