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Antisense rescue defines specialized and generalized functional domains for c-Fos protein
1Department of Cell Biology, Vanderbilt University, Medical School, Nashville, Tennessee 37232.
Abstract:
Serum induces the expression of a number of proteins with similar transcriptional properties, including those encoded by the proto-oncogenes c-fos and c-jun. This study employs a novel antisense rescue method to determine whether antisense-resistant genes (constructed by deletion of antisense RNA target sequences) can replace c-fos expression during serum-induced DNA synthesis. Immunoprecipitation studies and nuclease protection assays demonstrated that anti-fos RNA inhibited endogenous c-fos expression but did not inhibit expression of transfected antisense-resistant mutant c-fos genes. The results of nuclear-labelling and cellular-proliferation studies indicated that C terminally truncated Fos mutants, including FBR v-fos, could not rescue endogenous Fos, although full-length and minimally truncated c-fos expression vectors could restore serum-induced DNA synthesis in cells expressing anti-fos RNA. Overexpression of c-Jun protein (Jun) could not restore serum-induced DNA synthesis to cells expressing inducible anti-fos RNA despite equivalent transactivation of an AP-1 target gene. Thus, the antisense rescue method defines a specialized function for c-Fos protein which is distinct from the function(s) of Jun and/or transforming FBR v-Fos proteins.
Insights
This study reveals that c-Fos protein has a unique role in serum-induced DNA synthesis, distinct from c-Jun. Antisense rescue experiments demonstrate that only full-length c-Fos can restore this process.
Area of Science:
- Molecular Biology
- Cellular Biology
- Oncogenes
Background:
- Serum stimulation activates gene expression, including proto-oncogenes c-fos and c-jun.
- Understanding the specific roles of these proteins in cellular processes like DNA synthesis is crucial.
Purpose of the Study:
- To investigate the specific function of c-Fos in serum-induced DNA synthesis using a novel antisense rescue method.
- To determine if antisense-resistant c-fos genes can substitute for endogenous c-fos expression.
Main Methods:
- Developed and utilized a novel antisense rescue technique.
- Employed immunoprecipitation and nuclease protection assays to assess gene expression.
- Conducted nuclear-labeling and cellular proliferation studies.
Main Results:
- Anti-fos RNA successfully inhibited endogenous c-fos expression.
- Antisense-resistant mutant c-fos genes did not rescue serum-induced DNA synthesis.
- Full-length or minimally truncated c-fos restored DNA synthesis, but C-terminally truncated mutants did not.
- Overexpressed c-Jun could not restore DNA synthesis.
Conclusions:
- The study defines a specialized, non-redundant function for c-Fos protein in serum-induced DNA synthesis.
- This function is distinct from that of c-Jun and transforming FBR v-Fos proteins.
- The antisense rescue method is effective for dissecting specific protein functions.