Genetic analysis of adenine metabolism in Leishmania donovani promastigotes. Evidence for diploidy at the adenine phosphoribosyltransferase locus.
Mutant promastigotes of Leishmania donovani deficient in adenine phosphoribosyltransferase (AP-Rib transferase) have been isolated in medium containing 4-aminopyrazolopyrimidine. The generation of AP-Rib transferase-deficient mutants occurred in two discrete steps. In the first step, clones were isolated with 50% of wild-type levels of AP-Rib transferase activity. These cells were reselected, and colonies totally deficient in AP-Rib transferase were isolated. Wild-type and AP-Rib transferase-deficient cells contained equivalent amounts of other enzymes essential to adenine metabolism such as adenine deaminase and hypoxanthine-guanine phosphoribosyltransferase. Partially and totally AP-Rib transferase-deficient cells exhibited intermediate and complete resistance to cytotoxic adenine analogs, respectively. Nevertheless, wild-type and mutant cells could salvage adenine and utilize adenine as a purine source equally efficiently, suggesting that the adenine deaminase-hypoxanthine-guanine phosphoribosyl-transferase pathway plays an important role in promastigote adenine metabolism. Kinetic and thermal inactivation studies of purified AP-Rib transferase and isoelectric focusing of crude extracts from wild-type and partially AP-Rib transferase-deficient cells suggested that the latter cells possessed wild-type AP-Rib transferase activity at half the amount found in wild-type parental cells. These data suggest that L. donovani possesses two copies of the AP-Rib transferase structural gene and that these organisms might be diploid for the AP-Rib transferase locus.
D000225:Adenine / Q000378:metabolism*; D000228:Adenine Phosphoribosyltransferase / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D004171:Diploidy; D007041:Hypoxanthine Phosphoribosyltransferase / Q000378:metabolism; D007700:Kinetics; D007891:Leishmania / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D009154:Mutation; D010430:Pentosyltransferases / Q000235:genetics*
D010430:Pentosyltransferases; D000228:Adenine Phosphoribosyltransferase; D007041:Hypoxanthine Phosphoribosyltransferase; D000225:Adenine