Giardia intestinalis: molecular characterization of UDP-N-acetylglucosamine pyrophosphorylase.
The flagellated protozoan Giardia intestinalis is one of the most prevalent human-infective parasites with a worldwide distribution. This parasite must encyst to complete the life cycle and N-acetylgalactosamine is produced from endogenous glucose for cyst wall synthesis during the transformation. UDP-N-acetylglucosamine pyrophosphorylase in G. intestinalis (GiUAP, EC 2.7.7.23) is the fourth enzyme in the inducible pathway of N-acetylgalactosamine biosynthesis, catalysing the conversion of N-acetylglucosamine-1-P to UDP-N-acetylglucosamine. In this study the gene GiUAP was cloned and sequenced from the Portland 1 strain using PCR techniques. It has an ORF of approximately 1.3 kb and contains no introns. BLAST and ClustalW analysis of the deduced amino acid sequence revealed significant similarities to other eukaryotic UAPs with putative active sites identified. Southern hybridization showed that GiUAP exists as a single-copy gene and it was shown to have two transcripts by RT-PCR and Northern hybridization. RLM-RACE identified both 5' and 3' untranslated regions and suggested the transcripts exist as a 5'-capped mRNA, with the use of two tandem polyadenylation sites to generate two unusually long giardial 3' untranslated regions of approximately 522 bp and approximately 3 kb. Moreover, a recombinant protein (rGiUAP) was expressed in E. coli and subjected to physical characterizations. Surprisingly the results obtained in this study were significantly different from those reported for the GiUAP in MR4 strain, suggesting this gene is under different transcription control in different strains of G. intestinalis. This report describes the molecular characterization of GiUAP and provides an opportunity to explore the control of gene expression during encystation of the parasite.
Mok
Myth T S
MT
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney 2052, Australia. mythmok@mythmok.com
Tay
Enoch
E
Sekyere
Eric
E
Glenn
Wendy K
WK
Bagnara
Aldo S
AS
Edwards
Michael R
MR
D020413:3' Untranslated Regions / Q000235:genetics; D020121:5' Untranslated Regions / Q000235:genetics; D000116:Acetylgalactosamine / Q000096:biosynthesis; D000818:Animals; D001483:Base Sequence; D002473:Cell Wall / Q000201:enzymology / Q000235:genetics; D018628:Gene Dosage; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D016829:Giardia lamblia / Q000201:enzymology* / Q000235:genetics; D005873:Giardiasis / Q000201:enzymology / Q000235:genetics; D005947:Glucose / Q000378:metabolism; D006801:Humans; D007438:Introns / Q000235:genetics; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism; D016366:Open Reading Frames / Q000502:physiology*; D026723:Polyadenylation / Q000502:physiology; D016133:Polymerase Chain Reaction / Q000379:methods; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D015800:Protozoan Proteins; D009713:Nucleotidyltransferases; C023226:UDPacetylglucosamine pyrophosphorylase; D005947:Glucose; D000116:Acetylgalactosamine