CHARACTERIZATION AND EXPRESSION OF RAF ONCOGENES IN NORMAL AND TUMOR CELLS

Raf-1基因位于染色体3p25上，靠近特定的位点。 肾细胞癌、小细胞肺癌和混合性癌的改变 腮腺肿瘤，与冯·希佩尔·林道有遗传联系 疾病基因。A-RAF-1位于Xpll.2-11.4易位附近 滑膜肉瘤t(X；18)的断裂点和Wiskott-Aldrich基因座 和诺里综合症。用Northern杂交技术获得胎儿和胎儿的RNA 成年小鼠组织表明RAF-1在所有组织中都有表达， 尽管稳态水平在不同组织之间变化约为10倍， 在小脑、横纹肌和胎儿脑中含量最高。 A-RAF在泌尿生殖系统组织中优先表达，最高表达 在卵巢和附睾处的水平。我们已经开始描述 单个人类RAF基因的启动子。基因组DNA测序 克隆、引物延伸和S1核酸酶已被用来鉴定 RAF-I和A-RAF RNA的5‘端。RAF-I启动子具有 看家基因，因为它富含GC(类似HTF)，缺乏共识的TATA 或CAAT盒，并具有异质RNA起始点。A-RAF启动子 显示受调控基因的特征，因为它包含TFIID结合 位点(TATA-box)和与以下几个结合位点相同的序列 转录因子。报告基因的融合顺序缺失 两个基因外显子1两侧的序列均为阳性和阴性 对报告基因表达水平的影响。DNA序列 负责基本启动子活性的基因被定义为瞬时 转染法、DNase I足迹法和凝胶滞留分析。

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.