MOLECULAR-CLONING OF INDIAN TOMATO LEAF CURL VIRUS GENOME FOLLOWING ASIMPLE METHOD OF CONCENTRATING THE SUPERCOILED REPLICATIVE FORM OF VIRAL-DNA

DNA-A and DNA-B components of the genome of a whitefly transmitted vir us causing yellowing and leaf curl in tomato (ITLCV) were cloned follo wing a simple procedure for isolation of the double stranded replicati ve form of viral DNA from infected tomato plants. The method is based on extraction of total DNA from infected plants followed by concentrat ion of the double stranded replicative form of viral DNA by an alkalin e denaturation procedure identical to that used for isolation of plasm id DNA from Escherichia coli. The attempted cloning of DNA showed that 95% of the transformants contained plasmids with an insert of either DNA-A (2.75 kb) or DNA-B (2.55 kb). Cloned DNA-A and DNA-B when used a s probes could detect DNA-A/DNA-B in total nucleic acid obtained from fresh diseased tissue. Bath DNA-A and DNA-B are needed for infection a nd they have a common region of 166 bases with about 94% nucleotide se quence homology, a characteristic of all bipartite geminiviruses. Comp arison of the amino acid sequence of the putative coat protein product of ITLCV with some other mono- and bipartite geminiviruses revealed a maximum of 86% homology with Indian cassava mosaic virus.