Molecular cloning of clover yellow mosaic virus RNA: Identification of coat protein coding sequences in vivo and in vitro

Abstract

We have prepared double-stranded cDNA from clover yellow mosaic virus (CYMV) RNA and have created a library of cloned CYMV cDNAs in plasmid vectors. Cloned fragments ranging from 0.2 to 2.0 kbp have been mapped relative to the CYMV genome and to each other by digestion with restriction enzymes and by colony hybridization. Together, these cloned DNAs constitute almost 90% of the CYMV genome. Two overlapping plasmids whose inserts originate from the 3′ portion of CYMV RNA arrest the synthesis of CYMV coat protein in vitro. A 0.44-kbp PstI fragment from one of these anneals to three CYMV-specific RNAs comprising the genomic RNA and two subgenomic RNAs of 2.1 and 1.0 kb isolated from polyribosomes from CYMV-infected broad bean leaves. RNA was enriched in the 1.0-kb subgenomic species by fractionating RNA extracted from purified preparations of CYMV or from polyribosomes isolated from infected leaves. Such RNA fractions directed the synthesis of CYMV coat protein in vitro indicating that the 1.0-kb subgenomic RNA is likely to be the coat protein messenger in vivo.