|Natarajan, Savithiry - Savi|
|Van beek, Nikolai|
Submitted to: Virus Research
Publication Type: Peer reviewed journal
Publication Acceptance Date: 3/13/2007
Publication Date: 4/27/2007
Citation: Dhar, A.K., Lakshman, D.K., Natarajan, Allnutt, F.C.T. and van Beek, N.A.M. 2007. Functional characterization of putative promotor elements from infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimp and in insect and fish cell lines. Virus Research. 127(1):1-8. Interpretive Summary: Viruses infecting eukaryotes are generally explored as sources of stronger, constitutive, organ or tissue non-specific promoter and enhancer elements. Promoters are DNA sequences located upstream of expressed gene (messenger RNA or mRNA), known to facilitate RNA polymerase binding and initiation of transcription. Enhancers are DNA sequences upstream of promoters (or some times downstream of genes) and enhance transcription of genes in an orientation independent manner. Promoter and enhancer sequences from simian virus 40 (SV 40) early and late genes, cytomegalovirus (CMV), cauliflower mosaic virus (CaMV) etc., are commonly used in cross-kingdom gene expression studies of eukaryotic organisms. We have tested the effectiveness of two putative promoter regions of shrimp infectious hypodermal and hematopoietic necrosis virus (IHHNV) tagged with the luciferase gene in bacteria, insect cells, fish cells, and shrimp tail muscles in vitro. Our results demonstrated that both P2 and P61 promoters of IHNNV are constitutive promoters that can drive gene expression in both prokaryotes and eukaryotes. In absence of genome sequencing data, information on stronger promoters of Rhizoctonia solani, a plant pathogenic fungus, are lacking. In future, the feasibility of the above IHHNV promoters will be tested in the gene expression experiments on R. solani to elucidate roles of specific genes in plant disease development.
Technical Abstract: Infectious hypodermal and hematopoietic necrosis virus (IHHNV) of shrimp contains a linear single-stranded DNA genome of approximately 4.1 kb with three putative open reading frames (ORFs) namely, the left ORF, middle ORF and the right ORF on the same DNA strand. Whereas the left ORF codes for non-structural protein and the right ORF codes for capsid protein, the role of the middle ORF is not known. Two putative promoter regions, P2 and P61, were detected upstream of the left ORF and right ORF, respectively. We evaluated the activities of these two promoters via luciferase reporter constructs with or without a transcriptional enhancer element in bacteria, insect cells, fish cells, and shrimp tail muscle. In bacteria the P61 promoter was stronger than the P2 promoter, while in insect and fish cells the P2 promoter was stronger. In shrimp, there was no significant difference in luciferase expression driven by these two promoters. Surprisingly, the presence of the SV 40 enhancer element suppressed P2 promoter activity in bacteria and insect cells, while P61 was only suppressed in shrimp. In fish cells, the SV 40 enhancer element dramatically increased the activities of both promoters. P2 and P61 were found to be constitutive promoters that can drive gene expression in both prokaryotes and eukaryotes.