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Transcriptome profiling reveals higher vertebrate orthologous of intra-cytoplasmic pattern recognition receptors in grey bamboo shark.

Krishnaswamy Gopalan T, Gururaj P, Gupta R, Gopal DR, Rajesh P, Chidambaram B, Kalyanasundaram A, Angamuthu R - PLoS ONE (2014)

Bottom Line: Few of the candidate genes were selected to analyze their expression levels in various tissues by real-time PCR and also localization of a receptor by in-situ PCR to validate the prediction.We also predicted the domains structures of some of the identified pattern recognition receptors, their phylogenetic relationship with lower and higher vertebrates and the complete downstream signaling mediators of classical dsRNA signaling pathway.The generated transcriptome will be a valuable resource to further genetic and genomic research in elasmobranchs.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Biotechnology and Translational Research Platform for Veterinary Biologicals, Tamil Nadu Veterinary and Animal Sciences University, Chennai, Tamil Nadu, India.

ABSTRACT
From an immunologist perspective, sharks are an important group of jawed cartilaginous fishes and survey of the public database revealed a great gap in availability of large-scale sequence data for the group of Chondrichthyans the elasmobranchs. In an attempt to bridge this deficit we generated the transcriptome from the spleen and kidney tissues (a total of 1,606,172 transcripts) of the shark, Chiloscyllium griseum using the Illumina HiSeq2000 platform. With a cut off of > = 300 bp and an expression value of >1RPKM we used 43,385 transcripts for BLASTX analysis which revealed 17,548 transcripts matching to the NCBI nr database with an E-value of < = 10(-5) and similarity score of 40%. The longest transcript was 16,974 bases with matched to HECT domain containing E3 ubiqutin protein ligase. MEGAN4 annotation pipeline revealed immune and signalling pathways including cell adhesion molecules, cytokine-cytokine receptor interaction, T-cell receptor signalling pathway and chemokine signaling pathway to be highly expressed in spleen, while different metabolism pathways such as amino acid metabolism, carbohydrate metabolism, lipid metabolism and xenobiotic biodegradation were highly expressed in kidney. Few of the candidate genes were selected to analyze their expression levels in various tissues by real-time PCR and also localization of a receptor by in-situ PCR to validate the prediction. We also predicted the domains structures of some of the identified pattern recognition receptors, their phylogenetic relationship with lower and higher vertebrates and the complete downstream signaling mediators of classical dsRNA signaling pathway. The generated transcriptome will be a valuable resource to further genetic and genomic research in elasmobranchs.

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Percentage of nucleotide and amino acid identity of the Chiloscyllium griseum Toll-like receptor 3 (shTLR3) signaling pathway mediators with sequences of other species.The full-length coding sequence of the C. griseum TRAF3 (1728 bp), TBK1 (1529 bp), IRF3 (1374 bp), IRF7 (1515 bp) and Mx (partial 353 bp) sequences was aligned with the other sequences representing these signaling molecules types from different lower and higher order species using Clustal W in the Megalign Module of Lasergene (V 7.1.0). Note: These signaling molecules revealed extreme diversity and the identity revealed close relationship with the higher vertebrate TLRs rather than the teleost. The TBK1 molecule was divergent among the signaling mediators.
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pone-0100018-g011: Percentage of nucleotide and amino acid identity of the Chiloscyllium griseum Toll-like receptor 3 (shTLR3) signaling pathway mediators with sequences of other species.The full-length coding sequence of the C. griseum TRAF3 (1728 bp), TBK1 (1529 bp), IRF3 (1374 bp), IRF7 (1515 bp) and Mx (partial 353 bp) sequences was aligned with the other sequences representing these signaling molecules types from different lower and higher order species using Clustal W in the Megalign Module of Lasergene (V 7.1.0). Note: These signaling molecules revealed extreme diversity and the identity revealed close relationship with the higher vertebrate TLRs rather than the teleost. The TBK1 molecule was divergent among the signaling mediators.

Mentions: With respect to the downstream mediators of the TLR3 signaling pathway we could identify the full-length TRAF-3 (4672 bp with a predicted ORF of 1728 bp coding for 575 amino acids) from transcriptome and BLAST analysis revealed 53 to 58.5% amino acid (aa) identity to various vertebrates (avian species). The downstream signalling mediator of TRAF3 is TBK1 and the annotated shTBK1 was 1529 nt long coding for 512 aa which BLAST matched to Ceratotherium simum simum (44.3% aa identity). TBK1 mediates the signals either through IRF3 or IRF7. The IRF3 coding sequence was 1374 nt with a predicted protein of 457 aa long and more related Xenopus laevis with an identity of 28.5 to 40.2% at aa level with different species. The annotated IRF7 sequence was 1515 nt long that could be translated to a 504 aa protein which was more related to Channa argus and revealed 27.1 to 34.7% aa identity with different species compared. We could also show the presence of a partial transcript in C. griseum transcriptome that was 353 nt long which could be translated to a 117 aa peptide that BLAST matched with Mx protein (Figure 11) (Data S1).


