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Cloning and characterization of ADP-ribosylation factor 1b from the olive flounder Paralichthys olivaceus

Cloning and characterization of ADP-ribosylation factor 1b from the olive flounder Paralichthys... Small GTPases are well known as one of the signal transduction factors of immune systems. The ADP-ribosylation factors (ARFs) can be classified into three groups based on the peptide sequence, protein molecular weight, gene structure, and phylogenetic analysis. ARF1 recruits coat proteins to the Golgi membranes when it is bound to GTP. The class I duplicated ARF gene was cloned and characterized from the olive flounder (Paralichthys olivaceus) for this study. PoARF1b contains the GTP-binding motif and the switch 1 and 2 regions. PoARF1b and PoARF1b mutants were transfected into a Hirame natural embryo cell to determine the distribution of its GDP/GTP-bound state; consequently, it was confirmed that PoARF1b associates with the Golgi body when it is in a GTP-binding form. The results of the qPCR-described PoARF1b were expressed for all of the P. olivaceus tissues. The authors plan to study the gene expression patterns of PoARF1b in terms of immunity challenges. Keywords: ADP-ribosylation factor, Olive flounder, GTPase, Duplication, Immune system, Golgi complex Background investigate the relation between cytoskeleton remodeling The aquatic culture of the olive flounder (Paralichthys and the olive flounder immune system. olivaceus) has been widespread in Korea. The farming of The ADP-ribosylation factor (ARF) proteins are small juvenile olive flounder, however, has caused a lot of GTP-binding proteins, and they are involved in mem- problems due to the occurrence of various diseases brane dynamics and the regulation of actin cytoskeleton (Ototake and Matsusato 1986; Park 2009). The juvenile organization (D’Souza-Schorey and Chavrier 2006; flounder is difficult to manage and is weak against dis- Myers and Casanova 2008). The ARF can be classified eases, and the mortality rates have been economically into three groups based on the peptide sequence, protein damaging (Jee et al. 2001). molecular weight, gene structure, and phylogenetic ana- Small GTPases are well known as one of the signal lysis, as follows: class I including ARF1, ARF2, and transduction factors of immune systems (Narumiya ARF3; class II including ARF4 and ARF5; and class III 1996; Scheele et al. 2007). Some documents indicated including only ARF6 (Myers and Casanova 2008; that small GTPases are related to virus infection in the Tsuchiya et al. 1991). The class I and class II ARFs are shrimp (Wu et al. 2007; Liu et al. 2009; Zhang et al. mainly associated with the Golgi complex, although they 2010). Also, the small GTPases of zebra fish have pro- also function in endosomal compartments (Myers and vided a firm basis of an innate immune system in verte- Casanova 2008). In addition, ARF proteins were identi- brates (Salas-Vidal et al. 2005). The authors therefore fied as activators of phospholipase D (PLD) (Luo et al. studied on ADP-ribosylation factor, which is a member 1998). ARF1 was shown to recruit coat proteins to the of the GTP-binding proteins, from the olive flounder to Golgi membranes when it is bound to GTP (Balch et al. 1992). The hydrolysis and binding of GTP by ARF1 were originally linked to the assembly and disassembly of vesicle coats (Nie and Randazzo 2006). * Correspondence: hyunghl@pknu.ac.kr The radiation of teleosts has been attributed to a Department of Biotechnology, Pukyong National University, Busan 608-737, South Korea genome-DNA event during the evolution of the teleosts Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Son et al. Fisheries and Aquatic Sciences (2017) 20:10 Page 2 of 7 (Venkatesh 2003). Although a number of ARFs, from The phylogenetic tree was constructed using the those of micro-organisms to those of mammals, have Neighbor-Joining Method by MEGA6 (Tamura et al. been studied, a lack of studies on the duplicated ARF 2013). Different DNA and protein sequences in Ensembl genes in the olive flounder persists. The authors there- sequence database were used to carry out phylogenetic fore isolated and characterized one of the class I dupli- tree generation, sequence alignments, and database cated ARF genes. searching (Additional file 1) (Flicek et al. 2011). Methods Tissue distribution of PoARF1b by qPCR analysis cDNA cloning and phylogenetic analysis of Paralichthys The tissue distribution of PoARF1b in different tissues olivaceus ARF1b was measured by RT-qPCR using a LightCycler 480 Total RNA was extracted by using GeneAll® Hybrid-R™ Real-Time PCR System (Roche, Mannheim, Germany) Total RNA (GeneAll Biotechnology Co., Ltd., Korea) fol- with LightCycler 480 SYBR green master I (Roche). The