The overall aim of this research is the determination of the functions of SV40 small-t antigen and of two cellular proteins with which it associates. Major effort will be placed on investigations of the metabolic patterns of cells that express small-t antigen, because recent studies have shown that antimitochondrial drugs and ionophores modify growth patterns observed in CV-1 cells which relate to the presence of small-t antigen. Plasma membrane functions, such as transport and membrane potential, will also be examined and related to small-t antigen. We will also pursue the observation that one of the cellular proteins, 32K, appears to be binding S-adenosylmethionine (SAM). This raises the possibility that the 32K protein may be an enzyme which uses SAM as a substrate, and may provide a major clue concerning the function of at least one of the cellular proteins and, indirectly, the function of small-t antigen. Purifications of small-t antigen and the cellular proteins will be continued and scaled-up, with particular emphasis on purification of the soluble small-t antigen expressed by a bacterial clone. Isolation of small-t antigen which retains the ability to interact with the cellular proteins is a priority. Additional efforts include development of hybridoma cell lines that produce antibodies that recognize the small-t antigen cellular protein complex efficiently, or that recognize the unique region of small-t antigen. If the latter is obtained, attempts to identify and active protein fragment from small-t antigen in infected cells will be made. Potential function of such a fragment is also being approached using a bacterial clone that contains DNA sequences only from this region of small-t antigen, and from which small-t unique sequences can be expressed in bacteria.
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