Complementary DNA cloning and analysis of carnation mottle virus RNA

Abstract

A 3850-base pair (bp) cDNA fragment, representing most of the carnation mottle virus (CarMV) genome, was molecularly cloned in Escherichia coli using a modification of the plasmid-primer method of Okayama and Berg (H. Okayama and P. Berg (1982), Mol. Cell. Biol. 2, 161-170). Poly(dT)-tailed pUC8 was used to prime cDNA synthesis from polyadenylated CarMV RNA template. Oligo(dC) was added to the cDNA 3' end, and second-strand synthesis was specifically primed using an oligo(dG)-tailed linker fragment. cDNA inserts of 3250 and 1950 by were identified from a clone bank as apparently overlapping and were fused at a common BglII restriction site to produce pCarMV-1C. Confirmation that this plasmid harbored CarMV cDNA sequences was achieved by Southern blot hybridization with a randomly primed cDNA probe. Nick-translated pCarMV-1C was used to probe virion and total single-stranded RNA extracts from infected Chenopodtium quinoa leaves by "Northern" blot hybridization. Besides the full-length genomic RNA, two additional species of approximately 1600 and 1750 bases were associated with CarMV. Based on a series of Northern blots with nick-translated subclones derived from pCarMV-1C as probes, these subgenomic RNAs were found to originate from the 3' domain of the virus genome.