Isolation and characterization of the gene encoding the surface membrane 3'-nucleotidase/nuclease of Leishmania donovani.
Leishmania donovani and related trypanosomatid protozoa possess an externally 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 to fungal and plant class-I single-strand-specific nucleases, is thought 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 tryptic peptide fragments yielded amino-acid sequence information (Pept-1 to Pept-4) which provided the basis for the preparation of oligonucleotide primers used for PCR amplification of an approx. 300-bp DNA fragment. This fragment was cloned, sequenced and used to probe a genomic L. donovani cosmid library. Nucleotide sequence analysis of a 4.5-kb SmaI fragment, isolated from a cosmid clone, revealed an open reading frame (ORF) of 1434 nt encoding a 477-amino-acid protein. Pept-1 to Pept-4 were mapped onto the ORF-deduced protein sequence. Peptides corresponding to Pept-1 to Pept-4 were synthesized and used to immunize rabbits. The resulting anti-peptide antibodies recognized the 43-kDa protein on Western blots and immunoprecipitated the native 3'-nucleotidase activity from L. donovani membrane extracts. Further, the ORF-deduced protein 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 to a gene for the L. donovani 3'-nucleotidase/nuclease. In Northern blots a nucleotide probe specific for the 3'-NT/Nu gene hybridized to a single 2.5-kb messenger RNA. Results of Southern blot analyses were consistent with the 3'-NT/Nu being encoded by a single copy gene. These data constitute the first report of the gene for this unique trypanosomatid surface membrane enzyme.
Debrabant
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A
Cell Biology Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-0425, USA.
Gottlieb
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Dwyer
D M
DM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology*; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000032:analysis; D017125:Genes, Protozoan / Q000235:genetics*; D007893:Leishmania donovani / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009708:Nucleotidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D016366:Open Reading Frames / Q000235:genetics; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D016053:RNA, Protozoan / Q000032:analysis; D016415:Sequence Alignment; D017421:Sequence Analysis
D016054:DNA, Protozoan; D010446:Peptide Fragments; D016053:RNA, Protozoan; D009708:Nucleotidases; C040470:3'-nucleotidase