CHARACTERIZATION AND EXPRESSION OF RAF ONCOGENES IN NORMAL AND TUMOR CELLS

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There are three active raf proto-oncogenes in man: raf-1, A-raf, and B-raf. raf-I 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-I 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 36 adult and fetal mouse tissues indicate that (1) raf-1 is expressed in all tissues, although steady state levels vary five- to tenfold between tissues; (2) B-raf is expressed at highest levels in the cerebrum; and (3) A-raf is expressed preferentially in urogenital tissues with highest levels in the 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-1 and A-raf RNA'S. The raf-1 promoter has features of a housekeeping gene in that it is GC-rich (HTF-like), lacks consensus TATA or CAAT boxes (but has a TTAA sequence), contains heterogeneous RNA start sites, and four potential SP1 sites; consistent with its ubiquitous expression pattern. In addition, an octamer-binding motif (ATTTCAT) is located at -700 base pairs. The A-raf promoter displays features of a regulated gene in that it contains a TFIID binding site (TATA-box), sequences identical to binding sites for transcription factors EF-IA/PEA-3 (GGAAGTG), glucocorticoid receptor (TGTTCT), and the SV40 core enhancer (GTGGTTTG). Both raf-1 and A-raf promoters direct 60-90% of reporter gene expression relative to the SV40 early promoter in COS 7 cells. Sequentially deleted sequences flanking exon 1 of both genes show positive and negative effects on the level of reporter gene expression.