Development and use of cDNA probes in studies of barley yellow dwarf virus
1990
Lister, R.M. | Barbara, D.J. | Kawata, E.E. | Ueng, P.P. | Fattouh, F. | Larkins, B.A. (Purdue University, West Lafayette, IN, (USA))
Libraries of cDNA clones were produced from the RNAs of the MAV, RPV, and Purdue (P)-PAV isolates of barley yellow dwarf virus (BYDV) in bacteriophage gtll, by random priming. Screening with antisera to the isolates allowed selection of the recombinants capable of expressing capsid protein. With MAV, subcloning into the plasmid pUC18, followed by restriction endonuclease mapping, identified overlapping inserts collectively representing at least 85 % (a total of 5.1 kbp) of the BYDV genome. Immunologically positive clones shared a common region of approximately 1000 bp, located between 750 bp and 1750 bp from the 3' terminus of the genome. Nucleotide sequencing of this region is under way. Similar strategies for genome analysis are being used with the other isolates. Sequence homology among various isolates of BYDV is being examined by using selected cDNA clones as probes in viral nucleic acid hybridization studies. For example, hybridization specificity with MAV cDNAs was influenced by the origin of the clones within the genome. Those from the putative coat protein-coding region hybridized well only to MAV-like isolates; those from elsewhere hybridized also with PAV-like isolates. None hybridized significantly to RPV. When used in diagnosis, the sensitivity of detection was related to probe size; the larger clones (to 2.2 kbp) detected as little as 70 pg of purified virus (1.4 ng/ml in a 50-u1 sample). Studies on infected plant tissue indicated that the sensitivity of virus detection in plant extracts by dot-blot hybridization can be greater than that of ELISA, although inhibition of the reaction by sap constituents occurs at low dilutions. Preparations from frozen or air-dried tissue reacted well, suggesting the possibility of diagnosis of frozen-stored or dry-shipped samples by use of appropriate cDNA probes.
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