The transcriptome of metamorphosing flatfish

Background Flatfish metamorphosis denotes the extraordinary transformation of a symmetric pelagic larva into an asymmetric benthic juvenile. Metamorphosis in vertebrates is driven by thyroid hormones (THs), but how they orchestrate the cellular, morphological and functional modifications associated with maturation to juvenile/adult states in flatfish is an enigma. Since THs act via thyroid receptors that are ligand activated transcription factors, we hypothesized that the maturation of tissues during metamorphosis should be preceded by significant modifications in the transcriptome. Targeting the unique metamorphosis of flatfish and taking advantage of the large size of Atlantic halibut (Hippoglossus hippoglossus) larvae, we determined the molecular basis of TH action using RNA sequencing. Results De novo assembly of sequences for larval head, skin and gastrointestinal tract (GI-tract) yielded 90,676, 65,530 and 38,426 contigs, respectively. More than 57 % of the assembled sequences were successfully annotated using a multi-step Blast approach. A unique set of biological processes and candidate genes were identified specifically associated with changes in morphology and function of the head, skin and GI-tract. Transcriptome dynamics during metamorphosis were mapped with SOLiD sequencing of whole larvae and revealed greater than 8,000 differentially expressed (DE) genes significantly (p < 0.05) up- or down-regulated in comparison with the juvenile stage. Candidate transcripts quantified by SOLiD and qPCR analysis were significantly (r = 0.843; p < 0.05) correlated. The majority (98 %) of DE genes during metamorphosis were not TH-responsive. TH-responsive transcripts clustered into 6 groups based on their expression pattern during metamorphosis and the majority of the 145 DE TH-responsive genes were down-regulated. Conclusions A transcriptome resource has been generated for metamorphosing Atlantic halibut and over 8,000 DE transcripts per stage were identified. Unique sets of biological processes and candidate genes were associated with changes in the head, skin and GI-tract during metamorphosis. A small proportion of DE transcripts were TH-responsive, suggesting that they trigger gene networks, signalling cascades and transcription factors, leading to the overt changes in tissue occurring during metamorphosis.

Polymorphisms at the five exons of gowth hormone gene in the Algarvia Goat. Possible association with milk traits

The present preliminary study attempts to establish associations between milk production traits and genetic polymorphisms at the GH gene in the Algarvia goat. The DNA of 108 goats of the indigenous Portuguese Algarvia breed was evaluated.

Production and characterisation of gilthead sea bream (Sparus auratus) recombinant parathyroid hormone related protein.

The production and puriWcation of gilthead sea bream recombinant parathyroid hormone related protein [sbPTHrP(1–125)] using an Escherichia coli system and one step puriWcation process with continuous elution gel electrophoresis is reported. The cDNA encoding sbPTHrP(1–125) was cloned into a prokaryotic expression vector pET-11a. The recombinant plasmid was used to transfect E. coli BL21(DE3) pLysS and sbPTHrP(1–125) synthesis was induced by addition of 1mM isopropyl- -D-thiogalactopyranoside. The rapid one step isolation method gave pure sbPTHrP(1–125) as judged by SDS–PAGE and yielded up to 40mg/L of culture medium (3.3mg protein/g of bacteria). The bioactivity of recombinant sbPTHrP(1–125) assessed using an in vitro scale bioassay was found to be equipotent to PTHrP(1–34) in stimulating cAMP accumulation. Assessment of the immunological reactivity of the isolated protein by Western blot revealed it cross-reacts with antisera speciWc for the N-terminal and C-terminal region of PTHrP. In a radioimmunoassay speciWc for piscine N-terminal (1–34 aa) PTHrP, the recombinant sbPTHrP(1–125) was equipotent with PTHrP(1–34) in displacing labelled 125I-PTHrP(1–36) PTHrP from the antisera. The availability of recombinant sbPTHrP will allow the development of region speciWc assays and studies aimed at deWning post-secretory processing of this protein and its biological activity in Wsh.

Five gonadotrophin-releasing hormone receptors in a teleost fish: isolation, tissue distribution and phylogenetic relationships

Gonadotrophin-releasing hormone (GnRH) is the main neurohormone controlling gonadotrophin release in all vertebrates, and in teleost fish also of growth hormone and possibly of other adenohypophyseal hormones. Over 20 GnRHs have been identified in vertebrates and protochoordates and shown to bind cognate G-protein couple receptors (GnRHR). We have searched the puffer fish, Fugu rubripes, genome sequencing database, identified five GnRHR genes and proceeded to isolate the corresponding complementary DNAs in European sea bass, Dicentrachus labrax. Phylogenetic analysis clusters the European sea bass, puffer fish and all other vertebrate receptors into two main lineages corresponding to the mammalian type I and II receptors. The fish receptors could be subdivided in two GnRHR1 (A and B) and three GnRHR2 (A, B and C) subtypes. Amino acid sequence identity within receptor subtypes varies between 70 and 90% but only 50–55% among the two main lineages in fish. All European sea bass receptor mRNAs are expressed in the anterior and mid brain, and all but one are expressed in the pituitary gland. There is differential expression of the receptors in peripheral tissues related to reproduction (gonads), chemical senses (eye and olfactory epithelium) and osmoregulation (kidney and gill). This is the first report showing five GnRH receptors in a vertebrate species and the gene expression patterns support the concept that GnRH and GnRHRs play highly diverse functional roles in the regulation of cellular functions, besides the ‘‘classical’’ role of pituitary function regulation.

Genomic structure and expression of parathyroid hormone-related protein gene (PTHrP) in a teleost, Fugu rubripes

In this study we describe the isolation and characterisation of the parathyroid hormone-related protein (PTHrP) gene from the teleost Fugu rubripes. The gene has a relatively simple structure, compared with tetrapod PTHrP genes, composed of three exons and two introns, encompassing 2.25 kb of genomic DNA. The gene encodes a protein of 163 amino acids, with a putative signal peptide of 37 amino acids and a mature peptide of 126 amino acids. The overall homology with known tetrapod PTHrP proteins is low (36%), with a novel sequence inserted between positions 38 and 65, the absence of the conserved pentapeptide (TRSAW) and shortened C-terminal domain. The N-terminus shows greater conservation (62%), suggesting that it may have a hypercalcaemic function similar to that of tetrapod PTHrP. In situ localisation and RT–PCR have demonstrated the presence of PTHrP in a wide range of tissues with varying levels of expression. Sequence scanning of overlapping cosmids has identified three additional genes, TMPO, LDHB and KCNA1, which map to human chromosome 12, with the latter two mapping to 12p12-11.2. PTHrP in human also maps to this chromosome 12 sub-region, thus demonstrating conservation of synteny between human and Fugu.