This application is a proposal to perform pilot studies to determine the role of the 3'-untranslated region (3'UTR) of the transforming growth factor-alpha (TGF-alpha) mRNA in the regulated expression of this cytokine gene. There is a substantial recent literature implicating the 3'UTR in the stability and translational efficiency of various mRNAs. We hypothesize that the 3'UTR of TGF-alpha function in the regulated expression of this multifunctional cytokine gene. We propose to perform our studies by using the hamster TGF-alpha model. Our laboratory is currently studying the molecular mechanisms of oral carcinogenesis by using the hamster check pouch oral cancer model. We have recently cloned the DNA sequence encoding the mature TGF-alpha peptide as well as the 5'UTR from the Syrian hamster. Furthermore, we have found that the hamster TGF-alpha 3'UTR is about 1-kb larger than that of human and rat. We proposed to clone the large (about 5-kb) hamster TGF-alpha 3'UTR by a novel modification of the polymerase chain reaction (PCR). Messenger RNA will be isolated from a hamster oral carcinoma cell line (HCPC-1) which is known to express TGF-alpha. Specific primers will be designed using our cloned sequence of the hamster TGF-alpha cDNA. PCR will be used to amplified the desired products. Specific PCR products will be cloned into a bacteriophagemid vector (pBlueScript II KS-). We will further determine the actual genetic sequence of the hamster TGF-alpha 3'UTR. It is anticipated that by the end of this project we will have obtained the full length hamster TGF-alpha 3'UTR. We will compare it with the human and rat TGF-alpha UTR's and determine where the extra 1-kb of hamster TGF-alpha sequence resides. More importantly, the availability of the hamster TGF- alpha 3'UTR will allow us to begin test the hypothesis that this portion of the TGF-alpha mRNA is involved in the regulated expression of this multifunctional cytokine gene, in normal and disease processes.
