Identification and characterization of purine nucleoside transporters from Crithidia fasciculata

To initiate a molecular dissection into the mechanism by which purine transport is up-regulated in Crithidia, genes encoding nucleoside transporters from Crithidia fasciculata were cloned and functionally characterized. Sequence analysis revealed CfNT1 and CfNT2 to be members of the equilibrative nu...

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Bibliographic Details
Published in:Molecular and biochemical parasitology 2005-03, Vol.140 (1), p.1-12
Main Authors: Liu, Wei, Arendt, Cassandra S., Gessford, Sarah K., Ntaba, Dziwe, Carter, Nicola S., Ullman, Buddy
Format: Article
Language:English
Subjects:
Adenosine - metabolism
Amino Acid Sequence
Animals
Cloning, Molecular
Crithidia fasciculata
Crithidia fasciculata - genetics
Crithidia fasciculata - growth & development
Crithidia fasciculata - metabolism
Culture Media
Genome, Protozoan
Guanosine - metabolism
Hypoxanthine - metabolism
Inosine - metabolism
Molecular Sequence Data
Nucleoside
Nucleoside Transport Proteins - biosynthesis
Nucleoside Transport Proteins - genetics
Nucleoside Transport Proteins - metabolism
Open Reading Frames
Protozoan Proteins - biosynthesis
Protozoan Proteins - genetics
Protozoan Proteins - metabolism
Purine Nucleosides - metabolism
Purine starvation
Purines
Sequence Alignment
Transport
Transporters
Xanthine
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Summary:To initiate a molecular dissection into the mechanism by which purine transport is up-regulated in Crithidia, genes encoding nucleoside transporters from Crithidia fasciculata were cloned and functionally characterized. Sequence analysis revealed CfNT1 and CfNT2 to be members of the equilibrative nucleoside transporter family, and the genes isolated encompassed polypeptides of 497 and 502 amino acids, respectively, each with 11 predicted membrane-spanning domains. Heterologous expression of CfNT1 cRNA in Xenopus laevis oocytes or CfNT2 in nucleoside transport-deficient Leishmania donovani demonstrated that CfNT1 is a novel high affinity adenosine transporter that also recognizes inosine, hypoxanthine, and pyrimidine nucleosides, while CfNT2 is a high affinity permease specific for inosine and guanosine. Southern blot analysis revealed that CfNT2 is present as a single copy within the C. fasciculata genome. Starvation of parasites for purines increased CfNT2 transport activity by an order of magnitude, although Northern blot analysis indicated CfNT2 transcript levels increased by
ISSN:0166-6851
1872-9428
DOI:10.1016/j.molbiopara.2004.11.018