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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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Transcript annotation: Chiloscyllium griseium transcripts that revealed BlastX matches to NCBI database.A & B- E-value and distribution of the similarity scores; C&D- Length and GC distribution of the transcripts that revealed matches and no matches to NCBI database; E- BlastX top hit species matches of the C. griseum transcripts identified in this study.
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pone-0100018-g001: Transcript annotation: Chiloscyllium griseium transcripts that revealed BlastX matches to NCBI database.A & B- E-value and distribution of the similarity scores; C&D- Length and GC distribution of the transcripts that revealed matches and no matches to NCBI database; E- BlastX top hit species matches of the C. griseum transcripts identified in this study.

Mentions: The assembled transcripts are compared against the NCBI non-redundant (nr) protein database using BLASTX program. Out of total 43,385 transcripts (Table S3), 17,548 (40.5%) transcripts revealed significant BLASTX matches with E-value of< = 10−5 and similarity score > = 40% (Table S4). More than 70% of the BlastX hit transcripts have confidence levels of at least 1E-50 and 68% of the assembled transcripts have a similarity of more than 60% at protein level with the existing proteins at NCBI database. The longest transcript with ID = 3212109 revealed BLASTX hit matches to HECTD4 gene that encodes HECT domain containing E3 ubiqutin protein ligase (NCBI Acc. No. 410047398 & Protein ID - XP_003314026.2 of Pan troglodytes). Based on the BLASTX hits, the organisms name for the top hit were extracted and the top 15 organisms is listed in Figure 1 which included matches to Gallus gallus (1715 transcripts −9.77%), Xenopus tropicalis (1271 transcripts –7.24%), Anolis carolinensis (1098 transcripts −6.26%), Danio rerio (922 transcripts – 5.25%), Callorhinchus milli (915 transcripts – 5.21%), Taeniopygia guttata (865 transcripts– 4.93%) and Homo sapiens (786 transcripts – 4.48%) (Table S5). The identified 915 transcripts matching to Callorhinchus milli (ghost shark) are listed in Table S6.Among the top hits, 11sequences matched to Chiloscyllium griseum, which includes NADH dehydrogenase genes (ND1, ND2, ND3, ND4, ND5), cytochrome (COX1, COX2, COX3, CYTB), and ATP synthase F0 subunit 6 (ATP6). It is interesting to note that, out of the 13 gene sequence information available on C. griseum at NCBI, we could detect 11 sequences in our transcriptome data. We further annotated the transcripts with BLASTX hits against UniProt database. A total of 8,716 transcripts matched to already reported sequences in UniProt. The transcripts with complete UniProt annotation including protein named and ID, organism name, Gene ontology (GO) and eggNOG annotation, pathway, protein localization and other relevant information are provided in Table S7, Table S8 & Table S9.


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)

Transcript annotation: Chiloscyllium griseium transcripts that revealed BlastX matches to NCBI database.A & B- E-value and distribution of the similarity scores; C&D- Length and GC distribution of the transcripts that revealed matches and no matches to NCBI database; E- BlastX top hit species matches of the C. griseum transcripts identified in this study.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0100018-g001: Transcript annotation: Chiloscyllium griseium transcripts that revealed BlastX matches to NCBI database.A & B- E-value and distribution of the similarity scores; C&D- Length and GC distribution of the transcripts that revealed matches and no matches to NCBI database; E- BlastX top hit species matches of the C. griseum transcripts identified in this study.
Mentions: The assembled transcripts are compared against the NCBI non-redundant (nr) protein database using BLASTX program. Out of total 43,385 transcripts (Table S3), 17,548 (40.5%) transcripts revealed significant BLASTX matches with E-value of< = 10−5 and similarity score > = 40% (Table S4). More than 70% of the BlastX hit transcripts have confidence levels of at least 1E-50 and 68% of the assembled transcripts have a similarity of more than 60% at protein level with the existing proteins at NCBI database. The longest transcript with ID = 3212109 revealed BLASTX hit matches to HECTD4 gene that encodes HECT domain containing E3 ubiqutin protein ligase (NCBI Acc. No. 410047398 & Protein ID - XP_003314026.2 of Pan troglodytes). Based on the BLASTX hits, the organisms name for the top hit were extracted and the top 15 organisms is listed in Figure 1 which included matches to Gallus gallus (1715 transcripts −9.77%), Xenopus tropicalis (1271 transcripts –7.24%), Anolis carolinensis (1098 transcripts −6.26%), Danio rerio (922 transcripts – 5.25%), Callorhinchus milli (915 transcripts – 5.21%), Taeniopygia guttata (865 transcripts– 4.93%) and Homo sapiens (786 transcripts – 4.48%) (Table S5). The identified 915 transcripts matching to Callorhinchus milli (ghost shark) are listed in Table S6.Among the top hits, 11sequences matched to Chiloscyllium griseum, which includes NADH dehydrogenase genes (ND1, ND2, ND3, ND4, ND5), cytochrome (COX1, COX2, COX3, CYTB), and ATP synthase F0 subunit 6 (ATP6). It is interesting to note that, out of the 13 gene sequence information available on C. griseum at NCBI, we could detect 11 sequences in our transcriptome data. We further annotated the transcripts with BLASTX hits against UniProt database. A total of 8,716 transcripts matched to already reported sequences in UniProt. The transcripts with complete UniProt annotation including protein named and ID, organism name, Gene ontology (GO) and eggNOG annotation, pathway, protein localization and other relevant information are provided in Table S7, Table S8 & Table S9.

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