TRANSFORMATION PROPERTIES OF THE HTLV-1 TAX GENE

人类逆转录病毒HTLV-1的感染已被证明与 随着人类特定恶性肿瘤的发展，成人T细胞 白血病（ATL）。 通过使用体外转化试验， 先前已经证明，来自HTLV-的反式激活基因（tax） 1具有转化活性。 具体来说，税收能够使 大鼠胚胎成纤维细胞（REF），并与ras癌基因合作， 这项拟议研究的长期目标是两个 折 [1]绘制Tax蛋白中的转换功能， 确定它是独立于，还是由于， 税的功能。 [2]为了鉴定表达与细胞增殖有关的细胞基因， 由税收监管。 这些细胞基因代表了 反式激活蛋白 他们的身份将使我们了解 HTLV-1诱导转化发生的机制。 一系列Tax基因羧基端进行性缺失突变体 已经衍生出，当 测定它们反式激活HTLV-1 LTR或人LTR的能力， 白细胞介素2受体基因。 这些突变体将被检测其 能够使REF永生化，并与RAS合作来转换REF。 这样，我们将确定Tax的转换属性是否与 或者如果它们定位于另一个结构域， 蛋白质。 将通过以下方法鉴定其表达受Tax调节的细胞基因： 两种互补的方法。 比较二维蛋白质凝胶 将对正常REF和REF细胞系进行电泳 通过Tax永生化以鉴定差异表达的蛋白质。 将对凝胶纯化的蛋白质进行测序，并将信息用于 产生一系列合成的寡核苷酸探针， 对应于待克隆的Tax调节蛋白的基因。 在 此外，cDNA文库将从正常REF分离的mRNA制备 和税收永生化的REF细胞系。 差减杂交将是 用于识别受Tax调控的基因，但也表达于 通过二维蛋白质凝胶分析检测的低水平 法 一旦确定这些基因将被分析，以确定 它们在多种人类恶性肿瘤的发展中可能发挥的作用。

Infection by the human retrovirus HTLV-1 has been shown to be associated with the development of a particular malignancy in man, adult T-cell leukemia (ATL). Through the use of an in vitro transformation assay we have previously demonstrated that the transactivation gene (tax) from HTLV- 1 has transformation activity. Specifically, tax was able to immortalize rat embryo fibroblasts (REF) and to cooperate with the ras oncogene to transform REF. The long term objectives of this proposed study are two fold. [1] To map the transformation function in the Tax protein, and determine whether it is separate from, or due to, the transactivation function of Tax. [2] To identify cellular genes whose expression is regulated by Tax. These cellular genes represent the targets of the transactivator protein. Their identification will give insight into the mechanism(s) by which HTLV-1 induced transformation occurs. A series of progressive deletion mutants of the carboxy-terminus of Tax have been derived that show differential transactivation activity when assayed for their ability to transactivate the HTLV-1 LTR or the human interleukin-2 receptor gene. These mutants will be assayed for their ability to immortalize REF and to cooperate with ras to transform REF. In this way we will determine if the transforming properties of Tax are linked to its transactivating activity, or if they are localized to another domain of the protein. Cellular genes whose expression is regulated by Tax will be identified by two complementary approaches. Comparative two-dimensional protein gel electrophoresis will be performed on normal REF and the REF cell lines immortalized by tax to identify proteins that are differentially expressed. Gel purified protein will be sequenced and the information will be used to generate a series of synthetic oligonucleotide probes that will allow the genes corresponding to the Tax-regulated proteins to be cloned. In addition, cDNA libraries will be made from mRNA isolated from normal REF and a tax immortalized REF cell line. Subtraction hybridization will be used to identify the genes that are regulated by Tax, but expressed at too low a level to be detected by the two-dimensional protein gel analysis method. Once identified these genes will be analyzed to determine what role they may play in the development of a variety of human malignancies.