lowing the manufacturer’s instructions from 12 tissues, total RNA was extracted from the brain, gullet, eye, including the brain, eye, gullet, heart, liver, stomach, heart, stomach, liver, kidney, spleen, pyloric ceca, muscle, kidney, spleen, pyloric ceca, intestine, and gill muscle, intestine, and gill from healthy P. olivaceus spec- tissues, of healthy Paralichthys olivaceus. And then, we imens. cDNA was synthesized with random hexamer carried out 5′- and 3′-rapid amplification of cDNA ends primers and oligo(dT)18 using the PrimeScript™ 1st (RACE) by using SMART™ RACE cDNA Amplification strand cDNA Synthesis Kit (TaKaRa), according to the Kit (Clontech laboratories, Inc.) according to the manu- manufacturer’s instructions. The specific primer for in- facturer’s instructions. For obtaining full-length cDNA ternal control was used 18s rRNA (Table 1) (Ahn et al. sequence, new gene-specific sense and antisense primers 2008). The quantitative real-time PCR followed program: were designed (Table 1). The primers were used to PCR pre-incubation at 95 °C for 5 min, 45 cycles at 95 °C for for obtaining full-length cDNA sequence. Nucleotide se- 10 s, 60 °C for 10 s, and 72 °C for 10 s. The qPCR reac- quences and deduced amino acid sequence aligned with tion mixture consists of the following elements: 10 μlof their respective homologues using Genetyx 7.0 software 2× SYBR (Roche), 7.5 μl of SYBR water (Roche), 1 μl (GENETYX Corporation, Tokyo, Japan) and sequence each of sense and antisense primers, and 0.5 μl diluted alignment editor (BioEdit) (Hall 2011). first-strand cDNA (diluted at 1:20). The ΔΔCt method Table 1 Oligonucleotide primers used in PCR amplification of ARF1b of P. olivaceus; F, Forward; R, reverse Primer name 5'-3' sequence Information DgARF1b-F1 ATGGGDRMYWTBKCYWSC Primers for cDNA library screening DgARF1b-F2 GACVACCATCYTGTACAARCTCAAAC DgARF1b-R1 CAYTTBVYDTTYYTBAGCTKG DgARF1b-R2 CRTCYTCWGMCARCATTCGCATC 3′GSP-PoARF1b-F1 GTTGAGACAGTAGAGTACAGGAACATCAG 3′GSP-PoARF1b-F2 GGAGCGAATCGGTGAGGCGAGAGAGGAGC 5′GSP-PoARF1b R1 CAGTTCCGCCGGGTGAGGTCGTGCAGGC 5′GSP-PoARF1b R2 GCTCCTCTCTCGCCTCACCGATTCGCTCC PoARF1b-RT-F GCAGCAAAGTACTTCAAAGCCC Primers for qPCR PoARF1b-RT-R CTCAGCCAGCATTCGCATC Po18S rRNA-RT-For2 ATGGCCGTTCTTAGTTGGTG GenBank accession no. EF126037.1 Po18S rRNA-RT-Rev2 CACACGCTGATCCAGTCAGT PoARF1b-ORF F ATGGGACAGTTCTTTAGCCTGTTTAAAG Primers for the construction of pEGFP CI PoARF1b-ORF R TCATTTTATGTTCTTGAGCTGGTTTGAAAG Xhol-PoARF1b-F CCGCTCGAGCTATGGGACAGTTCTTTA Xpnl-PoARF1b-R CGGGGTACCTCATTTTATGTTCTTGAGCTG PoARF1bT30N-F CTGCATGCTGGAAAGAACACCATCCTGTACAAA Primers for the site-directed mutation PoARF1bT30N-R TTTGTACAGGATGGTGTTCTTTCCAGCATGCAG PoARF1bQ70L-F GTCGGTGGTCTGGACAAGATCAGGCCACTC PoARF1bQ70L-R GAGTGGCCTGATCTTGTCCAGACCACCGAC Son et al. Fisheries and Aquatic Sciences (2017) 20:10 Page 3 of 7 −ΔΔCt was applied to calculate the data, and 2 method 180 amino acid residues and the molecular weight is ap- was applied to calculate the relative quantitative value proximately 20,561 Da (Fig. 1a). PoARF1b contains (Giulietti et al. 2001). GTP-binding motif, switch 1 and 2 regions (Fig. 1a) (Pasqualato et al. 2002). The GTP-binding motif is Statistics shaded with gray box and conserved sequence in other All qPCR data were statistically analyzed using SPSS 21 ARFs. The switch 1 and 2 regions were indicated with program (SPSS, Chicago, IL, USA). One-way ANOVA blue and red letters. The switch regions were signifi- was used to study the PoARF1b expression, followed by cantly assumed to conformational change classical struc- Duncan’s Multiple Range test. A p value with p < 0.05 tural GDP/GTP switch that bind tightly to GTP but was considered to be significant (Sokal and Rohlf 1969). poorly or not at all to the GDP nucleotide (Pasqualato et al. 2002). Cell culture and transfection The aligned amino acid sequence appeared in Hirame natural embryo cell line (HINAE) was grown in PoARF1b had well conserved domains such as GTP- Leibovitz’s L-15 medium (Gibco BRL, Grand Island, NY) binding motif, switch 1 and 2 regions, and shared the containing 10% fetal bovine serum (Gibco) and 1% anti- high homology with ARFs from other species (Fig. 1b). biotics (Gibco) at 20 °C (Kasai and Yoshimizu 2001). It showed 90% homology with ARF1b from Takifugu The transfection was performed using PolyPlus (JetPrime, rubripes. New York, NY, USA) kit to transient transfection of PoARF1b and its mutants according to the manufacturer’s Phylogenetic tree of PoARF1b instructions in six-well test plates. The PoARF1b and mu- To determine the evolutionary relationship of PoARF1b tants were observed by EGFP fluorescence signal under with other ARFs, phylogenetic tree was performed using confocal microscopy after 48 h post-transfection. the Ensembl sequence data using the Neighbor-Joining