A major objective in the study of the biochemical mechanisms underlying development of the transformed phenotype, and ultimately of tumor growth, is the identification of normal cell growth regulating proteins, and of the amino acid structure of their active sites. The adenovirus E1A gene is an excellent model for such a study, as the products of this small gene induce both gene expression and host cell immortalization. Recent mutational analysis has revealed that there are at least three largely independent functional domains in the E1A products, two of which are required for the induction of proliferation and cellular transformation (for review, see Moran and Mathews, Cell, 48: 177-178). One of the two domains involved in the control of cell growth has been subjected to detailed genetic analysis but the other, which is required for the induction of DNA synthesis in quiescent cells, has not yet been studied in any detail. The initial specific aims of this project are to define the functional boundaries of this domain, and to correlate its function with the requirement for specific amino acid residues within this region. To accomplish these aims, a combination of random and site-directed mutagenesis will be used to delete nonessential regions, and to introduce single amino acid substitutions into the active site. The isolation of missense mutations impairing the DNA synthesis induction function will provide the tools needed to accomplish the remaining specific aims: to correlate the ability to induce DNA synthesis with other biological activities of the E1A products (such as nuclear localization and the activation of virus early gene expression), and to identify the primary cellular targets responsive to regulation by the E1A domains involved in cell cycle control. To accomplish the last aim, cells expressing E1A products defective in one or the other functions involved in control of cell growth will be probed in an attempt to identify cellular products which are regulated selectively in response to one or the other of these E1A functions. At least one cellular product, a DNA replication factor known as proliferating cell nuclear antigen (PCNA), is already known to be induced by E1A independently of either of the functional domains which are not required for the induction of DNA synthesis. The regulation of this product will be studied in detail to determine whether there are elements in the PCNA promoter which make it specifically responsive to the amino acid domain of E1A required for the induction of DNA synthesis.
