Rat selenoprotein W (SeW) promoter activity was investigated using different
concentrations of cadmium, copper, and zinc. Two fragments (404bp and
1265bp) of the SeW promoter, containing a single metal response element
(MRE), were ligated into the multiple cloning site of a pGL3-Basic reporter
plasmid. The constructs were transfected into cultured rat C6 (glial) and L8
(myoblast) cells and promoter activity measured by means of luciferase
reporter gene fused to the SeW promoter fragments in the reporter plasmid.
With post-transfection exposure of these cell lines to these metals, copper and
zinc, but not cadmium, significantly increased promoter activity of the
unmutated 1265bp (not 404bp) construct (p<0.05) only in the C6 cells.
Mutation of the MRE sequence abolished promoter response to metal exposure
but did not eliminate promoter activity. The results suggest that SeW
expression in glial cells can be increased on exposure to copper and zinc and
that this response is dependent on the MRE sequence present in the SeW
promoter.
To understand transcriptional regulation of the SeW gene, we used in vitro
binding assays to identify transcription factors that may be involved in the
transcriptional regulation of the SeW gene. Using protein from rat C6 (glial)
cell nuclear extracts, oligonucleotides containing putative regulatory elements
in the SeW promoter, and antibodies, we were able to show that the specificity
protein 1(Sp1) transcription factor binds to the Sp1 consensus sequence in the
SeW promoter as well as the MRE. However, the MRE, GRE, AP-1 and LF-A1
did not yield any specific binding. Although, competition analysis showed
specific binding at the TFII-1 site, super-shift analysis using anti-TFII-1
antibody did not yield any super-shifted band. Therefore the SeW gene may be
a target for Sp1 whose interaction with the SeW promoter may activate or
repress the transcription of SeW. / Graduation date: 2003
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/30187 |
| Date | 13 February 2003 |
| Creators | Amantana, Adams |
| Contributors | Whanger, Philip D. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
Page generated in 0.002 seconds