Method by MEGA (version 6) with bootstrapping 2000 Site mutation of PoARF1b times (Flicek et al. 2011; Tamura et al. 2013). The result PoARF1b(T30N) and PoARF1b(Q70L) were performed of phylogenetic tree was contained fish which was using QuikChange II Site-Directed Mutagenesis Kit grouped with the tetrapod and human. The ARF tree (Agilent Technologies), according to the manufacturer’s consists of three major groups: (i) class I, (ii) class II, instructions (Wang and Malcolm 1999). For PoARF1b and (iii) class III. This result indicated that PoARF1b is mutants, we used specific primers (Table 1). The closely related to ARF1b of class I (Fig. 2). pEGFP-C1 (Clontech) was used to construct the green fluorescent protein-fused PoARF1b and PoARF1b Tissue distribution of PoARF1b by qPCR analysis mutants. Real-time PCR showed tissue distribution of PoARF1b. The Golgi body in HINAE was stained using GOLGI The results of qPCR-described PoARF1b was expressed ID® Green assay kit containing a Golgi apparatus- for all mRNA transcripts in different organs which in- selective dye. clude the brain, gullet, eye, heart, stomach, liver, kidney, spleen, pyloric ceca, muscle, intestine, and gill (Fig. 3). Results and Discussion The expression of the PoARF1b gene was the highest Cloning and sequence analysis of PoARF1b level in the gill and the lowest level in the muscle. To identify the initial sequence of PoARF1b, we ob- tained databases of other ARF1b by using Ensembl se- Site mutation analysis of PoARF1b quence data. These sequences were used to design the To determine the distribution of PoARF1b, PoARF1b, forward and reverse primers (Table 1). The initial se- and PoARF1b mutants, those were constructed into quence was obtained from PCR amplification of olive pEGFP-C1 (Clontech) and transfected into HINAE cell. flounder cDNA including the brain, eye, gullet, heart, The punctuate morphology of PoARF1b-EGFP resem- liver, muscle, stomach, kidney, spleen, pyloric ceca, bled the Golgi complex distribution in HINAE cells intestine, and gill. The partial sequence was used for (Fig. 4b). This result examines PoARF1b that may act in isolation of full-length flounder ARF1b by using 3′ the Golgi body like as human ARF1 complexed with and 5′ GeneRace with flounder ARF1b specific GDP (Amor et al. 1994). Also, PoARF1b mutants examine primers (Table 1). As the result, the full nucleotide the distribution which depends on each GDP or GTP- sequence of PoARF1b is 1677 bp (GenBank accession binding form. The mutants designed PoARF1b(T30N) no. KX668134). and PoARF1b(Q70L) (Fig. 4a), according to other re- The sequence comprised a 108 bp 5′-untranslated re- ports (Chavrier and Goud 1999; Teal et al. 1994). gion (5′-UTR), a 544 bp coding region, and a 1025 bp PoARF1b(T30N) was designed by exchanging the Thr 3′-untranslated region (3′-UTR). Also, the PoARF1b has amino acid at position 30 with Asn amino acid. It Son et al. Fisheries and Aquatic Sciences (2017) 20:10 Page 4 of 7 Fig. 1 a Cloning analysis of PoARF1b. GTP-binding site is shaded with gray box; switch 1 region is indicated with blue letter; switch 2 region is indicated with red letter. b Amino acid sequence analysis of ARFs. The identical conserved amino acid residues are shaded in black. The letters in front of ARF are species names for acronym. Tr Takifugu rubripes, Tn Tetraodon nigroviridis, Ol Oryzias latipes, Xm Xiphophorus maculatus, Ga Gasterosteus aculeatus, On Oreochromis niloticus, Po Paralichthys olivaceus,and Hs Homo sapiens was expected to function in a dominant-negative manner Conclusion and retain the GDP-binding form. PoARF1b(Q70L) was The small GTPases regulate multiple signaling processes designed by replacing the Gln at position 70 with Leu. It including cell growth, survival, and differentiation was expected to function in a dominant-positive manner (Johnson and Chen 2012). The ARF1 function of the and keep the GTP-binding form. The result of Golgi complex may be important and plays a significant PoARF1b(T30N) showed a clearly disassembled punc- role in the secretory pathway (Radhakrishna and tuate morphology (Fig. 4a). When PoARF1b is in a Donaldson 1997). In this paper, Paralichthys olivaceus GDP-binding form, it examines to disassociate from ARF (PoARF) was cloned. The deduced amino acid se- the Golgi complex. On the other hand, the result of quence of PoARF contains the GTP-binding motif, and PoARF1b(Q70L) examined more expanded morphology the switch 1 and 2 regions are present as mammalian than that of normal PoARF1b and PoARF1b(T30N) ARF. The PoARF is highly conserved in the other amino (Fig. 4a). When PoARF1b is in a GTP-binding form, it acid sequences from teleosts and humans. The PoARF is shows to associate from the Golgi complex. indicated from approximately 76 to 85% of the overall Son et