The cloning and inducible expression of the rainbow trout ERp57 gene

Sever, Lital; Bols, Niels C.; Dixon, Brian

doi:10.1016/j.fsi.2012.11.001

Cited by 16 publications

(25 citation statements)

References 40 publications

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“…Adult fish ($800 g) were anesthetized in 1 ml/L of 2-phenoxyethanol (Sigma Aldrich St. Louis, MO). Blood was drawn from the caudal sinus as previously described (Sever et al, 2013) and tissues samples were collected in RNA later (2.5 mM Na citrate, 5.3 M (NH 4 ) 2 SO 4 , 0.01 M EDTA, pH 5.2).…”

Section: Fishmentioning

confidence: 99%

“…The reaction parameters were 95°C for 5 min followed by 30 cycles of (95°C 40 s, 50°C 30 s, 72°C 2 min) with a final 15 min 72°C using a BioRad DNA Engine thermocycler (BioRad, Mississauga Ontario). An estimated 1200 bp PCR product was purified using a Qiagen gel extraction kit (Mississauga, ON), then ligated into pGEM-T Easy vector, subcloned into XL1-Blue MRF 0 E. coli competent cells and sequenced at The Centre for Applied Genomics (Toronto, ON, Canada as described in Sever et al (2013).…”

Section: Cloning Of Rainbow Trout Calnexin Cdnamentioning

confidence: 99%

“…The full 5 0 end of the calnexin transcript was obtained using RACE adapted from Frohman et al (1988) and described in detail in Sever et al (2013). Briefly, cDNA synthesis used four lg of total RNA derived from peripheral blood leukocytes and gene specific primer1 (GSP1) 5 0 CATGTCCTCCACCTCCCA 3 0 .…”

Section: Generation Of Full Length Cdnamentioning

confidence: 99%

“…However, no studies have been carried out to assess its role during MHC class I folding, MHC class I receptor assembly in the ER or its possible association with viral glycoproteins mainly due to the lack of available antibodies. In rainbow trout, besides calnexin, most of the genes encoding molecular chaperones involved in this pathway have been fully characterised such as tapasin (Landis et al, 2006), calreticulin (Kales et al, 2004) and recently ERp57 (Sever et al, 2013). In this work two unique cDNA clones for calnexin from trout peripheral blood leukocytes are reported and their regulation under ER stress induced conditions is described.…”

Section: Introductionmentioning

confidence: 99%

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Rainbow trout (Oncorhynchus mykiss) contain two calnexin genes which encode distinct proteins

Sever

Bols

et al. 2014

Developmental & Comparative Immunology

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Calnexin (IP90/P88) is an integral membrane protein of the endoplasmic reticulum that binds newly synthesized N-linked glycoproteins during their folding in the ER including MHC class I molecule. This manuscript reports the identification of two unique cDNA clones of calnexin in rainbow trout. Both encode putative mature proteins of 579 and 592 aa respectively in addition to a 24 aa signal peptide. Sequence analysis revealed that only one of the two cDNA clones encodes a putative ER retention signal, K/QEDDL, followed by a serine phosphorylation site conserved with mammalian homologs. Amino acid sequence alignment illustrated conservation of the calnexin luminal domain, which consists of a globular and a P domain, in both copies. Southern blotting revealed that there are at least two copies of the calnexin gene in the trout genome and northern blotting showed a wide tissue distribution of an estimated 3 kbp calnexin transcript with an additional minor transcript of 2.3 kbp expressed only in head kidney, spleen PBLs and strongly in RTS11. Importantly, the smaller transcript was predominantly upregulated in RTS11 after a 24h treatment with the calcium ionophore A23187. In western blots, calnexin was detected primarily as a 120 kDa protein and upon A23187 treatment; a 100 kDa band was most prominently expressed. These results suggest that in salmonids there are two differentiated versions of the calnexin gene which encode proteins that may have diverged to perform unique biological functions.

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Section: Fishmentioning

confidence: 99%

Section: Cloning Of Rainbow Trout Calnexin Cdnamentioning

confidence: 99%

Section: Generation Of Full Length Cdnamentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Rainbow trout (Oncorhynchus mykiss) contain two calnexin genes which encode distinct proteins

Sever

Bols

et al. 2014

Developmental & Comparative Immunology

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“…For rainbow trout cell lines examined to date, expression is seen of antiviral proteins, such as Mx (DeWitte‐Orr et al ., ), and components of the endogenous pathway of antigen presentation, such as the major histocompatibility complex I alpha chain (MH Iα) (Kawano et al . ) and ERp57 (Sever, Bols & Dixon ). In response to the viral mimic, poly inosinic: poly cytidylic (poly I:C), some of these are induced whereas others are constitutively expressed (DeWitte‐Orr et al .…”

Section: Introductionmentioning

confidence: 99%

Development and use of an Arctic charr cell line to study antiviral responses at extremely low temperatures

Semple

et al. 2017

Journal of Fish Diseases

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Arctic charr (Salvelinus alpinus) are the northernmost distributed freshwater fish and can grow at water temperatures as low as 0.2 °C. Other teleost species have impaired immune function at temperatures that Arctic charr thrive in, and thus, charr may maintain immune function at these temperatures. In this study, a fibroblastic cell line, named ACBA, derived from the bulbus arteriosus (BA) of Arctic charr was developed for use in immune studies at various temperatures. ACBA has undergone more than forty passages at 18 °C over 3 years, while showing no signs of senescence-associated β-galactosidase activity and producing nitric oxide. Remarkably, ACBA cells survived and maintained some mitotic activity even at 1 °C for over 3 months. At these low temperatures, ACBA also continued to produce MH class I proteins. After challenge with poly I:C, only antiviral Mx proteins were induced while MH proteins remained constant. When exposed to live viruses, ACBA was shown to permit viral infection and replication of IPNV, VHSV IVa and CSV at 14 °C. Yet at the preferred temperature of 4 °C, only VHSV IVa was shown to replicate within ACBA. This study provides evidence that Arctic charr cells can maintain immune function while also resisting infection with intracellular pathogens at low temperatures.

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Immune stimulation of rainbow trout reveals divergent regulation of MH class II-associated invariant chain isoforms

et al. 2019

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The cloning and inducible expression of the rainbow trout ERp57 gene

Cited by 16 publications

References 40 publications

Rainbow trout (Oncorhynchus mykiss) contain two calnexin genes which encode distinct proteins

Rainbow trout (Oncorhynchus mykiss) contain two calnexin genes which encode distinct proteins

Development and use of an Arctic charr cell line to study antiviral responses at extremely low temperatures

Immune stimulation of rainbow trout reveals divergent regulation of MH class II-associated invariant chain isoforms

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