FUNCTIONAL DISSECTION OF THE AUTOGRAPHA-CALIFORNICA NUCLEAR POLYHEDROSIS-VIRUS ENHANCER ELEMENT HR5

The hr5 enhancer element of Autographa californica nuclear polyhedrosi s virus stimulates transcription from baculovirus-delayed early promot ers in the presence of the viral transactivator, IE1. Gel retardation analyses of interactions between a fragment of hr5 and extracts prepar ed from cells transfected with an IE1-encoding plasmid revealed the pr esence of three DNA-protein complexes. In order to better define the f unctional domains of the hr5 enhancer, we constructed a set of plasmid s containing partial deletions in the enhancer element. These construc ts were tested for both in vitro DNA binding activity and enhancer fun ction in transient assays. The results indicated that the minimum sequ ence required for DNA-protein interactions was half of the conserved 2 4-bp palindrome that is contained within a 60-bp direct repeat (DR60). However, the minimum sequence required for enhancer function was a co mplete copy of DR60. Template challenge experiments indicated that IE1 bound with equal affinity with a complete or a half copy of DR60. The deletion analyses were confirmed by in vitro binding and transient ex pression assays with synthetic oligonucleotides. (C) 1994 Academic Pre ss, Inc.