Experiments will be carried out to isolate and characterize the DNA sequences involved in resistance to nucleoside and other analogs in mammalian cells, utilizing techniques of biochemistry, somatic cell genetics, and molecular biology.
The specific aims of this project are to 1) further characterize the cellular components affected by the thymidine analog 5-hydroxymethyl-2'- deoxyuridine (hmdUrd), and isolate the human DNA sequences coding for these cellular components, 2) utilize mutant cell lines resistant to 5-fluorouracil (FUra) and arabinosylcytosine (araCyt) to isolate the human DNA sequences coding for CTP synthetase (EC 6.3.4.2), and 3) utilize mutant cell lines resistant to the glutamine analog 6-diazo-5-oxo-L-norleucine (DON) to isolate the DNA sequences coding for GMP synthetase (EC 6.3.5.2). The formation of hmdUrd in DNA by exposure to ionizing radiation may contribute to the cytotoxic effects of the radiation. Isolation and characterization of the human gene coding for resistance to hmdUrd may provide insight into the response of human cells to radiation damage. Populations of cells with high intracellular levels of CTP have increased resistance to FUra and araCyt. This mechanism may be the cause of a significant portion of the FUra and araCyt resistance observed in human tumors. Isolation and characterization of the human gene coding for CTP synthetase will provide a means of determining the control of its expression in normal and tumor cells. Renewed interest in the glutamine analog, DON, as a chemotherapeutic agent provides the rationale for the isolation and characterization of the DNA sequences coding for a GMP synthetase activity that is resistant to inhibition by DON.
