This project examines two aspects of the polyma middle T (MT) antigen: (1) how structure is related to function within the protein and (2) how the MT antigen interacts with cellular components to exert its regulatory effect on the cell. Recombinant DNA techniques are used to overproduce the MT antigen and the protein is purified from the resulting strains. Purified proteins are then be used as reagents for raising banks of monoclonal antibodies, for structural studies, and as affinity reagents for studying interactions with host cell proteins. To complement these biochemical approaches, genetic studies will be done using retrovirus vectors which transduce the individual T antigen. A library of point mutants of MT antigen which have conditionally or absolutely lost transforming function will be made using the virus vectors. Interesting mutations will be charcterized biochemically and sequenced at the DNA level to yield structure function data for the protiens. Together, the biochemical and genetic approaches will eventually allow us to overproduce mutant T antigens so that they can be purified for further study. Such a situation should yield valuable information about the manner in which polyoma MT antigens alter a cell to a transformed state. In parallel, we will purify and raise antisera to host proteins with which MT antigen and its complex wth pp60 C- src interact. These sera will be used to purify the interactive proteins and determine their biochemical properties and subcellular localizations.
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