Recombination-Activating Gene 1 and 2 (RAG1 and RAG2) in Flounder (Paralichthys Olivaceus)
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Recombination-activating gene 1 and 2 (RAG1 and RAG2) in flounder (Paralichthys olivaceus) 1,2 1, 1 1 1 XIANLEI WANG ; XUNGANG TAN *; PEI-JUN ZHANG ; YUQING ZHANG and PENG XU 1Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, China 2National Oceanographic Center, 88 Xuzhou Road, Qingdao, Shandong 266071, China *Corresponding author (Email, [email protected]) During the development of B and T lymphocytes, Ig and TCR variable region genes are assembled from germline V, D, and J gene segments by a site-specific recombination reaction known as V(D)J recombination. The process of somatic V(D)J recombination, mediated by the recombination-activating gene (RAG) products, is the most significant characteristic of adaptive immunity in jawed vertebrates. Flounder (Paralichthys olivaceus) RAG1 and RAG2 were isolated by Genome Walker and RT-PCR, and their expression patterns were analysed by RT-PCR and in situ hybridization on sections. RAG1 spans over 7.0 kb, containing 4 exons and 3 introns, and the full-length ORF is 3207 bp, encoding a peptide of 1068 amino acids. The first exon lies in the 5′-UTR, which is an alternative exon. RAG2 full-length ORF is 1062 bp, encodes a peptide of 533 amino acids, and lacks introns in the coding region. In 6-month- old flounders, the expression of RAG1 and RAG2 was essentially restricted to the pronephros (head kidney) and mesonephros (truck kidney). Additionally, both of them were mainly expressed in the thymus. These results revealed that the thymus and kidney most likely serve as the primary lymphoid tissues in the flounder. [Wang X, Tan X, Zhang P-J, Zhang Y and Xu P 2014 Recombination-activating gene 1 and 2 (RAG1 and RAG2) in flounder (Paralichthys olivaceus). J. Biosci. 39 849–858] DOI 10.1007/s12038-014-9469-1 1. Introduction two tightly linked genes in a single murine genomic DNA fragment (Schatz et al. 1989). Both of RAG1 and RAG2 are The mammalian immune system can be divided into the innate essential for V(D)J recombination (Mombaerts et al. 1992; immune system and the adaptive immune system. T and B Shinkai et al. 1992), and somatic V(D)J recombination is lymphocytes are the main features of the adaptive immune mediated by the RAG gene products, a process that occurs system and originate from common pluripotent stem cells specifically in immature lymphocytes. During this process, a (Ikuta et al. 1992). The primary immunoglobulin (Ig) and complex of RAG1 and RAG2 proteins specifically recog- antigen receptor (TCR) repertoires for B and T cells are as- nizes and binds to recombination signal sequences (RSS), sembled from the component V (variable), D (diversity), and J which flank the V, D, and J gene segments, to initiate (joining) gene segments by site-specific recombination, termed recombination by introducing DNA double-stranded breaks V(D)J recombination (Schatz et al. 1992), to form continuous (DSBs) (Shlyakhtenko et al. 2009). The recruitment of DSB functional genes as the B and T cells mature. repair proteins, including DNA PKcs, KU70, Ku80, XRCC4 V(D)J recombination is activated by the recombination- and DNA ligase 4, then mediates the imprecise joining of the activating genes, RAG1 and RAG2 (Oettinger et al. 1990), two coding ends into a coding joint, whereas the precise Keywords. Flounder; RAG1; RAG2; thymus Supplementary materials pertaining to this article are available on the Journal of Biosciences Website at http://www.ias.ac.in/jbiosci/ dec2014/supp/Wang.pdf http://www.ias.ac.in/jbiosci J. Biosci. 39(5), December 2014, 849–858, * Indian Academy of Sciences 849 Published online: 20 October 2014 850 Xianlei Wang et al. joining of the two signal ends produces a signal joint (Du cultured flounder. Although flounder RAG2 had been Pasquier 1992; Grawunder et al. 1998). Also, the RAG1- recombinant-expressed in E. coli (Wang et al. 2012), the RAG2 complex plays a role in immunoglobulin allelic organ- and lymphocyte-specific characterization of RAGs pairing, and in determining which recombination methods has never been performed in flounder. Thus, to elucidate will be used to repair the broken ends generated during the ontogeny of the lymphocytes and lymphoid organs in V(D)J recombination (Matthews and Oettinger 2009). flounder, RAG1 and RAG2 were cloned from flounder, and The RAG genes have been cloned from a variety of the organ- and lymphocyte-specific expression was species (Schatz et al. 1989; Carlson et al. 1991; Greenhalgh investigated. et al. 1993; Hansen 1997; Bernstein et al. 1996; Hansen and Kaattari 1995, 1996; Willett et al. 1997; Peixoto et al. 2000; 2. Materials and methods Corripio-Miyar et al. 2007; Zhang et al. 2012; Covello et al. 2013), and a striking feature of the RAG locus was that the RAG genes are highly conserved between species. In fact, in 2.1 Ethics statement all of the vertebrates for which the entire locus has been studied, RAG1 and RAG2 are tightly linked and