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MOLECULAR CHARACTERIZATION AND SEQUENCE PHYLOGENETIC ANALYSIS OF SURFACE ANTIGEN 3 (SAG3) GENE OF LOCAL INDIAN ISOLATES (CHENNAI AND IZATNAGAR) OF Toxoplasma gondii.

Sudan V, Tewari AK, Singh H - Rev. Inst. Med. Trop. Sao Paulo (2015 May-Jun)

Bottom Line: The surface antigen 3 (SAG3) of two local Indian isolates were cloned and sequenced before being compared with the available published sequences.The two Indian isolates used in the present study are known to vary between themselves, as far as homologies related to other genes are concerned, but they were found to be 100% homologous as far as SAG3 locus is concerned.The findings are important from the point of view of molecular phylogeny.

View Article: PubMed Central - PubMed

Affiliation: Department of Parasitology, College of Veterinary Sciences & Animal Husbandry, U. P. Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan, Mathura, India.

ABSTRACT

Context and objective: The molecular characterization of local isolates of Toxoplasma gondii is considered significant so as to assess the homologous variations between the different loci of various strains of parasites.

Design and setting: The present communication deals with the molecular cloning and sequence analysis of the 1158 bp entire open reading frame (ORF) of surface antigen 3 (SAG3) of two Indian T. gondii isolates (Chennai and Izatnagar) being maintained as cryostock at the IVRI.

Method: The surface antigen 3 (SAG3) of two local Indian isolates were cloned and sequenced before being compared with the available published sequences.

Results: The sequence comparison analysis revealed 99.9% homology with the standard published RH strain sequence of T. gondii. The strains were also compared with other established published sequences and found to be most related to the P-Br strain and CEP strain (both 99.3%), and least with PRU strain (98.4%). However, the two Indian isolates had 100% homology between them.

Conclusion: Finally, it was concluded that the Indian isolates were closer to the RH strain than to the P-Br strain (Brazilian strain), the CEP strain and the PRU strains (USA), with respect to nucleotide homology. The two Indian isolates used in the present study are known to vary between themselves, as far as homologies related to other genes are concerned, but they were found to be 100% homologous as far as SAG3 locus is concerned. This could be attributed to the fact that this SAG3 might be a conserved locus and thereby, further detailed studies are thereby warranted to exploit the use of this particular molecule in diagnostics and immunoprophylactics. The findings are important from the point of view of molecular phylogeny.

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Related in: MedlinePlus

Release of SAG3 insert by restriction digestion of insTA cloning vector of thetwo Indian isolates on 1.5% agarose gel. Lane M: Marker 100 bp DNA ladder plus(MBI Fermentas); Lane IZN: Insert release after PstI andEcoRI digestion of vector containing Izatnagar isolate; LaneCHEN: Insert release after PstI and EcoRI digestion of vector containing Chennaiisolate; Lane Uncut Plasmid: Undigested recombinant insTA cloning vector.
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f02: Release of SAG3 insert by restriction digestion of insTA cloning vector of thetwo Indian isolates on 1.5% agarose gel. Lane M: Marker 100 bp DNA ladder plus(MBI Fermentas); Lane IZN: Insert release after PstI andEcoRI digestion of vector containing Izatnagar isolate; LaneCHEN: Insert release after PstI and EcoRI digestion of vector containing Chennaiisolate; Lane Uncut Plasmid: Undigested recombinant insTA cloning vector.

Mentions: PCR amplification, molecular cloning and molecular characterization of the SAG3gene of Indian isolates: The whole ORF of the SAG3 gene was amplified from thecDNA of Indian isolates of T. gondii using the specific forward andreverse primers. The amplicons were resolved as a single band of 1158 bp (Fig. 1). It was further purified for ligation inInsTAclone PCR cloning vector. The selection of positive colonies was performed bycolony PCR using the specific primers and also by restriction enzyme digestion of therecombinant plasmids with PstI and EcoRI for therelease of insert. The results of restriction enzyme digestion (Fig. 2) as well as colony PCR (Fig.3) were checked by agarose gel electrophoresis.


