Molecular Cloning of Complementary DNA Sequences of Citrus Tristeza Virus RNA

Abstract

SUMMARYComplementary 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.

A New Israeli Tobamovirus Isolate Infects Tomato Plants Harboring Tm-22 Resistance Genes

“…For RNA extraction, purified viral preparations served as the source material as described previously [24] with modifications [25]. Briefly, virions were incubated with RQ RNase-free DNase I (Promega) for 1 h at 37°C, followed by proteinase K (Sigma) treatment at a final concentration of 200 μg/ml for 1 h at 37°C.…”

Molecular cloning and nucleotide sequencing of the coat protein gene of citrus tristeza virus

“…Complementary DNA libraries to CTV RNA have been constructed previously (Rosner et al, 1983;Calvert, 1987). The first library, cloned from an Israeli isolate of CTV, yielded cDNA probes capable of differentiating CTV isolates (Rosner & Bar-Joseph, 1984;Rosner et al, 1986); two cDNA libraries of Florida isolates have been prepared and the library of the severe isolate T36 yielded a partial (70~) map of the viral genome (Calvert, 1987).…”

“…Generally, fractions 4 to 8 were highly enriched with virus and were chosen for RNA isolation and peptide microsequencing analysis. RNA was extracted from virus as described (Rosner et al, 1983) and was analysed on non-denaturing 1% agarose gels (Maniatis et al, 1982). One predominant ethidium bromide-staining band was observed near the top of the gel at the approximate location of the 21 kbp molecular size standard.…”

Nucleotide sequences and expression inEscherichia coli of the coat protein genes from two strains of melon necrotic spot virus

“…On the other hand, amino acid homologies of CPs a m o n g distinct members of carmoviruses were from 31~o to 34~o. It appears that amino acid homologies of CPs between strains and distinct members of carmoviruses correspond well to those observed among strains and distinct members of potyviruses [18,23], and among strains and distinct members of luteoviruses [11,19]. Thus, it is probably possible to differentiate between strains and distinct species of carmoviruses based on the amino acid sequence homologies of their CPs and to use this information for taxonomic purposes.…”