Expression profile and in silico characterization of novel RTF2h gene under oxidative stress in Indian catfish, Clarias magur (Hamilton 1822).

During the investigation of genes involved in the hypoxia tolerance, novel transcript, Replication Termination Factor 2 homologue (RTF2h), was found to be differentially expressed in brain of Clarias magur (previous name, C. batrachus) whose function was still undefined. Thus, present study was aimed to examine the transcriptional response of novel RTF2h gene, for its possible involvement in hypoxia tolerance in C. magur. Novel transcripts expressed under hypoxic stress were identified from ESTs obtained from SSH libraries of C. magur. Homology analysis of novel transcript (JK487668) revealed it to have RING superfamily signature and was closely related with RTF2 homologue. To characterize the expression pattern of CmRTF2h gene in different tissues of C. magur, qRT-PCR analysis was performed which showed significant increased expression, in spleen following short-term hypoxia exposure (12 H; 2.33-fold), while after long-term hypoxia exposure, significant up-regulation was found three tissues: in spleen (6.57-fold), liver (2.31-fold) and head kidney (2.99-fold) and was down-regulated in brain (3.52-fold). Further, the consensus sequence, obtained from SSH EST sequence and transcripts from hypoxia induced transcriptome data, through multiple sequence alignments, homology modeling and phylogentic analysis together, confirmed it as a novel CmRTF2h gene. In-silico protein-protein interaction and docking studies suggested its closely related function to that of RTF2. The present study reports the expression pattern of CmRTF2h under hypoxia. The up-regulated expression of CmRTF2h under hypoxic conditions may contribute to defense mechanism against induced DNA damage, which in turn may of adaptive significance for hypoxia tolerance in C. magur.

Hypoxia induced altered expression of heat shock protein genes (Hsc71, Hsp90α and Hsp10) in Indian Catfish, Clarias batrachus (Linnaeus, 1758) under oxidative stress.

Heat shock proteins (Hsps) are typically associated with stress response and tolerance. The Indian catfish, Clarias batrachus, is a freshwater air-breathing hypoxia tolerant teleost and is potentially important catfish species for aquaculture and for its economic value as food. The present study aimed at determining the transcriptional response of three Hsps, CbHsc71, CbHsp90α and CbHsp10 in hypoxia tolerant Indian catfish, C. batrachus, under experimental and natural hypoxia. The expression profile of above three genes were studied under different periods of hypoxia, through qRT-PCR. Primers were designed from ESTs obtained through SSH libraries constructed from hypoxia treated fishes. The Hsp10 ESTs and deduced protein was in silico characterized for its ORF and for its physical and chemical properties, respectively, using GeneScan, blastp, scanprosite, superfamily and other softwares. A phylogenetic tree was constructed based on deduced amino acid sequences of Hsc71, Hsp90α, Hsp90ß of Homo sapiens and other fishes along with CbHsp10 protein in MEGA4. The deduced protein sequences of CbHsp10 was found to have characteristic Hsp10 family signatures, and it is proposed for inclusion of methionine in the consensus sequences of Hsp10 family signature, after the "proline" residue. At transcription level, these genes were found to be differentially regulated under hypoxia stress, in different tissues of C. batrachus. The CbHsc71 and CbHsp90α were up-regulated after short and long-term hypoxia, whereas CbHsp10 was significantly down-regulated after short-term hypoxia. The differential expression of these Hsps may play a role in protection and survival under hypoxia induced oxidative stress in C. batrachus.

Molecular characterization and expression analysis of a novel cystatin-like gene in a hypoxia-tolerant Indian catfish, Clarias batrachus [Linnaeus, 1758].

A novel member of Cystatin superfamily was identified from Indian catfish, Clarias batrachus, in response to oxidation stress induced by environmental hypoxia. Integrated genomic approaches, expression profiling and computational techniques showed that CbCystatin had putative cystatin/monelin like domain and might be a transmembrane and/or intermediate protein in signaling pathways. CbCystatin was found to be clustered into family 2 Cystatins. At transcriptional level, its expression was significantly up-regulated in response to short as well as long periods (more than 20 fold) of hypoxia, suggesting its positive association with oxygen concentrations lower than physiological concentrations.

Draft genome assembly of Tenualosa ilisha, Hilsa shad, provides resource for osmoregulation studies.

