Enhanced thymidine uptake causes the lowered thymidine requirement of D. discoideum auxotroph HPS 401

Abstract

Dictyostelium discoideum strain HPS 401 contains a spontaneous mutation that lowers the amount of thymidine required for cell growth relative to that of the auxotrophic parental strain HPS 400. Growth studies in defined medium show that as little as 8 μg thymidine/ml supports maximal growth of HPS 401, whereas 50 μg/ml is required by HPS 400. In contrast, both strains require over 40 μg thymidylate/ml to achieve maximal growth. HPS 401 exhibits thymidineless death when grown without thymidine; relative viability decreases to <0.01 % after 190 h incubation. Assays for enzymes related to thymidine metabolism reveal that none of the strains tested (HPS 401, HPS 400, and prototrophic HPS 83 cells) contain detectable thymidine phosphorylase activity and that the specific activity of thymidine kinase is the same in these three strains. Thin-layer chromatography of extracts from cells grown on radiolabeled thymidine shows that there is no detectable conversion of thymidine to thymine in any of these strains. These analyses show that HPS 401 has rapid intracellular accumulation of thymidine, while only slight uptake is observed with HPS 400 or wild-type strains. HPS 401 also shows greater uptake of uridine in comparison to HPS 400 and wild-type cells. Thymidylate uptake was the same for all three strains. Thus, the mutation giving rise to the HPS 401 phenotype selectively increases the uptake of thymidine into the cell, where it can be efficiently utilized for DNA synthesis by the “salvage” pathways of nucleotide metabolism.