al. Fisheries and Aquatic Sciences (2017) 20:10 Page 5 of 7 Fig. 2 Phylogenetic tree of PoARF1b with other ARF family. Phylogenetic tree of ARF sequences was inferred using the Neighbor-Joining Method by MEGA (version 6) with bootstrapping 2000 times. The degree of confidence for each branch point is indicated by bar. The box indicates PoARF1b. The Ensembl accession numbers used in the alignment are shown in Additional file 1 Fig. 3 Tissue distribution of PoARF1b by qPCR analysis. Total RNA was isolated from various tissues of P. olivaceus. PoARF1b normalized against 18S rRNA expression. Mean ± standard deviation (n = 3) are shown. Means denoted by the same letter did not differ significantly (p > 0.05) while different letters (a, b, c, d, e)atthe top of the bars indicate statistically significant differences (p < 0.05) between tissues determined by one-way ANOVA followed by Duncan’s Multiple Range test Son et al. Fisheries and Aquatic Sciences (2017) 20:10 Page 6 of 7 Fig. 4 a Punctuate morphology of PoARF1b and its mutants. The amounts of plasmids (2 μg) were transfected into HINAE cells in 6 wells. EGFP, control; PoARF1b-EGFP, wild-type; PoARF1b(T30N)-EGFP, negative mutant; and PoARF1b(Q70L)-EGFP, positive mutant. b Intracellular distribution of the Golgi complex in HINAE cells using GOLGI ID. Bar means 50 μm identities from the other ARF isozymes (data not Further studies are needed to be carried out to explain shown). PoARF shares approximately 85% with the iden- the highest expression of PoARF1b in gill. tity of Oreochromis niloticus ARF1b (OnARF1b) and ap- proximately 79% with the identity of Gasterosteus Additional file aculeatus ARF1b (GaARF1b). Also, PoARF shares 76% with the identity of Homo sapiens ARF1 (HsARF1). In Additional file 1: Sequences used in sequence and phylogenetic analysis of PoARF1b. (TIF 493 kb) addition, the phylogenetic tree showed that PoARF is more closely related to ARF1b than ARF1a. These re- sults indicate that PoARF is PoARF1b. OnARF1b, which Abbreviations ARF: ADP-ribosylation factor; HINAE: Hirame natural embryo cell line; shares a high percentage with the identity of PoARF1b, RACE: Rapid amplification of cDNA ends; UTR: Untranslated region shares 76% with the identity of HsARF1. As is known, the PoARF1b is expressed in all of the Funding tissues of the olive flounder. The PoARF1b mRNA has a This work was supported by a Research Grant of Pukyong National University (year 2015). high expression level in the gill and a low expression level in the muscle. This finding resembles the ARF1 ex- Availability of data and materials pression from the shrimp (Marsupenaeus japonicus), The dataset(s) supporting the conclusions of this article is(are) included which shows the lowest expression level in the muscle within the article. (Ma et al. 2010). It will be necessary to further study Authors’ contributions why such an outcome. S-HS carried out the whole process, participated in the whole experiments The Golgi-binding distribution of PoARF1b depends and drafted the manuscript. J-HJ carried out the cDNA cloning and on the GTP- or GDP-bound state. The PoARF1b-EGFP phylogenetic analysis. J-KC participated in its statistical analysis. HHL participated in the design of the study and coordination. All authors read showed a punctuate morphology that resembles the and approved the final manuscript. morphology of the Golgi body in HINAE cells (Fig. 4). The GOLGI ID of the Golgi bodies can be detected Competing interests using a microscopy. The authors declare that they have no competing interests. PoARF1b(T30N) showed a clearly disassembled punc- Consent for publication tuate morphology, and PoARF1b(Q70L) showed a more Not applicable expanded morphology; these results resemble those of the mammalian ARF1. The results of this study indicate Ethics approval and consent to participate that PoARF1b functions within the Golgi complex. Not applicable Son et al. Fisheries and Aquatic Sciences (2017) 20:10 Page 7 of 7 Publisher’sNote Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular Springer Nature remains neutral with regard to jurisdictional claims in evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013;30(12):2725–9. published maps and institutional affiliations. Teal SB, Hsu VW, Peters PJ, Klausner RD, Donaldson JG. An activating mutation in ARF1 stabilizes coatomer binding to Golgi membranes. J Biol Chem. Author details 1994;269(5):3135–8. Department of Biotechnology, Pukyong National University, Busan 608-737, Tsuchiya M, Price SR, Tsai SC, Moss J, Vaughan M. Molecular identification of South Korea. Department of Aquatic Life Medicine, Pukyong National ADP-ribosylation factor mRNAs and their expression in mammalian cells. University, Busan 608-737, South Korea. J Biol Chem. 1991;266(5):2772–7. Venkatesh B. Evolution and diversity of fish genomes. Curr Opin Genet Dev. Received: 17 November 2016 Accepted: 27 March 2017 2003;13(6):588–92. Wang W, Malcolm BA. Two-stage PCR protocol allowing introduction of multiple mutations, deletions and insertions using QuikChange Site-Directed Mutagenesis. Biotechniques. 1999;26(4):680–2. Wu W, Zong R, Xu J, Zhang X. 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Cloning and characterization of ADP-ribosylation factor 1b from the olive flounder Paralichthys olivaceus

