Structure and Expression of Genes for Flavivirus Immunogens.
Annual summary rept. Sep 82-Aug 83,
MASSACHUSETTS UNIV AMHERST DEPT OF BIOCHEMISTRY
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Banks of cloned Japanese encephalitis virus cDNA and monoclonal antibodies specific to virus structural proteins are being used to characterize the structure and expression of the flavivirus genome. Fragmented RNA has been used as template for initial cDNA synthesis in an effort to produce a statistically complete bank of cDNA in a single round of cloning. For ease in establishing physical and functional maps of the genome, intact RNA is also used but in successive rounds of primer-extended synthesis to clone the genome in a few overlapping fragments. The genomic bank derived from the fragmented RNA consists of 40 clones with inserts ranging from 0.2 - 1.6 kb with an average length of about 1 kb. Assuming random fragmentation of the RNA, a bank of DNA inserts of this size need have only 27 members for a 90 probability that any given genomic sequence is present. In the second strategy cloning of cDNA transcribed from the 3-terminus of the intact RNA has yielded an insert that is somewhat more than 10 of the genome. A biological expression system designed to detect protein coding regions in cloned DNA has been established. The system involves cloning of open-reading frame DNA into an out-of-phase beta-galactosidase gene to successfully restore lacZ function.