Development of an In Vitro Assay for the Generation of Novel Oncogenes

Abstract

Molecular analysis of the human trk oncogene, a transforming gene isolated from a colon carcinoma biopsy, revealed the existence of a novel member of the tyrosine kinase family. This locus, which we now designate as the trk proto-oncogene, was generated by the replacement of the coding sequence of the extracellular ligand binding domain by non - muscle tropomyosin sequences. The present studies were initiated to investigate whether the human trk gene can recombine with other cellular sequences and how these events contribute to the ontogeny of cancer. We have observed that transfection of indicator NIH3T3 cells with either the entire coding sequences or just the tyrosine kinase domain of the human trk proto-oncogene results in the frequent generation of novel trk oncogenes. These transforming genes express proteins of different molecular weights that can be precipitated with antibodies specific for the tyrosine kinase domain of the trk gene. Like the trk oncogene and proto-oncogene products, these novel oncogenes also coded for proteins having tyrosine kinase activity. Analysis of the messenger RNA and proteins of these trk oncogenes indicated that most of them had acquired additional sequences which were distinct from tropomyosin. We have determined that these additional sequences could come from the carrier DNA as well as the trk kinase domain itself. We have determined that some of these trk oncogenes were generated by direct recombination with cellular sequences. These studies represent the first reproducible system for the generation of oncogenes by recombination of cellular sequences.