STRUCTURE-FUNCTION ANALYSIS OF THE AUTOGRAPHA-CALIFORNICA MULTINUCLEOCAPSID NUCLEAR POLYHEDROSIS-VIRUS HOMOLOGOUS REGION PALINDROMES

Homologous regions (hrs), which are present at eight dispersed locatio ns on the Autographa californica multinucleocapsid nuclear polyhedrosi s virus genome, are composed of repeated imperfect palindromes within directly repeated sequences. Hrs act as transcriptional enhancers of R NA polymerase Ii-mediated transcription and as origins of DNA replicat ion when incorporated into plasmids and tested in transient replicatio n assays. To characterize the physical structure of these elements and to determine the role that mismatched nucleotides may play in hr func tion, oligonucleotides containing a consensus mismatched ''imperfect'' palindrome and a closely related ''perfect'' palindrome were synthesi zed. These sequences were cloned into individual plasmids and tested f or their ability to form cruciform structures using nuclease P1 assays and two-dimensional (2-D) gel analyses of topoisomers. The perfect pa lindrome formed a cruciform structure and the energy requirement for i ts formation was predicted to occur under physiological conditions. In contrast, the construct containing an imperfect palindrome did not fo rm a cruciform under these conditions. Both hr constructs were found t o bind IE-1 in electrophoresis mobility shift assays and act as enhanc ers when cis-linked to the baculovirus 39K early gene promoter. Howeve r, a single oligonucleotide containing the palindrome sequence did not bind IE-1 when annealed under conditions conducive to hairpin formati on. (C) 1996 Academic Press, Inc.