Cloning and expression analysis of two ROR-γ homologues (ROR-γa1 and ROR-γa2) in rainbow trout Oncorhynchus mykiss

Milena M. Monte, Tiehui Wang, Maria M. Costa, Nor Omaima Harun, Chris J. Secombes

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Abstract

This paper describes the cloning and characterisation of two retinoid-related orphan receptor (ROR)-γ homologues (ROR-γa1 and -γa2) in rainbow trout (Oncorhynchus mykiss). The coding region predicted for both homologues consists of 1410 base pairs (bp), which translate into two 469 amino acid (aa) proteins. The trout ROR-γs revealed a high conservation of both DNA- and ligand-binding domains (functional regions of the nuclear receptor family), and shared a high homology to mammalian ROR-γt. A phylogenetic tree containing ROR family members confirmed that both trout homologues clustered within the ROR-γ group. Both results suggested that these molecules are likely to be ROR-γ homologues, more similar to the mammalian splice variant ROR-γt than the full length ROR-γ. Expression analysis of tissues obtained from healthy fish revealed highest constitutive expression of trout ROR-γ in muscle, followed by the brain, heart and skin. This suggests that these genes may play an important role in such tissues. In vitro studies, using trout cell lines, demonstrated that ROR-γ is induced significantly by LPS and down-regulated by the presence of PolyI:C and recombinant interferon (IFN)-γ. Moreover, analysis of this gene in head kidney macrophages and mixed primary leucocyte cultures indicated that differences were apparent between the different cell types/sources used, indicating that its expression may be cell-type dependent. Additional studies to investigate the regulation of this gene in vivo demonstrated that its expression was significantly higher in vaccinated vs unvaccinated fish following bacterial (Yersinia ruckeri) challenge but it was down-regulated after a viral (VHSV) infection. This suggests a potential role of trout ROR-γ, a putative T(H)17 transcription factor, in protection against extracellular bacteria.
Original languageEnglish
Pages (from-to)365-374
Number of pages10
JournalFish & Shellfish Immunology
Volume33
Issue number2
Early online date24 May 2012
DOIs
Publication statusPublished - Aug 2012

Fingerprint

orphan
Cloning
rainbow
molecular cloning
Oncorhynchus mykiss
Genes
Fish
receptors
Tissue
Macrophages
Retinoids
trout
Cytoplasmic and Nuclear Receptors
Cell culture
Interferons
Muscle
Conservation
Brain
Bacteria
Skin

Keywords

  • phylogeny
  • yersinia ruckeri
  • receptors, cytoplasmic and nuclear
  • animals
  • fish diseases
  • yersinia infections
  • amino acid sequence
  • Oncorhynchus mykiss
  • cloning, molecular
  • gene expression profiling
  • sequence alignment
  • cells, cultured
  • rhabdoviridae infections
  • bacterial vaccines
  • molecular sequence data
  • gene expression regulation
  • novirhabdovirus
  • cell line

Cite this

Cloning and expression analysis of two ROR-γ homologues (ROR-γa1 and ROR-γa2) in rainbow trout Oncorhynchus mykiss. / Monte, Milena M.; Wang, Tiehui; Costa, Maria M.; Harun, Nor Omaima; Secombes, Chris J.

In: Fish & Shellfish Immunology, Vol. 33, No. 2, 08.2012, p. 365-374.

Research output: Contribution to journalArticle

Monte, Milena M. ; Wang, Tiehui ; Costa, Maria M. ; Harun, Nor Omaima ; Secombes, Chris J. / Cloning and expression analysis of two ROR-γ homologues (ROR-γa1 and ROR-γa2) in rainbow trout Oncorhynchus mykiss. In: Fish & Shellfish Immunology. 2012 ; Vol. 33, No. 2. pp. 365-374.
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abstract = "This paper describes the cloning and characterisation of two retinoid-related orphan receptor (ROR)-γ homologues (ROR-γa1 and -γa2) in rainbow trout (Oncorhynchus mykiss). The coding region predicted for both homologues consists of 1410 base pairs (bp), which translate into two 469 amino acid (aa) proteins. The trout ROR-γs revealed a high conservation of both DNA- and ligand-binding domains (functional regions of the nuclear receptor family), and shared a high homology to mammalian ROR-γt. A phylogenetic tree containing ROR family members confirmed that both trout homologues clustered within the ROR-γ group. Both results suggested that these molecules are likely to be ROR-γ homologues, more similar to the mammalian splice variant ROR-γt than the full length ROR-γ. Expression analysis of tissues obtained from healthy fish revealed highest constitutive expression of trout ROR-γ in muscle, followed by the brain, heart and skin. This suggests that these genes may play an important role in such tissues. In vitro studies, using trout cell lines, demonstrated that ROR-γ is induced significantly by LPS and down-regulated by the presence of PolyI:C and recombinant interferon (IFN)-γ. Moreover, analysis of this gene in head kidney macrophages and mixed primary leucocyte cultures indicated that differences were apparent between the different cell types/sources used, indicating that its expression may be cell-type dependent. Additional studies to investigate the regulation of this gene in vivo demonstrated that its expression was significantly higher in vaccinated vs unvaccinated fish following bacterial (Yersinia ruckeri) challenge but it was down-regulated after a viral (VHSV) infection. This suggests a potential role of trout ROR-γ, a putative T(H)17 transcription factor, in protection against extracellular bacteria.",
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AU - Wang, Tiehui

AU - Costa, Maria M.

AU - Harun, Nor Omaima

AU - Secombes, Chris J.

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