Retrovirus promoter-trap vectors will be employed as insertional mutagens to isolate genes which prevent activated ras oncogenes from transforming cultured cells. The vector expresses an activated ras oncogene and also contain histidinol dehydrogenase (his) coding sequences inserted into the 3' long terminal repeat (LTR). His sequences do not interfere with virus replication or integration. Thus, when the virus is passaged, duplication of the LTRs places his sequences in the 5'LTR just 30 nt. from the flanking cellular DNA. Selection for histidinol resistance generates cell clones in which the provirus has integrated downstream of transcriptionally active promoters, disrupting cellular genes in the process. Cells which resist transformation by ras oncogenes (because of genetically dominant activities) will by infected by U3HisTKras and 104 -105 independent histidinol resistant clones (representative of all expressed sites) will be isolated, and pools of clones will be injected into nude mice. Transformants which emerge will be analyzed to determine if genes which normally prevent ras from transforming were disrupted as a result of provirus integration. Retrovirus promoter-trap vectors will also be used to analyze transcriptional control by growth factors, oncogenes and antioncogenes. The strategy uses promoter-trap retroviruses to force the integration of a reporter gene or selectable marker into a large collection of chromosomal sites and selecting for clones in which expression of the transduced marker gene is regulated by growth factors, by the myc and E1A oncogenes and by the Rb1 and WT1 anti-oncogenes. Genes inserted in U3 which permit selections both for and against expression (xanthine-guanine phosphoribosyltransferase, thymidine kinase, CD4) are expected to facilitate the isolation of cell clones in which the provirus has become linked to regulated promoters. Cellular sequences flanking proviruses will be amplified by polymerase chain reaction, cloned and analyzed for promoter elements and disrupted genes. Finally, cell lines containing provirus reporter genes fused to promoters which are regulated by growth factors, oncogenes or anti-oncogenes will be used to develop biological assays for functional studies of transcription factors encoded by oncogene and anti-oncogene proteins and of signal transduction pathways which regulate transcription.
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