Structure and Expression of Genes for Flavivirus Immunogens.
Annual summary rept. Sep 83-Aug 84,
MASSACHUSETTS UNIV AMHERST DEPT OF BIOCHEMISTRY
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The Japanese encephalitis virus JEV genome is being cloned for detailed structure-function studies. Full-length cDNA transcripts have been obtained via reverse transcription of the 12 kb plus strand RNA genome. Synthesis was from a 3 -specific synthetic primer. Second strand synthesis yielded double-stranded cDNA ranging in size from 500 bp to 10 kb. A variety of plasmids with cDNA inserts of up to 5.0 kb have been developed. At least 8 kb of unique DNA is contained in three plasmids accounting forr 75 of the genome. Much of the remaining sequences will likely be identified in a bank of over 100 cDNA clones currently being characterized. A functional map off the genome is being developed by in vivo expression analyses and DNA sequencing. Protein coding regions are identified by fusion of viral open reading frame ORF sequences with the E. coli lacZ gene in plasmid and phage expression vectors. Screening for inidivdual JEV-lacZ hybrid proteins uses antibody probes for specific viral peptides. One cDNA clone containing 1.45 kb was subjected to extensive sequence and expression analyses. ORF elements of various sizes have been identified by both methods however, only one is thus fare considered large enough to accommodate a protein of potential relevance.