Structure and Functional Studies of DEN-2 Virus Genome.
Progress rept. no. 3, 1 Sep 83-31 Aug 84,
KANSAS UNIV MEDICAL CENTER KANSAS CITY DEPT OF BIOCHEMISTRY
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We showed the presence of polyAtail at the 3 end of DEN-2 RNA by direct sequence analysis of RNA as well as oligo dT-primed cDNA synthesis by reverse transcriptase. Double-stranded cDNA was inserted into pUC13-1 vector and was used for the transformation of E.coli JM83 host. Ampicillin-resistant clones were screened for DEN-2 cDNA inserts Eleven clones containing inserts of 0.95 kb - 1.95 kb were isolated and cDNAs were prepared in large scale. Hybridization of cDNA probes to DEN-2 RNA proved that the cDNAs are virus-specific. Hybridization to other serotypes of Dengue Viruses as well as to other flavivirus RNAs showed that these clones are type specific. The cDNA clones were mapped for restriction sites using several restriction endonucleases. A physical map of the clones containing restriction enzyme cleavage sites was constructed. DNA sequence analysis of the clones are currently being carried out to locate the open reading frame regions coding sequences for viral antigens among the clones.