TRANSFORMATION PROPERTIES OF THE HTLV-1 TAX GENE

感染人类逆转录病毒HTLV-1已被证明与 随着人类一种特殊的恶性肿瘤的发展，成人T细胞 白血病(ATL)。通过使用体外转化试验，我们 此前已证明HTLV的反式激活基因(Tax)- %1具有转化活性。具体地说，税收能够永垂不朽 大鼠胚胎成纤维细胞(REF)与ras癌基因的协同作用 变换参考这项拟议研究的长期目标有两个 收牌。[1]定位Tax蛋白中的转化功能，以及 确定它是否独立于或归因于转录激活 税收的功能。[2]鉴定其表达的细胞基因 受税收管制的。这些细胞基因代表了 反式激活蛋白。他们的身份将让我们深入了解 HTLV-1诱导转化的机制(S)。 Tax羧基末端的一系列渐进性缺失突变体 已派生出在以下情况下显示差异激活活性的 检测它们反式激活HTLV-1 LTR或人类的能力 白介素2受体基因。这些突变体将被检测其 有能力使裁判永垂不朽，并与RAS合作转变裁判。在……里面 这样，我们就可以确定税收的转化属性是否相互关联 它的反式激活活性，或者如果它们被定位到另一个域 蛋白质的含量。 表达受税收调控的细胞基因将通过 两种相辅相成的方法。比较二维蛋白质凝胶 将对正常REF和REF细胞系进行电泳法 通过税收使之永垂不朽，以识别差异表达的蛋白质。 凝胶纯化蛋白将被测序，这些信息将被用于 产生一系列人工合成的寡核苷酸探针，使 与待克隆的税收调控蛋白相对应的基因。在……里面 此外，还将从正常参考系中分离出mRNA，并构建其cDNA文库 和一个不朽的税收参考细胞系。减法杂交将是 用来识别受税收监管但也在 通过二维蛋白质凝胶分析检测低a水平 方法。一旦确定，将对这些基因进行分析以确定 它们可能在多种人类恶性肿瘤的发展过程中发挥作用。

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.