We constructed a cDNA library with mRNA isolated from Sf9 cells infected with
Spodoptera littoralis nucleopolyhedrovirus type B (SpliNPV-B) and identified the lef-3
gene from this library. Northern blot analysis showed that SpliNPV-B lef-3 mRNA was
expressed as a 1.6 Kb transcript at 5 hours post infection (p.i.), reached high levels at
24 hours p.i., and remained highly expressed at 56 hours p.i.. Transcriptional mapping
showed that lef-3 transcription started from two initiation sites (the distal and the
proximal transcription initiation sites) located approximately 9 nucleotides apart. The
sequences that modulate lef-3 expression were investigated by transient expression
assays using a reporter gene under transcriptional control of the lef-3 promoter.
Deletion analysis of the 5'-flanking region demonstrated that sequences up to 584 bases
5' of the distal transcription initiation site affected the level of reporter activity,
indicating that this region contains transcription regulators. A region that was sufficient
to direct basal level of promoter activity, the minimal promoter, was identified. This
region encompasses the two transcription initiation sites, two TATA boxes, and a GATA
motif. Mutations in the GATA motif resulted in substantial decrease in the level of
reporter activity, suggesting that the GATA motif is an important element in the
regulation of lef-3 gene expression. The sequence of a 2.6-kb region (mu 42.8-46.8)
encompassing the lef-3 gene and flanking sequences was determined. Alignment of the
predicted amino acid sequence of the LEF-3 polypeptide of SpliNPV-B with the putative
sequences of AcMNPV and OpMNPV LEF-3 revealed low levels of sequence conservation
(26% and 21% amino acid sequence identity, respectively). This low level of sequence
conservation corroborates the view that, within the genus Nucleopolyhedrovirus,
SpliNPV-B is distantly related to AcMNPV and OpMNPV. / Graduate
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/8225 |
| Date | 05 June 2017 |
| Creators | Wolff, Jose Luiz Caldas |
| Contributors | Levin, David Bernard |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Rights | Available to the World Wide Web |
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