Genetic manipulation of two fowlpox virus late transcriptional regulatory elements influences their ability to direct expression of foreign genes.
Fowlpox virus (FWPV) is currently used as a vector to express foreign genes of various poultry and mammalian pathogens. However, due to limited information available about the primary structure of FWPV promoters required for an optimal transcriptional efficiency, the full potential of FWPV as an expression vector has not been completely realized. To dissect such transcriptional regulatory elements at the molecular level, we selected two FWPV promoters dictating contrasting levels of expression of acidic-type inclusion body protein gene (P190) and A15L vaccinia virus homolog of FWPV (P180) for site-directed mutagenesis studies. The transcriptional activity of mutated promoters was analyzed based on their ability to transcribe a reporter gene, lacZ, and translation of the resultant mRNA into functional protein. Replacement of the spacer sequences of P180 with those of P190 resulted in a five-fold increase in mRNA and a 17.6-fold increase in protein over those with its parental promoter, P180. Similarly, replacement of a thymidine after the start codon with guanosine resulted in a 2.3-fold increase in lacZ mRNA and a seven-fold increase in protein. Combining these substitutions in P180SG produced a maximum increase in mRNA and protein of 6.7- and 29.9-fold, respectively, over concentrations with its parental P180 promoter. The promoter activity of P180SG was comparable to that of the strongest natural promoter, P190. The amount of protein per transcript generated by the mutated promoters of P180 increased to at least three times that with the parental P180. In contrast, similar replacements in P190 resulted in a 40-50% reduction in mRNA and protein in all the mutated promoters. We discuss the significance of spacer sequence and the purine after the start codon in the context of a high level of expression.
Srinivasan
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Department of Veterinary Pathobiology, College of Veterinary Medicine, Veterinary Medicine Basic Sciences Building, 2001 South Lincoln Avenue, University of Illinois, Urbana, 61802-6178, USA. vishi@rics.bwh.harvard.edu
Schnitzlein
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WM
Tripathy
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DN
D000818:Animals; D019677:Artificial Gene Fusion; D021901:DNA, Intergenic; D005587:Fowlpox virus / Q000235:genetics*; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D005822:Genetic Vectors; D007763:Lac Operon; D016297:Mutagenesis, Site-Directed; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D050436:Regulatory Elements, Transcriptional / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics
D021901:DNA, Intergenic; D012333:RNA, Messenger; D011994:Recombinant Proteins; D001616:beta-Galactosidase