are Experiments with flounder were performed according to the convergently transcribed (Willett et al. 1997). The genomic regulations of local and central governments. All of the structure of RAG1 was not conserved. A different number of experiments were approved by the Institutional Animal Care introns was found in the teleost RAG1 genomic coding and Use Committee of Institute of Oceanology, Chinese region; zebrafish and Japanese pufferfish each contain two Academy of Science. introns in their RAG1 coding regions (Willett et al. 1997; Peixoto et al. 2000), whereas rainbow trout has one intron in 2.2 Animals the RAG1 coding region (Hansen and Kaattari 1995). In contrast, other vertebrates, such as humans, rabbits, mice, Flounder were cultured at the Institute of Oceanology, Chi- chickens and Xenopus, have no introns in their RAG1 coding nese Academy of Sciences and a fish farm (Homey Group, region. No introns were found in the RAG2 coding region of Rongcheng City, Shandong Province) under controlled con- any of the organisms studied till date (Peixoto et al. 2000). ditions (a 14 h light:10 h dark photoperiod; temperature of The thymus, kidney and spleen are the major lymphoid 15±1°C; seawater; aeration). The fish were fed a commercial organs of teleosts, and the ontogeny of these organs in particle diet twice daily. teleosts has been widely studied in different species, includ- ing yellowtail, flounder, seabream and turbot (Willett et al. 1997; Zapata et al. 2006; Hansen and Zapata 1998; Romano 2.3 Isolation of flounder RAG1 and RAG2 genes et al. 1999; Josefsson and Tatner 1993; Pulsford et al. 1994; Padrós and Crespo 1996), using morphological methods. The genomic sequences of flounder the RAG1 and RAG2 However, immunity genes are also good markers for the genes were isolated using degenerate PCR and study of the development of the immune system. Genes, GenomeWalker methods (Zhang et al. 2006). Based on the such as Ikaros, Ig, RAG1 and RAG2, involved in the differ- conserved regions of the RAG1 and RAG2 nucleotide se- ent developmental stages of lymphocytes have been used to quences from human, mouse, chicken, Xenopus, shark, follow the development of lymphoid organs in fish (Lam zebrafish, trout and pufferfish (the RAG1 GenBank acces- et al. 2002; Danilova et al. 2004). The expression of RAG1 sion numbers are M29474, P15919, P24271, Q91829, and RAG2 are independent of one another (Carlson et al. AAB17267.1, U71093, Q91187 and AAD20561.1, respec- 1991; Greenhalgh et al. 1993; Chun et al. 1991), and yet tively; the RAG2 GenBank accession numbers are P55895, both genes are expressed together in maturing B and T M64796, P25022, Q91830, AY172838, U71094, Q91193 lymphocytes. Therefore, the expression of the RAG genes and AAD20562.1, respectively), nested degenerate PCR can be used to monitor the appearance and location of pre-B primers (R1F1, R1R1, R1F2, R1R2; R2F1, R2R1, R2F2 and pre-T cells during the development of the immune and R2R2) were designed to amplify a region from exon 4 system (Oettinger et al. 1990; Greenhalgh et al. 1993). of the RAG1 gene and a 5′ region of the RAG2 gene using Flounder is an important economic fish species in mari- genomic DNA as the template. The PCR program was 5 min culture and has been extensively cultured under intensive at 94°C, 40 cycles of 40 s at 94°C, 40 s at 58°C, and 1 min systems in the northern coastal regions of China. However, (for RAG1) or 2 min (for RAG2) at 72°C, followed by 10 min the frequent occurrence of diseases has caused enormous at 72°C. The remaining portions of RAG1 and RAG2 were losses in recent years. Therefore, to maintain the stable, isolated by several rounds of PCR using RAG1- or RAG2- healthy and sustainable development of aquaculture, more specific primers (R1F3, R1F4, R1R3, R1R4, R1R5, R1R6, attention is being given to the immune mechanisms in R1R7, R1R8; R2F3, R2F4, R2F5, R2F6, R2R3, R2R4, J. Biosci. 39(5), December 2014 Recombination-activating genes from flounder 851 R2R5 and R2R6; table 1) together with the adapter primers and are in the units of the number of amino acid substitutions (AP1 and AP2) (Clontech, USA) from the flounder per site. The analysis involved 11 amino acid sequences for GenomeWalker libraries (Clontech, USA) (Zhang et al. RAG1 and 10 amino acid sequences for RAG2. All positions 2006). All of the fragments were cloned into the pUCm-T containing gaps and missing data were eliminated. There were vector (Sangon, Shanghai, China) and sequenced. a total of 973 for RAG1 and 508 positions for RAG2 in the final dataset. 2.4 Prediction of lymphocyte-specific transcription factors binding sites 2.6 Reverse transcription-polymerase chain reaction (RT-PCR) To analysis lymphocyte specific transcription factors bind- ing sites in the alternatively 5′-UTR of RAG1,P-match Total RNA extraction was performed using the TRIzol re- program was used (http://www.gene-regulation.com/cgi- agent, following the manufacturer’s instructions (Invitrogen, bin/pub/programs/pmatch/bin/p-match.cgi) and database CA, USA).