Synthesis and functional studies on nucleic acids. Problems related to synthetic ras genes

Abstract

Synthesis of a gene for an oncoprotein, ras p21 and its functions are described. Point mutations in hot spots of ras genes have been found in human cancer cells and produce activated p21 which result in transformation of cultured NIH3T3 cells. To produce normal and activated p21 in quantity for biochemical and structural studies, genes encoding these proteins were synthesized and expressed in E. coli. Normal and activated RAS proteins were tested for their GTPase activity and three dimensional structures were determined by X-ray crystallography. Transforming activities of the synthetic genes have been tested by transfecting their expression vectors to NIH3T3 cells and the synthetic activated genes were found to transform these cells indicating that the product of the activated gene is responsible for these malignant growth of the cells. These activities were proved to be inhibited by transfecting designed ribozyme genes. These synthetic genes were used to investigate mutagenesis of damaged bases such as 7,8-dihydro-8-oxoguanine and thymine photodimers, by introducing the damaged base in hot spots of the oncogene. These unnatural bases in the ras gene were found to be mutagenic and cause malignant growth of NIH3T3 cells.