MOLECULAR CHARACTERIZATION AND SEQUENCE PHYLOGENETIC ANALYSIS OF SURFACE ANTIGEN 3 (SAG3) GENE OF LOCAL INDIAN ISOLATES (CHENNAI AND IZATNAGAR) OF Toxoplasma gondii.

Sudan V, Tewari AK, Singh H - Rev. Inst. Med. Trop. Sao Paulo (2015 May-Jun)

Release of SAG3 insert by restriction digestion of insTA cloning vector of thetwo Indian isolates on 1.5% agarose gel. Lane M: Marker 100 bp DNA ladder plus(MBI Fermentas); Lane IZN: Insert release after PstI andEcoRI digestion of vector containing Izatnagar isolate; LaneCHEN: Insert release after PstI and EcoRI digestion of vector containing Chennaiisolate; Lane Uncut Plasmid: Undigested recombinant insTA cloning vector.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4544243&req=5

f02: Release of SAG3 insert by restriction digestion of insTA cloning vector of thetwo Indian isolates on 1.5% agarose gel. Lane M: Marker 100 bp DNA ladder plus(MBI Fermentas); Lane IZN: Insert release after PstI andEcoRI digestion of vector containing Izatnagar isolate; LaneCHEN: Insert release after PstI and EcoRI digestion of vector containing Chennaiisolate; Lane Uncut Plasmid: Undigested recombinant insTA cloning vector.
Mentions: PCR amplification, molecular cloning and molecular characterization of the SAG3gene of Indian isolates: The whole ORF of the SAG3 gene was amplified from thecDNA of Indian isolates of T. gondii using the specific forward andreverse primers. The amplicons were resolved as a single band of 1158 bp (Fig. 1). It was further purified for ligation inInsTAclone PCR cloning vector. The selection of positive colonies was performed bycolony PCR using the specific primers and also by restriction enzyme digestion of therecombinant plasmids with PstI and EcoRI for therelease of insert. The results of restriction enzyme digestion (Fig. 2) as well as colony PCR (Fig.3) were checked by agarose gel electrophoresis.

Bottom Line: The surface antigen 3 (SAG3) of two local Indian isolates were cloned and sequenced before being compared with the available published sequences.The two Indian isolates used in the present study are known to vary between themselves, as far as homologies related to other genes are concerned, but they were found to be 100% homologous as far as SAG3 locus is concerned.The findings are important from the point of view of molecular phylogeny.

View Article: PubMed Central - PubMed

Affiliation: Department of Parasitology, College of Veterinary Sciences & Animal Husbandry, U. P. Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan, Mathura, India.

ABSTRACT

Context and objective: The molecular characterization of local isolates of Toxoplasma gondii is considered significant so as to assess the homologous variations between the different loci of various strains of parasites.

Design and setting: The present communication deals with the molecular cloning and sequence analysis of the 1158 bp entire open reading frame (ORF) of surface antigen 3 (SAG3) of two Indian T. gondii isolates (Chennai and Izatnagar) being maintained as cryostock at the IVRI.

Method: The surface antigen 3 (SAG3) of two local Indian isolates were cloned and sequenced before being compared with the available published sequences.

Results: The sequence comparison analysis revealed 99.9% homology with the standard published RH strain sequence of T. gondii. The strains were also compared with other established published sequences and found to be most related to the P-Br strain and CEP strain (both 99.3%), and least with PRU strain (98.4%). However, the two Indian isolates had 100% homology between them.

Conclusion: Finally, it was concluded that the Indian isolates were closer to the RH strain than to the P-Br strain (Brazilian strain), the CEP strain and the PRU strains (USA), with respect to nucleotide homology. The two Indian isolates used in the present study are known to vary between themselves, as far as homologies related to other genes are concerned, but they were found to be 100% homologous as far as SAG3 locus is concerned. This could be attributed to the fact that this SAG3 might be a conserved locus and thereby, further detailed studies are thereby warranted to exploit the use of this particular molecule in diagnostics and immunoprophylactics. The findings are important from the point of view of molecular phylogeny.

Show MeSH
Related in: MedlinePlus