Structure–Function Analysis of theAutographa californicaMultinucleocapsid Nuclear Polyhedrosis Virus Homologous Region Palindromes
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文摘
Homologous regions (hrs), which are present at eight dispersed locations on theAutographa californicamultinucleocapsid nuclear polyhedrosis virus genome, are composed of repeated imperfect palindromes within directly repeated sequences.Hrsact as transcriptional enhancers of RNA polymerase II-mediated transcription and as origins of DNA replication when incorporated into plasmids and tested in transient replication assays. To characterize the physical structure of these elements and to determine the role that mismatched nucleotides may play inhrfunction, oligonucleotides containing a consensus mismatched “imperfect” palindrome and a closely related “perfect” palindrome were synthesized. These sequences were cloned into individual plasmids and tested for their ability to form cruciform structures using nuclease P1 assays and two-dimensional (2-D) gel analyses of topoisomers. The perfect palindrome formed a cruciform structure and the energy requirement for its formation was predicted to occur under physiological conditions. In contrast, the construct containing an imperfect palindrome did not form a cruciform under these conditions. Bothhrconstructs were found to bind IE-1 in electrophoresis mobility shift assays and act as enhancers when cis-linked to the baculovirus 39K early gene promoter. However, a single oligonucleotide containing the palindrome sequence did not bind IE-1 when annealed under conditions conducive to hairpin formation.