|DHAR, ARUN - Viracine Therapeutics Corporation
|KAIZER, KRISTA - Viracine Therapeutics Corporation
Submitted to: Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/1/2010
Publication Date: 8/13/2010
Citation: Dhar, A.K., Kaizer, K.N., Lakshman, D.K. 2010. Transcriptional analysis of Penaeus stylirostris densovirus genes. Virology. 402:112-120.
Interpretive Summary: Promoters are genomic DNA sequences located upstream of expressed gene (messenger RNA or mRNA) and known to facilitate initiation of transcription. Viruses infecting eukaryotes are generally explored as sources of stronger, constitutive, organ or tissue non-specific promoters. Promoter sequences from simian virus 40, cytomegalovirus, cauliflower mosaic virus etc., are commonly used in cross-kingdom gene expression studies of eukaryotic organisms. However, availability and patent limitations, etc. often restrict their use in gene expression investigations. Earlier, we tested the effectiveness of two (left and right) potential promoter regions of Penaeus stylirostris densovirus (PstDNV) and found that both of them function in vitro in bacteria, insect (sf9), fish, and shrimp cells. In this investigation we have shown that a third (middle) potential promoter of the virus is functional in sf9 insect cells. In addition, we have precisely determined the transcription start and termination sites of the three promoters and found that left promoter had the highest activity followed by middle and right promoters. In absence of genome sequencing data, information on stronger promoters of Rhizoctonia solani and many other soilborne plant pathogenic fungi, are lacking. PstDNV promoters will be evaluated for gene expression experiments on soilborne pathogenic fungi to elucidate roles of specific genes in plant disease development.
Technical Abstract: Penaeus stylirostris densovirus (PstDNV) genome contains three open reading frames (ORFs), left, middle, and right, which encode a non-structural (NS) protein, an unknown protein, and a capsid protein (CP), respectively. Transcription mapping revealed that P2, P11 and P61 promoters transcribe the left, middle and right ORFs. NS transcript uses the D1/A1 donor/acceptor sites for splicing and has two alternate transcription termination sites (TTS) that were different from the previously predicted TTS. The transcription initiation site (TIS) and the TTS for the middle and the right ORFs conform to predicted sites. PstDNV transcript quantification in infected shrimp revealed that the NS and CP transcripts were expressed at an equivalent level and significantly higher than the middle ORF transcript. In vitro assay showed that P2 had the highest promoter activity followed by P11 and P61. Transcription mapping data provided new insights into PstDNV gene expression strategy.