RESUMEN
Aims: Study the role of glycine-arginine rich (GAR) domain of fibrillarin in Giardia lamblia. Study Design: Identifying a specific glycine arginine rich (GAR) sequence of Giardia fibrillarin which plays an important role in ribonucleoprotein particles complex formation with snoRNA during post transcriptional modifications of rRNA. The present study uses Electrophoretic Mobiliy Shift Assay (EMSA) to detect protein-RNA interactions. 32P labeled snoRNA incubated with purified fibrillarin or GAR domain truncated fibrillarin protein were separated by a non denaturing polyacrylamide gel where the band patterns suggested the interaction of the RNAs with both proteins. Place and Duration of Study: Department of Parasitology, National Institute of Cholera and Enteric Diseases, Indian Council of Medical Research, Kolkata, between January 2011 and July 2012. Methodology: Homology analysis of G. lamblia fibrillarin was performed with fibrillarins from Homo sapiens, Mus musculus and Arabidopsis thaliana to determine the conserved domain by ClustalW analysis. Cloning, expression and purification of fibrillarin and GAR domain truncated fibrillarin were done to study the role of GAR domain in snoRNA-fibrillarin binding by EMSA and Gradient EMSA. Immunoblot assay of purified GAR domain truncated fibrillarin was performed by using polyclonal antibody of purified recombinant giardia fibrillarin protein. Results: Gel retardation assay showed that snoRNA does not bind with GAR domain truncated fibrillarin to form any RNP complex. Similarly in gradient EMSA the GAR domain truncated fibrillarin does not bind with any of the in vitro transcribed snoRNA even at much higher concentration than they do for full length purified recombinant fibrillarin. Conclusion: The amino terminal conserved glycine arginine rich (GAR) domain is present in Giardia lamblia fibrillarin and is essential for binding with snoRNA of this protein.
RESUMEN
Aim: To determine the common genotypes of Giardia duodenalis causing diarrhea in the study region and to assess the extent of genetic polymorphism among them. Study Design: Stool samples were collected from the patients attending IDBG Hospital, Kolkata with diarrheal complaints through a systemic sampling technique and were screened for Giardia duodenalis. The G. duodenalis positive samples were subjected to molecular genotyping through ‘PCR - Direct DNA sequencing’ procedure. All the sequence data obtained were incorporated into MEGA 4 software for multiple alignment and validation followed by phylogenetic analysis. The genotyping data obtained are stored in Excel spreadsheets and incorporated into EpiInfo 3.1 for analyzing possible association of genotype outcome with common physical factors such as age, sex etc. Place and Duration of Study: Department of parasitology, National Institute of Cholera and Enteric Diseases, Kolkata, India from July 2009 to November 2011. Methodology: A total of 68 Giardia duodenalis positive stool samples were identified from the diarrhea patients attending IDBG hospital in the city and were subjected to multi-locus genotyping. Fragments of ß-giardin, Glutamate-dehydrogenase and Triosephosphateisomerase genes of Giardia were amplified from those samples with specific primers and sequenced. All the sequences were analyzed using MEGA 4 software for obtaining the genotyping results. Results: Multi-locus genotyping identified 13 isolates as assemblage A and 41 as assemblage B, whereas 14 of them could not be assigned in a particular group. Detailed phylogenetic analysis revealed that multiple genotypes were observed in those 14 isolates depending upon the marker loci. Conclusion: The study could produce a preliminary idea about the G. duodenalis genotypes found in Kolkata city. High percentage of mixed assemblages in the study population also revealed the presence of genetic diversity among a small population of diarrheal patient within a limited geographical boundary. It has also hypothesized the possibility of inter-assemblage genetic exchange among Giardia.