ISOLATION AND CHARACTERIZATION OF THE GENE ENCODING THE SURFACE-MEMBRANE 3'-NUCLEOTIDASE NUCLEASE OF LEISHMANIA-DONOVANI

Leishmania donovani and related trypanosomatid protozoa possess an ext ernally oriented surface membrane enzyme capable of hydrolyzing both 3 '-nucleotides and nucleic acids. By virtue of these activities, this 3 '-nucleotidase/nuclease (3'-NT/Nu), previously shown to be analogous t o fungal and plant class-I single-strand-specific nucleases, is though t to play a critical role in the salvage of purines, essential for the survival of these organisms. The 43-kDa 3'-NT/Nu was purified from L. donovani promastigotes and trypsin treated. Four of the released tryp tic peptide fragments yielded amino-acid sequence information (Pept-1 to Pept-4) which provided the basis for the preparation of oligonucleo tide primers used for PCR amplification of an approx. 300-bp DNA fragm ent. This fragment was cloned, sequenced and used to probe a genomic L . donovani cosmid library. Nucleotide sequence analysis of a 4.5-kb Sm aI fragment, isolated from a cosmid clone, revealed an open reading fr ame (ORF) of 1434 nt encoding a 477-amino-acid protein. Pept-1 to Pept -4 were mapped onto the ORF-deduced protein sequence. Peptides corresp onding to Pept-1 to Pept-4 were synthesized and used to immunize rabbi ts. The resulting anti-peptide antibodies recognized the 43-kDa protei n on Western blots and immunoprecipitated the native 3'-nucleotidase a ctivity from L. donovani membrane extracts. Further, the ORF-deduced p rotein shared significant sequence identity with the S1 and P1 fungal nucleases of Aspergillus oryzae and Penicillium citrinum, respectively . Cumulatively, these results demonstrated that the ORF corresponded t o a gene for the L, donovani 3'-nucleotidase/nuclease. In Northern blo ts a nucleotide probe specific for the 3'-NT/Nu gene hybridized to a s ingle 2.5-kb messenger RNA. Results of Southern blot analyses were con sistent with the 3'-NT/Nu being encoded by a single copy gene. These d ata constitute the first report of the gene for this unique trypanosom atid surface membrane enzyme.