BUDR DEPENDENCE, MALIGNANCY, AND DIFFERENTIATION

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The long term goal of this research is to elucidate the molecular mechanisms by which differentiation is regulated in melanoma cells. The experiments will involve the techniques of somatic cell genetics (cell hybridization and gene transfer) and the use of the thymidine analog 5- bromodeoxyuridine (BrdU) in combination with the techniques of recombinant DNA. The specific aims of the research relate to the extinction of pigmentation in somatic cell hybrids, the transaction of the fibroblast tyrosinase gene in hybrids, and the suppression of pigmentation by BrdU. The experiments with cell hybrids will focus on the chromosomal localization of the regulatory element causing extinction, the identification of the extinguisher locus, the occurrence of transactivation, the identification of DNA sequences (presumably upstream) in the tyrosinase gene that are involved in regulation, and the analysis of the way in which these sequences affect gene expression. The experiments with BrdU will focus on the identification of DNA sequences in the tyrosinase gene that are involved in suppression, the analysis of the way in which these sequences act, and the determination of the role of BrdU in the interactions between regulatory factors and regulatory regions of the tyrosinase gene. These experiments should lead to the identification of cis- and trans-acting regulatory elements involved in the regulation of tyrosinase gene expression, to the identification of factors binding to or produced by these elements, and to the isolation of the extinguisher locus. These experiments should contribute to the elucidation of the molecular mechanisms regulating differentiation in mammalian cells. The experiments also will contribute to our knowledge of the biology of melanoma cells. The information gained about regulatory mechanisms could be relevant to tumorigenesis, since malignancy (like differentiation) is affected by cell hybridization and by BrdU.