The protein encoded by the adenovirus E1A gene has several biological activities including the stimulation of transcription of some genes, the repression of transcription of other genes, and cell immortalization and transformation. This proposal will examine two aspects of the E1A. First, it is now generally accepted that nuclear proteins, of which E1A is an example, contain within their primary sequences discrete regions that target them to the nucleus. E1A is unusual in that it contains two nuclear targeting domains that are differentially used during development. The fine structure of these domains will be assessed as well as the exact time during development when each is used. The experiments will be performed in the convenient model system, oocytes and embryos of the frog Xenopus laevis. E1A mutants will be constructed such that certain amino acid residues are deleted from the protein. In addition, chimeric proteins will be constructed that fuse certain regions of E1A to B-galactosidase. All constructions will be performed at the DNA level, and the resulting mutant proteins will be expressed either in vitro or in vivo. The second aspect of E1A to be examined is its rate of turnover. E1A normally has a very short half-life within cells. This proposal will extend preliminary observations that E1A turnover is related to mRNA translation and that the amino terminal region of E1A is indispensible for instability. This proposal will assess the connection between E1A translation and instability. These experiments will also make use of deletion and chimeric E1A mutants similar to those described above. Protein turnover will be assayed in injected Xenopus oocytes and transfected somatic cells. E1A greatly influences growth control of cells. Therefore, a deciphering of the mechanism by which it enters the nucleus, where it exerts its effects, and the length of time it remains active within cells are of crucial importance for our understanding of cancer.
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