In the AcMNPV genome, ~28% of the genes are arranged divergently on opposite strands
with an intergenic region of <1 kbp. In this configuration, a bidirectional promoter generally
drives expression of both genes. However, no baculovirus bidirectional promoters have been
characterized in any detail. We chose the AcMNPV chiA/v-cath intergenic region to serve as a
model to characterize transcriptional regulation of bidirectional gene pairs during AcMNPV
infection. We sequentially truncated putative upstream regulatory regions of chiA and v-cath to
identify sequences essential for transcriptional initiation. Forty bp of the chiA gene 5’-flanking
region was sufficient to support chiA transcription at half the level of the AcΔCC+CC repair
virus. Interestingly, v-cath transcription from viruses containing only 40 bp of their upstream
5’-flanking region was found to be higher by 4-fold relative to the level of native expression.
Linker-scanning mutagenesis that inserted 5 bp linkers spanning the chiA/v-cath intergenic
region identified nucleotides critical for the transcriptional activation of both genes. From this,
nucleotides -36 to -45, of the v-cath gene were found to negatively regulate v-cath mRNA
expression. Quantitative RT-PCR studies revealed a 2-4 fold higher chiA mRNA expression
relative to v-cath possibly explaining why translation of CHIA can be detected 6 hours earlier
than V-CATH. This study identifies upstream regions of viral chiA and v-cath required for
initiation of transcription and provides the first insight into baculovirus mechanisms for
transcriptional regulation of interdependent gene pairs.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OGU.10214/3260 |
| Date | 10 January 2012 |
| Creators | Norris, Michael |
| Contributors | Krell, Peter |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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