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Structure/Function Analysis of ras Using Random Mutagenesis Coupled with Functional Screening Assays^*

Dana-Farber Cancer Institute and Department of Pathology, Harvard Medical School Boston, Massachusetts 02115

Address requests for reprints to: Dr. Geoffrey M. Cooper, Dana-Farber Cancer Institute, Department of Pathology, Harvard Medical School, 44 Binney Street, Boston, MA 02115.

Abstract

We review the use of functional assays for the ras protein, p21, that have allowed us to screen for mutant ras genes encoding proteins defective in either interactions with guanine nucleotides or transforming activity. GTP binding and GTP-dependent autokinase activities were assayed directly on lysed bacterial colonies expressing p21. Mutants encoding ras proteins deficient in these activities were isolated after randomly mutagenizing a v-ras^H expression vector. Transformation defective mutants were isolated by randomly mutagenizing a v-ras^H retroviral shuttle vector. NIH cells were then infected with a stock of nonreplicating mutagenized retroviruses and nontransformed infected colonies were isolated. The mutant ras genes were then rescued from these cells for analysis. Characterization of these mutants defines domains of p21 involved in both biochemical and biological activities and addresses the role of guanine nucleotide binding in p21 function.

FOOTNOTES

^* This research was supported by grants from the National Cancer Institute, a fellowship (to L.A.F.) from the Leukemia Society of America, and a faculty research award (to G.M.C.) from the American Cancer Society.

Received for publication September 5, 1986.

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