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Springer Journals
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Copyright © 2017 by The Author(s)
Subject
Life Sciences; Fish & Wildlife Biology & Management; Marine & Freshwater Sciences; Zoology; Animal Ecology
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Abstract

Small GTPases are well known as one of the signal transduction factors of immune systems. The ADP-ribosylation factors (ARFs) can be classified into three groups based on the peptide sequence, protein molecular weight, gene structure, and phylogenetic analysis. ARF1 recruits coat proteins to the Golgi membranes when it is bound to GTP. The class I duplicated ARF gene was cloned and characterized from the olive flounder (Paralichthys olivaceus) for this study. PoARF1b contains the GTP-binding motif and the switch 1 and 2 regions. PoARF1b and PoARF1b mutants were transfected into a Hirame natural embryo cell to determine the distribution of its GDP/GTP-bound state; consequently, it was confirmed that PoARF1b associates with the Golgi body when it is in a GTP-binding form. The results of the qPCR-described PoARF1b were expressed for all of the P. olivaceus tissues. The authors plan to study the gene expression patterns of PoARF1b in terms of immunity challenges. Keywords: ADP-ribosylation factor, Olive flounder, GTPase, Duplication, Immune system, Golgi complex Background investigate the relation between cytoskeleton remodeling The aquatic culture of the olive flounder (Paralichthys and the olive flounder immune system. olivaceus) has been widespread in Korea. The farming of The ADP-ribosylation factor (ARF) proteins are small juvenile olive flounder, however, has caused a lot of GTP-binding proteins, and they are involved in mem- problems due to the occurrence of various diseases brane dynamics and the regulation of actin cytoskeleton (Ototake and Matsusato 1986; Park 2009). The juvenile organization (D’Souza-Schorey and Chavrier 2006; flounder is difficult to manage and is weak against dis- Myers and Casanova 2008). The ARF can be classified eases, and the mortality rates have been economically into three groups based on the peptide sequence, protein damaging (Jee et al. 2001). molecular weight, gene structure, and phylogenetic ana- Small GTPases are well known as one of the signal lysis, as follows: class I including ARF1, ARF2, and transduction factors of immune systems (Narumiya ARF3; class II including ARF4 and ARF5; and class III 1996; Scheele et al. 2007). Some documents indicated including only ARF6 (Myers and Casanova 2008; that small GTPases are related to virus infection in the Tsuchiya et al. 1991). The class I and class II ARFs are shrimp (Wu et al. 2007; Liu et al. 2009; Zhang et al. mainly associated with the Golgi complex, although they 2010). Also, the small GTPases of zebra fish have pro- also function in endosomal compartments (Myers and vided a firm basis of an innate immune system in verte- Casanova 2008). In addition, ARF proteins were identi- brates (Salas-Vidal et al. 2005). The authors therefore fied as activators of phospholipase D (PLD) (Luo et al. studied on ADP-ribosylation factor, which is a member 1998). ARF1 was shown to recruit coat proteins to the of the GTP-binding proteins, from the olive flounder to Golgi membranes when it is bound to GTP (Balch et al. 1992). The hydrolysis and binding of GTP by ARF1 were originally linked to the assembly and disassembly of vesicle coats (Nie and Randazzo 2006). * Correspondence: hyunghl@pknu.ac.kr The radiation of teleosts has been attributed to a Department of Biotechnology, Pukyong National University, Busan 608-737, South Korea genome-DNA event during the evolution of the teleosts Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Son et al. Fisheries and Aquatic Sciences (2017) 20:10 Page 2 of 7 (Venkatesh 2003). Although a number of ARFs, from The phylogenetic tree was constructed using the those of micro-organisms to those of mammals, have Neighbor-Joining Method by MEGA6 (Tamura et al. been studied, a lack of studies on the duplicated ARF 2013). Different DNA and protein sequences in Ensembl genes in the olive flounder persists. The authors there- sequence database were used to carry out phylogenetic fore isolated and characterized