Analysis of the role of 5' promoter elements and 3' flanking sequences on the expression of a baculovirus polyhedron envelope protein gene.
The polyhedron envelope protein gene of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) is the third in a series of five open reading frames (ORFs 1-5) oriented in the same direction. Individual mRNAs initiate at conserved late gene promoter/mRNA start site (A/GTAAG) sequences located upstream of each ORF and the mRNAs co-terminate after the fifth ORF. To examine the influence of transcription from upstream promoter elements and the presence of an extensive 3' flanking sequence on the expression of the polyhedron envelope protein gene, the region was cloned into a phagemid vector and a BamHi site was inserted downstream of the ATG by site-directed mutagenesis and used for the insertion of a chloroamphenicol acetyl transferase (CAT) reporter gene. A set of clones were constructed in which individual or combinations of the late promoter elements from OFs 1, 2, and 3 were destroyed by site-directed mutagenesis. These plasmid constructs were transfected into Lymantria dispar cells infected with OpMNPV and cell extracts were assayed for CAT activity. Inactivation of the late promoter element immediately 5' of the polyhedron envelope protein gene led to a 96% decrease in CAT expression. Destruction of the ORF 2 late promoter element, or both the ORF 1 and ORF 2 late promoter elements, or deletion of the entire region containing ORFs 1 and 2 resulted in a 17 to 35% increase in CAT expression. In contrast, inactivation of the ORF 1 promoter alone resulted in no increase in CAT expression. Deletions of 3' flanking sequences of the polyhedron envelope protein gene caused major reduction in both CAT activity and steady-state levels of CAT mRNA.
Gross
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CH
Department of Agricultural Chemistry, Oregon State University, Corvallis 97331-7301.
Rohrmann
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GF
D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D004252:DNA Mutational Analysis; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D017384:Sequence Deletion; D014759:Viral Envelope Proteins / Q000235:genetics*; D015678:Viral Structural Proteins / Q000235:genetics*
D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D014759:Viral Envelope Proteins; D015678:Viral Structural Proteins