title: Structure/function analysis of ras using random mutagenesis coupled with functional screening assays.
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-rasH expression vector. Transformation defective mutants were isolated by randomly mutagenizing a v-rasH 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.
authors.affiliation: Dana-Farber Cancer Institute, Boston, Massachusetts.
authors.forename: L A
authors.initials: LA
authors.lastname: Feig
authors.forename: M
authors.initials: M
authors.lastname: Corbley
authors.forename: B T
authors.initials: BT
authors.lastname: Pan
authors.forename: T M
authors.initials: TM
authors.lastname: Roberts
authors.forename: G M
authors.initials: GM
authors.lastname: Cooper
mesh_terms: D000818:Animals; D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000235:genetics; D002461:Cell Line, Transformed; D011905:Genes, ras; D005820:Genetic Testing / Q000379:methods*; D006160:Guanosine Triphosphate / Q000378:metabolism; D009152:Mutagenicity Tests / Q000379:methods*; D009154:Mutation; D010641:Phenotype; D013539:Simian virus 40 / Q000235:genetics / Q000302:isolation & purification; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D006160:Guanosine Triphosphate