one of the class I dupli- tree generation, sequence alignments, and database cated ARF genes. searching (Additional file 1) (Flicek et al. 2011). Methods Tissue distribution of PoARF1b by qPCR analysis cDNA cloning and phylogenetic analysis of Paralichthys The tissue distribution of PoARF1b in different tissues olivaceus ARF1b was measured by RT-qPCR using a LightCycler 480 Total RNA was extracted by using GeneAll® Hybrid-R™ Real-Time PCR System (Roche, Mannheim, Germany) Total RNA (GeneAll Biotechnology Co., Ltd., Korea) fol- with LightCycler 480 SYBR green master I (Roche). The lowing the manufacturer’s instructions from 12 tissues, total RNA was extracted from the brain, gullet, eye, including the brain, eye, gullet, heart, liver, stomach, heart, stomach, liver, kidney, spleen, pyloric ceca, muscle, kidney, spleen, pyloric ceca, intestine, and gill muscle, intestine, and gill from healthy P. olivaceus spec- tissues, of healthy Paralichthys olivaceus. And then, we imens. cDNA was synthesized with random hexamer carried out 5′- and 3′-rapid amplification of cDNA ends primers and oligo(dT)18 using the PrimeScript™ 1st (RACE) by using SMART™ RACE cDNA Amplification strand cDNA Synthesis Kit (TaKaRa), according to the Kit (Clontech laboratories, Inc.) according to the manu- manufacturer’s instructions. The specific primer for in- facturer’s instructions. For obtaining full-length cDNA ternal control was used 18s rRNA (Table 1) (Ahn et al. sequence, new gene-specific sense and antisense primers 2008). The quantitative real-time PCR followed program: were designed (Table 1). The primers were used to PCR pre-incubation at 95 °C for 5 min, 45 cycles at 95 °C for for obtaining full-length cDNA sequence. Nucleotide se- 10 s, 60 °C for 10 s, and 72 °C for 10 s. The qPCR reac- quences and deduced amino acid sequence aligned with tion mixture consists of the following elements: 10 μlof their respective homologues using Genetyx 7.0 software 2× SYBR (Roche), 7.5 μl of SYBR water (Roche), 1 μl (GENETYX Corporation, Tokyo, Japan) and sequence each of sense and antisense primers, and 0.5 μl diluted alignment editor (BioEdit) (Hall 2011). first-strand cDNA (diluted at 1:20). The ΔΔCt method Table 1 Oligonucleotide primers used in PCR amplification of ARF1b of P. olivaceus; F, Forward; R, reverse Primer name 5'-3' sequence Information DgARF1b-F1 ATGGGDRMYWTBKCYWSC Primers for cDNA library screening DgARF1b-F2 GACVACCATCYTGTACAARCTCAAAC DgARF1b-R1 CAYTTBVYDTTYYTBAGCTKG DgARF1b-R2 CRTCYTCWGMCARCATTCGCATC 3′GSP-PoARF1b-F1 GTTGAGACAGTAGAGTACAGGAACATCAG 3′GSP-PoARF1b-F2 GGAGCGAATCGGTGAGGCGAGAGAGGAGC 5′GSP-PoARF1b R1 CAGTTCCGCCGGGTGAGGTCGTGCAGGC 5′GSP-PoARF1b R2 GCTCCTCTCTCGCCTCACCGATTCGCTCC PoARF1b-RT-F GCAGCAAAGTACTTCAAAGCCC Primers for qPCR PoARF1b-RT-R CTCAGCCAGCATTCGCATC Po18S rRNA-RT-For2 ATGGCCGTTCTTAGTTGGTG GenBank accession no. EF126037.1 Po18S rRNA-RT-Rev2 CACACGCTGATCCAGTCAGT PoARF1b-ORF F ATGGGACAGTTCTTTAGCCTGTTTAAAG Primers for the construction of pEGFP CI PoARF1b-ORF R TCATTTTATGTTCTTGAGCTGGTTTGAAAG Xhol-PoARF1b-F CCGCTCGAGCTATGGGACAGTTCTTTA Xpnl-PoARF1b-R CGGGGTACCTCATTTTATGTTCTTGAGCTG PoARF1bT30N-F CTGCATGCTGGAAAGAACACCATCCTGTACAAA Primers for the site-directed mutation PoARF1bT30N-R TTTGTACAGGATGGTGTTCTTTCCAGCATGCAG PoARF1bQ70L-F GTCGGTGGTCTGGACAAGATCAGGCCACTC PoARF1bQ70L-R GAGTGGCCTGATCTTGTCCAGACCACCGAC Son et al. Fisheries and Aquatic Sciences (2017) 20:10 Page 3 of 7 −ΔΔCt was applied to calculate the data, and 2 method 180 amino acid residues and the molecular weight is ap- was applied to calculate the relative quantitative value proximately 20,561 Da (Fig. 1a). PoARF1b contains (Giulietti et al. 2001). GTP-binding motif, switch 1 and 2 regions (Fig. 1a) (Pasqualato et al. 2002). The GTP-binding motif is Statistics shaded with gray box and conserved sequence in other All qPCR data were statistically analyzed using SPSS 21 ARFs. The switch 1 and 2 regions were indicated with program (SPSS, Chicago, IL, USA). One-way ANOVA blue and red letters. The switch regions were signifi- was used to study the PoARF1b expression, followed by cantly assumed to conformational change classical struc- Duncan’s Multiple Range test. A p value with p < 0.05 tural GDP/GTP switch that bind tightly to GTP but was considered to be significant (Sokal and Rohlf 1969). poorly or not at all to the GDP nucleotide (Pasqualato et al. 2002). Cell culture and transfection The aligned amino acid sequence appeared in Hirame natural embryo cell line (HINAE) was grown in PoARF1b had well conserved domains such as GTP- Leibovitz’s L-15 medium (Gibco BRL, Grand Island, NY) binding motif, switch 1 and 2 regions, and shared the containing 10% fetal bovine serum (Gibco) and 1% anti- high homology with ARFs from other species (Fig. 1b). biotics (Gibco) at 20 °C (Kasai and Yoshimizu 