Date of Award

Spring 1995

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

Committee Director

Timothy J. Bos

Committee Member

Mark Elliot

Committee Member

Richard Stenberg

Committee Member

William Wasilenko

Committee Member

Kenneth Somers

Abstract

The v-jun oncogene was initially identified as the causative agent for fibrosarcomas in chickens. Studies show that overexpression of v-Jun proteins transforms chicken embryo fibroblasts (CEF) in vitro, and forms tumors in chickens in vivo. The mechanisms for this are not clearly defined. Conceivably, overexpression of an unregulated transcription factor would cause cell transfonnation by illicit regulation of its target genes. In support of this, we show that in vivo v-Jun complexes exhibit differential binding to in vitro generated AP-1 and 'AP-1 like' target sequences, suggesting that the pattern of target gene expression is altered during cell transformation. With this in mind, we set out to identify genes associated with v-Jun induced cell transformation. We have isolated several clones by subtractive hybridization, and a modified differential display procedure. One of these is clone 4, showing strong sequence homology, both at nucleotide and amino acid level, to cysteine thiol proteases. Northern blot analysis shows that the steady state levels of clone 4 mRNA are 3 to 7 times higher in v-Jun transformed CEF (VJ-1), when compared to c-Jun overexpressing CEF (CJ-3), or normal CEF infected with vector sequences only (RCAS).

Another is clone 15-15, showing strong sequence identity to the chicken Apolipoprotein A1 (ApoA1) gene. Northern blot analysis demonstrates that the steady state levels of ApoA1 mRNA in RCAS is 3 to 10 times higher than in VJ-1 cells, indicating that v-Jun might repress this gene by transcriptional mechanisms. To investigate this possibility, we generated several ApoA1 reporter CAT constructs containing 5' deletions in the promotor, and tested them in VJ-1 and RCAS cells. Our findings suggest that three potential cis-acting sequences could regulate this promotor in normal RCAS CEF. Quite remarkably, none of these constructs were transcriptionally active in VJ-1 cells. DNA binding studies utilizing one of the potential cis-acting regions, suggests that a specific factor is present in normal nuclear extracts, but absent from v-Jun transformed extracts. This observation suggests that this specific factor may be a positive activator protein. In addition, actinomycin D studies demonstrate that the ApoA1 mRNA has a long half-life of up to 20 hours. We therefore propose that ApoA1 is positively regulated by at least three cis-acting sequences, and maintained at high steady state levels in normal CEF. Several possible mechanisms exist to explain ApoA1 repression in normal RCAS and VJ-1 cells. One possibility is the direct repressor mechanism, whereby a silencer region directly inhibits ApoA1 expression in normal cells. In VJ-1 cells however, a squelching mechanism could predominate. In this case, overexpressed v-Jun proteins would sequester and inactivate potential factors that positively regulate ApoA1 transcription, leading to repression.

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Comments

A Dissertation Submitted to the Faculties of Eastern Virginia Medical School and Old Dominion University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Microbiology and Immunology.

DOI

10.25777/02fy-jh53

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