CHARACTERIZATION AND EXPRESSION OF RAF ONCOGENES IN NORMAL AND TUMOR CELLS

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The raf-1 gene is located at chromosome 3p25 near sites specifically altered in renal cell carcinoma, small cell lung carcinoma, and mixed parotid gland tumors, and is genetically linked to the von Hippel Lindau disease gene. A-raf-1 is located at Xpll.2-11.4 near the translocation breakpoint in synovial sarcoma t(X;18) and the loci for Wiskott-Aldrich and Norrie syndromes. Northern hybridizations to RNA from fetal and adult mouse tissues indicate that raf-1 is expressed in all tissues, although steady state levels vary about tenfold between tissues with the highest levels in the cerebellum, striated muscle, and fetal brain. A-raf is expressed preferentially in urogenital tissues with the highest levels in the ovary and epididymis. We have begun to characterize the promoters for the individual human raf genes. DNA sequencing of genomic clones, primer extension, and Sl nuclease have been used to identify the 5' ends of raf-I and A-raf RNA's. The raf-I promoter has features of a housekeeping gene in that it is GC-rich (HTF-like), lacks consensus TATA or CAAT boxes, and has heterogeneous RNA start sites. The A-raf promoter displays features of a regulated gene in that it contains a TFIID binding site (TATA-box) and sequences identical to binding sites for several transcription factors. Fusion of a reporter gene to sequentially deleted sequences flanking exon 1 of both genes show positive and negative effects on the level of reporter gene expression. DNA sequences responsible for basal promoter activity have been defined by transient transfection assays, DNAse I footprinting, and gel retardation analyses.