2001). It showed 90% homology with ARF1b from Takifugu The transfection was performed using PolyPlus (JetPrime, rubripes. New York, NY, USA) kit to transient transfection of PoARF1b and its mutants according to the manufacturer’s Phylogenetic tree of PoARF1b instructions in six-well test plates. The PoARF1b and mu- To determine the evolutionary relationship of PoARF1b tants were observed by EGFP fluorescence signal under with other ARFs, phylogenetic tree was performed using confocal microscopy after 48 h post-transfection. the Ensembl sequence data using the Neighbor-Joining Method by MEGA (version 6) with bootstrapping 2000 Site mutation of PoARF1b times (Flicek et al. 2011; Tamura et al. 2013). The result PoARF1b(T30N) and PoARF1b(Q70L) were performed of phylogenetic tree was contained fish which was using QuikChange II Site-Directed Mutagenesis Kit grouped with the tetrapod and human. The ARF tree (Agilent Technologies), according to the manufacturer’s consists of three major groups: (i) class I, (ii) class II, instructions (Wang and Malcolm 1999). For PoARF1b and (iii) class III. This result indicated that PoARF1b is mutants, we used specific primers (Table 1). The closely related to ARF1b of class I (Fig. 2). pEGFP-C1 (Clontech) was used to construct the green fluorescent protein-fused PoARF1b and PoARF1b Tissue distribution of PoARF1b by qPCR analysis mutants. Real-time PCR showed tissue distribution of PoARF1b. The Golgi body in HINAE was stained using GOLGI The results of qPCR-described PoARF1b was expressed ID® Green assay kit containing a Golgi apparatus- for all mRNA transcripts in different organs which in- selective dye. clude the brain, gullet, eye, heart, stomach, liver, kidney, spleen, pyloric ceca, muscle, intestine, and gill (Fig. 3). Results and Discussion The expression of the PoARF1b gene was the highest Cloning and sequence analysis of PoARF1b level in the gill and the lowest level in the muscle. To identify the initial sequence of PoARF1b, we ob- tained databases of other ARF1b by using Ensembl se- Site mutation analysis of PoARF1b quence data. These sequences were used to design the To determine the distribution of PoARF1b, PoARF1b, forward and reverse primers (Table 1). The initial se- and PoARF1b mutants, those were constructed into quence was obtained from PCR amplification of olive pEGFP-C1 (Clontech) and transfected into HINAE cell. flounder cDNA including the brain, eye, gullet, heart, The punctuate morphology of PoARF1b-EGFP resem- liver, muscle, stomach, kidney, spleen, pyloric ceca, bled the Golgi complex distribution in HINAE cells intestine, and gill. The partial sequence was used for (Fig. 4b). This result examines PoARF1b that may act in isolation of full-length flounder ARF1b by using 3′ the Golgi body like as human ARF1 complexed with and 5′ GeneRace with flounder ARF1b specific GDP (Amor et al. 1994). Also, PoARF1b mutants examine primers (Table 1). As the result, the full nucleotide the distribution which depends on each GDP or GTP- sequence of PoARF1b is 1677 bp (GenBank accession binding form. The mutants designed PoARF1b(T30N) no. KX668134). and PoARF1b(Q70L) (Fig. 4a), according to other re- The sequence comprised a 108 bp 5′-untranslated re- ports (Chavrier and Goud 1999; Teal et al. 1994). gion (5′-UTR), a 544 bp coding region, and a 1025 bp PoARF1b(T30N) was designed by exchanging the Thr 3′-untranslated region (3′-UTR). Also, the PoARF1b has amino acid at position 30 with Asn amino acid. It Son et al. Fisheries and Aquatic Sciences (2017) 20:10 Page 4 of 7 Fig. 1 a Cloning analysis of PoARF1b. GTP-binding site is shaded with gray box; switch 1 region is indicated with blue letter; switch 2 region is indicated with red letter. b Amino acid sequence analysis of ARFs. The identical conserved amino acid residues are shaded in black. The letters in front of ARF are species names for acronym. Tr Takifugu rubripes, Tn Tetraodon nigroviridis, Ol Oryzias latipes, Xm Xiphophorus maculatus, Ga Gasterosteus aculeatus, On Oreochromis niloticus, Po Paralichthys olivaceus,and Hs Homo sapiens was expected to function in a dominant-negative manner Conclusion and retain the GDP-binding form. PoARF1b(Q70L) was The small GTPases regulate multiple signaling processes designed by replacing the Gln at position 70 with Leu. It including cell growth, survival, and differentiation was expected to function in a dominant-positive manner (Johnson and Chen 2012). The ARF1 function of the and keep the GTP-binding form. The result of Golgi complex may be important and plays a significant PoARF1b(T30N) showed a clearly disassembled punc- role in the secretory pathway (Radhakrishna and tuate morphology (Fig. 4a). When PoARF1b is in a Donaldson 1997). In this paper, Paralichthys olivaceus GDP-binding form, it examines to