Molecular characterization of a hyperinducible, surface membrane-anchored,class I nuclease of a trypanosomatid parasite

The 3'-nucleotidase/nuclease (5'-NT/NU) is a surface enzyme unique to trypa nosomatid parasites. These organisms lack the pathway for de novo purine bi osynthesis and thus are entirely dependent upon their hosts to supply this nutrient for their survival, growth, and multiplication. The 3'-NT/NU is in volved in the salvage of preformed purines via the hydrolysis of either 3'- nucleotides or nucleic acids. In Crithidia luciliae, this enzyme is highly inducible. For example, in these organisms purine starvation triggers an si milar to 1000-fold up-expression of 3'-NT/NU activity. In the present study , we cloned and characterized a gene encoding this intriguing enzyme from C . luciliae (Cl). Sequence analysis showed that the Cl 3'-NT/NU deduced prot ein possessed five regions, which we defined here as being characteristic o f members of the class I nuclease family. Further, we demonstrated that the Cl 3'-NT/NU-expressed protein possessed both 3'-nucleotidase and nuclease activities. Moreover, we showed that the dramatic up-expression of 5'-NT/NU activity in response to purine starvation of C. luciliae was concomitant w ith the similar to 100-fold elevation in steady-state mRNA specific for thi s gene. Finally, results of our nuclear run-on analyses demonstrated that s uch up-regulation in 3'-NT/NU enzyme activity was mediated at the posttrans criptional level.