TRANSFORMATION PROPERTIES OF THE HTLV-1 TAX GENE

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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.