This study provides the first high-quality draft genome assembly (762.5 Mb) of Tenualosa ilisha that is highly contiguous and nearly complete. We observed a total of 2,864 contigs, with 96.4% completeness with N50 of 2.65 Mbp and the largest contig length of 17.4 Mbp, along with a complete mitochondrial genome of 16,745 bases. A total number of 33,042 protein coding genes were predicted, among these, 512 genes were classified under 61 Gene Ontology (GO) terms, associated with various homeostasis processes. Highest number of genes belongs to cellular calcium ion homeostasis, followed by tissue homeostasis. A total of 97 genes were identified, with 16 GO terms related to water homeostasis. Claudins, Aquaporins, Connexins/Gap junctions, Adenylate cyclase, Solute carriers and Voltage gated potassium channel genes were observed to be higher in number in T. ilisha, as compared to that in other teleost species. Seven novel gene variants, in addition to claudin gene (CLDZ), were found in T. ilisha. The present study also identified two putative novel genes, NKAIN3 and L4AM1, for the first time in fish, for which further studies are required for pinpointing their functions in fish. In addition, 1.6 million simple sequence repeats were mined from draft genome assembly. The study provides a valuable genomic resource for the anadromous Hilsa. It will form a basis for future studies, pertaining to its adaptation mechanisms to different salinity levels during migration, which in turn would facilitate in its domestication.

Hypoxia influences expression profile of Pleckstrin homology-like domain, family A, member 2 in Indian catfish, Clarias batrachus (Linnaeus, 1758): a new candidate gene for hypoxia tolerance in fish.

Several physiologically important genes were found to be regulated by hypoxia at the transcriptional level. The Pleckstrin homology-like domain, family A, member 2 (PHLDA2) gene was previously identified as an imprinted gene. The present study was aimed to determine the structure of complete cDNA and the deduced protein of PHLDA2 along with analysing the changes in its mRNA expression in Clarias batrachus tissues under hypoxic conditions. The complete cDNA of CbPHLDA2 gene consisted of 1009 nucleotides with an open reading frame of 417 nucleotides. The deduced CbPHLDA2 protein of 139 amino acids shared high homology with PHLD2A of other fishes as well as that of vertebrates. Importantly, a single amino acid (asparagine/lysine) insertion was identified in the PH domain of CbPHLDA2 and other fishes, which was absent in other vertebrates studied. Furthermore, under normoxic conditions, CbPHLDA2 was constitutively expressed with varying levels in analysed tissues. Short- and long-term hypoxia exposure resulted in significant changes in the expression of CbPHLDA2 in liver, spleen, head kidney, brain and muscle in a time-dependent manner. The results suggested that CbPHLDA2 might play an important role for adaptive significance under hypoxia.

Molecular characterization and expression analysis of PPP1R3C in hypoxia-tolerant Indian catfish, Clarias batrachus (Linnaeus, 1758) under hypoxia.

Hypoxia is an important environmental stressor that leads to rapid adaptive changes in metabolic organization. However, the molecular mechanisms of hypoxia tolerance in fish remain largely unknown. The present work was focused on understanding the molecular mechanisms and signaling pathways that may lead to tolerance of Clarias batrachus to hypoxic stress. Protein phosphatase 1 regulatory subunit 3C (PPP1R3C) is a new hypoxia-inducible factor (HIF) targeted gene and is regulated by HIF-1 under hypoxic conditions. Overexpression of PPP1R3C increases glycogen accumulation through activation of several enzymes and processes. In this study, for the first time, full length cDNA of PPP1R3C from C. batrachus was characterized and its expression pattern in the brain, liver, muscle and spleen under short (progressive hypoxia; PH, 1h, 6h and 12h) and long-term (natural) hypoxic conditions was investigated. The complete cDNA of PPP1R3C was of 1499 bp, encoding 285 amino acid residues. The identified protein had a protein phosphatase 1 binding motif and a carbohydrate binding domain, thought to be involved in the regulation of glycogen metabolism. Short-term hypoxia exposure caused significant increase in PPP1R3C transcripts in the liver (6h; 6.96 fold and 12h; 3.91 fold) and muscle (progressive hypoxia; 3.46 fold), while, after long-term hypoxia exposure, significant up-regulation in the liver (7.77 fold) and spleen (6.59 fold) tissues was observed. No significant differences were observed in the brain for any time periods. Thus PPP1R3C may play an important role in the tolerance of C. batrachus to hypoxia.

Permanent genetic resources added to Molecular Ecology Resources Database 1 June 2012-31 July 2012.

This article documents the addition of 96 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Clarias batrachus, Marmota himalayana, Schizothorax richardsonii, Sitophilus zeamais and Syagrus romanzoffiana. These loci were cross-tested on the following species: Clarias dussumeri, Clarias gariepinus, Heteropneustus fossilis, Sitophilus granarius and Sitophilus oryzae.