disassociate from ARF (PoARF) was cloned. The deduced amino acid se- the Golgi complex. On the other hand, the result of quence of PoARF contains the GTP-binding motif, and PoARF1b(Q70L) examined more expanded morphology the switch 1 and 2 regions are present as mammalian than that of normal PoARF1b and PoARF1b(T30N) ARF. The PoARF is highly conserved in the other amino (Fig. 4a). When PoARF1b is in a GTP-binding form, it acid sequences from teleosts and humans. The PoARF is shows to associate from the Golgi complex. indicated from approximately 76 to 85% of the overall Son et al. Fisheries and Aquatic Sciences (2017) 20:10 Page 5 of 7 Fig. 2 Phylogenetic tree of PoARF1b with other ARF family. Phylogenetic tree of ARF sequences was inferred using the Neighbor-Joining Method by MEGA (version 6) with bootstrapping 2000 times. The degree of confidence for each branch point is indicated by bar. The box indicates PoARF1b. The Ensembl accession numbers used in the alignment are shown in Additional file 1 Fig. 3 Tissue distribution of PoARF1b by qPCR analysis. Total RNA was isolated from various tissues of P. olivaceus. PoARF1b normalized against 18S rRNA expression. Mean ± standard deviation (n = 3) are shown. Means denoted by the same letter did not differ significantly (p > 0.05) while different letters (a, b, c, d, e)atthe top of the bars indicate statistically significant differences (p < 0.05) between tissues determined by one-way ANOVA followed by Duncan’s Multiple Range test Son et al. Fisheries and Aquatic Sciences (2017) 20:10 Page 6 of 7 Fig. 4 a Punctuate morphology of PoARF1b and its mutants. The amounts of plasmids (2 μg) were transfected into HINAE cells in 6 wells. EGFP, control; PoARF1b-EGFP, wild-type; PoARF1b(T30N)-EGFP, negative mutant; and PoARF1b(Q70L)-EGFP, positive mutant. b Intracellular distribution of the Golgi complex in HINAE cells using GOLGI ID. Bar means 50 μm identities from the other ARF isozymes (data not Further studies are needed to be carried out to explain shown). PoARF shares approximately 85% with the iden- the highest expression of PoARF1b in gill. tity of Oreochromis niloticus ARF1b (OnARF1b) and ap- proximately 79% with the identity of Gasterosteus Additional file aculeatus ARF1b (GaARF1b). Also, PoARF shares 76% with the identity of Homo sapiens ARF1 (HsARF1). In Additional file 1: Sequences used in sequence and phylogenetic analysis of PoARF1b. (TIF 493 kb) addition, the phylogenetic tree showed that PoARF is more closely related to ARF1b than ARF1a. These re- sults indicate that PoARF is PoARF1b. OnARF1b, which Abbreviations ARF: ADP-ribosylation factor; HINAE: Hirame natural embryo cell line; shares a high percentage with the identity of PoARF1b, RACE: Rapid amplification of cDNA ends; UTR: Untranslated region shares 76% with the identity of HsARF1. As is known, the PoARF1b is expressed in all of the Funding tissues of the olive flounder. The PoARF1b mRNA has a This work was supported by a Research Grant of Pukyong National University (year 2015). high expression level in the gill and a low expression level in the muscle. This finding resembles the ARF1 ex- Availability of data and materials pression from the shrimp (Marsupenaeus japonicus), The dataset(s) supporting the conclusions of this article is(are) included which shows the lowest expression level in the muscle within the article. (Ma et al. 2010). It will be necessary to further study Authors’ contributions why such an outcome. S-HS carried out the whole process, participated in the whole experiments The Golgi-binding distribution of PoARF1b depends and drafted the manuscript. J-HJ carried out the cDNA cloning and on the GTP- or GDP-bound state. The PoARF1b-EGFP phylogenetic analysis. J-KC participated in its statistical analysis. HHL participated in the design of the study and coordination. All authors read showed a punctuate morphology that resembles the and approved the final manuscript. morphology of the Golgi body in HINAE cells (Fig. 4). The GOLGI ID of the Golgi bodies can be detected Competing interests using a microscopy. The authors declare that they have no competing interests. PoARF1b(T30N) showed a clearly disassembled punc- Consent for publication tuate morphology, and PoARF1b(Q70L) showed a more Not applicable expanded morphology; these results resemble those of the mammalian ARF1. The results of this study indicate Ethics approval and consent to participate that PoARF1b functions within the Golgi complex. Not applicable Son et al. Fisheries and Aquatic Sciences (2017) 20:10 Page 7 of 7 Publisher’sNote Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular Springer Nature remains neutral with regard to jurisdictional claims in evolutionary genetics analysis version 6.0. 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