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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29CA046436-03
Application #
3458610
Study Section
Molecular Biology Study Section (MBY)
Project Start
1988-03-01
Project End
1993-02-28
Budget Start
1990-03-01
Budget End
1991-02-28
Support Year
3
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Cold Spring Harbor Laboratory
Department
Type
DUNS #
065968786
City
Cold Spring Harbor
State
NY
Country
United States
Zip Code
11724
Abraham, S E; Carter, M C; Moran, E (1992) Transforming growth factor beta 1 (TGF beta 1) reduces cellular levels of p34cdc2, and this effect is abrogated by adenovirus independently of the E1A-associated pRB binding activity. Mol Biol Cell 3:655-65
Yaciuk, P; Moran, E (1991) Analysis with specific polyclonal antiserum indicates that the E1A-associated 300-kDa product is a stable nuclear phosphoprotein that undergoes cell cycle phase-specific modification. Mol Cell Biol 11:5389-97
Wang, H G; Draetta, G; Moran, E (1991) E1A induces phosphorylation of the retinoblastoma protein independently of direct physical association between the E1A and retinoblastoma products. Mol Cell Biol 11:4253-65
Yaciuk, P; Carter, M C; Pipas, J M et al. (1991) Simian virus 40 large-T antigen expresses a biological activity complementary to the p300-associated transforming function of the adenovirus E1A gene products. Mol Cell Biol 11:2116-24
Raychaudhuri, P; Bagchi, S; Devoto, S H et al. (1991) Domains of the adenovirus E1A protein required for oncogenic activity are also required for dissociation of E2F transcription factor complexes. Genes Dev 5:1200-11
Stein, R W; Corrigan, M; Yaciuk, P et al. (1990) Analysis of E1A-mediated growth regulation functions: binding of the 300-kilodalton cellular product correlates with E1A enhancer repression function and DNA synthesis-inducing activity. J Virol 64:4421-7
Pietenpol, J A; Stein, R W; Moran, E et al. (1990) TGF-beta 1 inhibition of c-myc transcription and growth in keratinocytes is abrogated by viral transforming proteins with pRB binding domains. Cell 61:777-85
Sturm, R A; Yaciuk, P (1989) DNA cleavage by restriction endonuclease PflMI is inhibited in recognition sites modified by dcm methylation. Nucleic Acids Res 17:3615