Previous DNA sequencing of BAC clones covering entire ovine MHC (OLA) region resulted in identification of approximately 130 functional genes in the region, of which 8 were predicted by computer software to be exclusively existed in sheep, but not in any other species known to date. In the present study, we successfully identified and cloned cDNA sequence of OaN2, OaN5, and OaN6 from representative sheep tissues, confirmed their existence in reality. The sequences obtained experimentally exactly identical to those predicted previously. The length of cDNA fragments for OaN2, OaN5, and OaN6 was 270 bp, 309 bp, and 205 bp, respectively, with GenBank accession number assigned as JF330782 (OaN2), JF330783 (OaN5), and JF330784 (OaN6). Northern analyses indicated that the mRNA transcripts of OaN2 were mainly seen in ovine mesenteric lymph nodes and spleen, while OaN5 was observed in only in mesenteric lymph nodes. In contrast, OaN6 transcripts were detected in all tissues except for liver and heart. Western blot showed that OaN2 protein expression level was detected in mesenteric lymph nodes, spleen, and liver, essentially consistent with that of mRNA transcripts. Immunohistochemistry analysis showed that OaN2 protein was highly expressed in ovine mesenteric lymph nodes, moderately expressed in, and not expressed in heart, liver, and pancreas, consistent with the results of Western blotting. The cloning and expression analysis of 3 novel genes provide a basis for revealing their specificities and would be helpful to further study of their expression profile and their potential functions.
[1] Gao J, Liu K, Liu H, Blair HT, Li G, Chen C, Tan P, Ma RZ. A complete DNA sequence map of the ovine major histocompatibility complex. BMC Genomics, 2010, 11(1): 466-474.
[2] Dawkins R, Leelayuwat C, Gaudieri S, Tay G, Hui J, Cattley S, Martinez P, Kulski J. Genomics of the major his-tocompatibility complex: haplotypes, duplication, retrovi ruses and disease. Immunol Rev, 1999, 167(1): 275-304.
[3] Kelley J, Walter L, Trowsdale J. Comparative genomics of major histocompatibility complexes. Immunogenetics, 2005, 56(10): 683-695.
[4] Liu H, Liu K, Wang J, Ma RZ. A BAC clone-based physical map of ovine major histocompatibility complex. Genomics, 2006, 88(1): 88-95.
[5] Margaron Y, Bostan L, Exposito JY, Malbouyres M, Trun-fio-Sfarghiu AM, Berthier Y, Lethias C. Tenascin-X in-creases the stiffness of collagen gels without affecting fibrillogenesis. Biophys Chem, 2010, 147(1-2): 87-91.
[6] Zimin AV, Delcher AL, Florea L, Kelley DR, Schatz MC, Puiu D, Hanrahan F, Pertea G, Van Tassell CP, Sonstegard TS, Marcais G, Roberts M, Subramanian P, Yorke JA, Salzberg SL. A whole-genome assembly of the domestic cow, Bos taurus. Genome Biol, 2009, 10(4): R42.
[7] Chessa B, Pereira F, Arnaud F, Amorim A, Goyache F, Mainland I, Kao RR, Pemberton JM, Beraldi D, Stear MJ, Alberti A, Pittau M, Iannuzzi L, Banabazi MH, Kazwala RR, Zhang YP, Arranz JJ, Ali BA, Wang ZL, Uzun M, Dione MM, Olsaker I, Holm LE, Saarma U, Ahmad S, Marzanov N, Eythorsdottir E, Holland MJ, Ajmone-Marsan P, Bruford MW, Kantanen J, Spencer TE, Palmarini M. Revealing the history of sheep domestication using retro-virus integrations. Science, 2009, 324(5926): 532-536.
[8] Liu K, Zhang P, Gao J, Liu H, Li G, Qiu Z, Zhang Y, Ren J, Tan P, Ma RZ. Closing a gap in the physical map of the ovine major histocompatibility complex. Anim Genet, 2010, 42(2): 204-207.
[9] Barcellos LF, May SL, Ramsay PP, Quach HL, Lane JA, Nititham J, Noble JA, Taylor KE, Quach DL, Chung SA, Kelly JA, Moser KL, Behrens TW, Seldin MF, Thomson G, Harley JB, Gaffney PM, Criswell LA. High-density SNP screening of the major histocompatibility complex in systemic lupus erythematosus demonstrates strong evidence for independent susceptibility regions. PLoS Genet, 2009, 5(10): e1000696.
[10] McKinnon E, Morahan G, Nolan D, James I. Association of MHC SNP genotype with susceptibility to type 1 dia-betes: a modified survival approach. Diabetes Obes Metab, 2009, 11(S1): 92-100.
[11] Vignal C, Bansal AT, Balding DJ, Binks MH, Dickson MC, Montgomery DS, Wilson AG. Genetic association of the major histocompatibility complex with rheumatoid arthritis implicates two non-DRB1 loci. Arthritis Rheum, 2009, 60(1): 53-62.