Transcriptome profiling reveals higher vertebrate orthologous of intra-cytoplasmic pattern recognition receptors in grey bamboo shark.

Krishnaswamy Gopalan T, Gururaj P, Gupta R, Gopal DR, Rajesh P, Chidambaram B, Kalyanasundaram A, Angamuthu R - PLoS ONE (2014)

Percentage of nucleotide and amino acid identity of the Chiloscyllium griseum Toll-like receptor 3 (shTLR3) signaling pathway mediators with sequences of other species.The full-length coding sequence of the C. griseum TRAF3 (1728 bp), TBK1 (1529 bp), IRF3 (1374 bp), IRF7 (1515 bp) and Mx (partial 353 bp) sequences was aligned with the other sequences representing these signaling molecules types from different lower and higher order species using Clustal W in the Megalign Module of Lasergene (V 7.1.0). Note: These signaling molecules revealed extreme diversity and the identity revealed close relationship with the higher vertebrate TLRs rather than the teleost. The TBK1 molecule was divergent among the signaling mediators.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4067322&req=5

pone-0100018-g011: Percentage of nucleotide and amino acid identity of the Chiloscyllium griseum Toll-like receptor 3 (shTLR3) signaling pathway mediators with sequences of other species.The full-length coding sequence of the C. griseum TRAF3 (1728 bp), TBK1 (1529 bp), IRF3 (1374 bp), IRF7 (1515 bp) and Mx (partial 353 bp) sequences was aligned with the other sequences representing these signaling molecules types from different lower and higher order species using Clustal W in the Megalign Module of Lasergene (V 7.1.0). Note: These signaling molecules revealed extreme diversity and the identity revealed close relationship with the higher vertebrate TLRs rather than the teleost. The TBK1 molecule was divergent among the signaling mediators.
Mentions: With respect to the downstream mediators of the TLR3 signaling pathway we could identify the full-length TRAF-3 (4672 bp with a predicted ORF of 1728 bp coding for 575 amino acids) from transcriptome and BLAST analysis revealed 53 to 58.5% amino acid (aa) identity to various vertebrates (avian species). The downstream signalling mediator of TRAF3 is TBK1 and the annotated shTBK1 was 1529 nt long coding for 512 aa which BLAST matched to Ceratotherium simum simum (44.3% aa identity). TBK1 mediates the signals either through IRF3 or IRF7. The IRF3 coding sequence was 1374 nt with a predicted protein of 457 aa long and more related Xenopus laevis with an identity of 28.5 to 40.2% at aa level with different species. The annotated IRF7 sequence was 1515 nt long that could be translated to a 504 aa protein which was more related to Channa argus and revealed 27.1 to 34.7% aa identity with different species compared. We could also show the presence of a partial transcript in C. griseum transcriptome that was 353 nt long which could be translated to a 117 aa peptide that BLAST matched with Mx protein (Figure 11) (Data S1).

Bottom Line: Few of the candidate genes were selected to analyze their expression levels in various tissues by real-time PCR and also localization of a receptor by in-situ PCR to validate the prediction.We also predicted the domains structures of some of the identified pattern recognition receptors, their phylogenetic relationship with lower and higher vertebrates and the complete downstream signaling mediators of classical dsRNA signaling pathway.The generated transcriptome will be a valuable resource to further genetic and genomic research in elasmobranchs.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Biotechnology and Translational Research Platform for Veterinary Biologicals, Tamil Nadu Veterinary and Animal Sciences University, Chennai, Tamil Nadu, India.

ABSTRACT
From an immunologist perspective, sharks are an important group of jawed cartilaginous fishes and survey of the public database revealed a great gap in availability of large-scale sequence data for the group of Chondrichthyans the elasmobranchs. In an attempt to bridge this deficit we generated the transcriptome from the spleen and kidney tissues (a total of 1,606,172 transcripts) of the shark, Chiloscyllium griseum using the Illumina HiSeq2000 platform. With a cut off of > = 300 bp and an expression value of >1RPKM we used 43,385 transcripts for BLASTX analysis which revealed 17,548 transcripts matching to the NCBI nr database with an E-value of < = 10(-5) and similarity score of 40%. The longest transcript was 16,974 bases with matched to HECT domain containing E3 ubiqutin protein ligase. MEGAN4 annotation pipeline revealed immune and signalling pathways including cell adhesion molecules, cytokine-cytokine receptor interaction, T-cell receptor signalling pathway and chemokine signaling pathway to be highly expressed in spleen, while different metabolism pathways such as amino acid metabolism, carbohydrate metabolism, lipid metabolism and xenobiotic biodegradation were highly expressed in kidney. Few of the candidate genes were selected to analyze their expression levels in various tissues by real-time PCR and also localization of a receptor by in-situ PCR to validate the prediction. We also predicted the domains structures of some of the identified pattern recognition receptors, their phylogenetic relationship with lower and higher vertebrates and the complete downstream signaling mediators of classical dsRNA signaling pathway. The generated transcriptome will be a valuable resource to further genetic and genomic research in elasmobranchs.

Show MeSH
Related in: MedlinePlus