Journal of Heredity 2003:94(1)
© 2003 The American Genetic Association 94:23-26
Molecular Organization of the Canine Major Histocompatibility Complex
From the Departments of Medicine, Immunology, and Microbiology, Thomas Jefferson University, Kimmel Cancer Center, 1025 Walnut St., Philadelphia, PA 19107.
Address correspondence to the author at the address above, or e-mail: John.Wagner@mail.tju.edu.
 |
Abstract |
|---|
 Top Abstract References |
|---|
The major histocompatibility complex (MHC) is composed of a tightly linked cluster of genes; in dogs, this is referred to as the dog leukocyte antigen (DLA) region. The canine MHC is located on chromosome 12, and several genes within the DLA region have been identified that have significant sequence similarity to their human counterparts. However, in order to characterize other loci in the DLA region, DNA sequencing has begun using a canine bacterial artificial chromosome (BAC) library. Initially 135 BAC clones were isolated from a BAC library using a mixture of human and canine probes. These BAC clones were screened with locus-specific primers in polymerase chain reactions (PCRs). Fifty-six BAC clones were subjected to FingerPrinted Contig (FPC) analysis and several overlapping clones were identified. One BAC clone RP81-231-G24 has been sequenced. Preliminary sequence analysis of this 150 kb clone indicates that it contains the region where the class I and class III regions are joined and encompasses DLA-12a, DLA-53, DLA-12, DLA-64, TNF-
, and a canine gene that appears to resemble the HLA class III gene HSPA1A (HSP70-1). The major histocompatibility complex (MHC) is a cluster of genes that are important in the immune response to infections and is associated with several autoimmune diseases. In humans, more diseases have been associated with the MHC than any other area of the genome. The dog serves as an important model for several human diseases, including prostate cancer (Cornell et al. 2000) and rheumatoid arthritis (Halliwell et al. 1983; Ollier et al. 2001). Many of the most common diseases in dogs, as well as humans, have an immune component. These diseases include hypothyroidism (Happ 1995); cancer (MacEwen 1990); autoimmune diseases such as systemic lupus erythematosus (Fournel et al. 1992; Teichner et al. 1990), myasthenia gravis (Dewey et al. 1997), diabetes (Gordon 1967), pemphigus vulgaris (Hurvitz and Feldman 1975), and Addison's disease (Willard et al. 1982); and allergic dermatitis (Zur et al. 2002). Drugs or vaccines can trigger autoimmune diseases, and that immune response is partially influenced by the genetic background of the subject. The genetic component of immunity may be especially important in purebred dogs that have a restricted gene pool. If the genetic component can be understood, it may be possible, for example, to control breeding so that certain diseases are not propagated.
In normal physiology, MHC gene products interact with bound peptides and present these antigens to T-cells via interactions with the T-cell receptors. This presentation of self and nonself antigens to the immune system is important in the regulation of the immune response.
In humans, MHC genes are divided into at least five regions: the classical class I, the extended class I, the classical class II, the extended class II, and the class III regions (Beck et al. 1999; Gruen et al. 1996). The function of many of these genes is unknown. However, in general several class I and class II gene products are involved in antigen presentation, while class III gene products play a role in complement and inflammation (Beck et al. 1999).
Early understanding of the canine MHC (called the dog leukocyte antigen [DLA]) was based on cellular, serological, and immunochemical analysis (summarized in Wagner et al. 1999). Later molecular analyses were performed on genes discovered due to strong DNA sequence similarity to their human counterparts. Recent efforts are under way to relate these genes to their protein products (Wagner et al. 2002). A list of known class I, II, and III genes and their polymorphism is shown in Tables 1–3.
|
As a result of previous studies it became apparent that in order to understand the large-scale organization of the DLA region and identify other loci not having similar DNA sequences to their human counterparts, sequencing multiple overlapping bacterial artificial chromosome (BAC) clones would be necessary, similar to what was done to characterize the human MHC (Beck et al. 1999). The general organization of the DLA region was known through large-scale mapping studies (Breen et al. 2001), but the distance between loci or the identification of all loci present would require sequencing individual BAC clones.
Initially a canine BAC library (Li et al. 1999) was screened with a mixture of human and canine class I, II, and III probes and 131 BAC clones were isolated. Using PCR screening with locus-specific primers, 56 clones were isolated containing class I, II, or III loci. These 56 BAC clones were "fingerprinted" or subjected to FingerPrinted Contig (FPC) analysis.
For FPC analysis, the gel image is digitally captured and imported into the software program (Soderlund et al. 2000). Contigs are generated based on shared fragments of identical size. This provides some information about how individual BAC clones may be related, but does not provide a physical map of the BAC clones. The goal is to determine which BAC clones overlap the least ("minimal tiling path") and sequence these BAC clones to obtain a detailed physical map of the DLA region.
BAC clone RP81-231-G24 was chosen for initial sequencing analysis because it was known to contain TNF-, a class III gene (Li et al. 1999), and DLA-64, a class I gene (Wagner JL, unpublished data), and because it was in the middle of a large contig (based on FPC analysis) that was composed of multiple BAC clones containing class I and class III genes.
Once the individual BAC clone to be sequenced is chosen, the BAC DNA is purified in large quantities. A process of shotgun sequencing of individual fragments of BAC DNA is performed (Green 2001; Inoue et al. 1997). This process initially involves sonicating the purified BAC DNA. The fragmented DNA is run on an agarose gel and fragments of 1–4 kb are extracted from the gel. These fragments are "blunt ended" and ligated into a vector such as PUC18. The ligated DNA fragments are transformed in competent Escherichia coli cells. Colonies of the transformed bacteria are grown in 96 well plates and the plasmid DNA from each colony is purified. Next, 500 ng of template is sequenced using M13 forward and reverse primers located on either side of the insert site. Approximately 500 bp of sequence data are obtained for each reaction. Data from the initial 300 sequencing reactions are analyzed for E. coli contamination (>90% BAC DNA is acceptable) as well as the quality of the sequence data and representation of the BAC DNA (i.e., the number of similar DNA fragments in terms of sequence in the first 300 reactions). The PHRED program (CodonCode Corp., Dedham, MA) is used to determine the quality of the sequencing data. If the initial sequence data are of sufficient quality and representation, about 3,000 total sequencing reactions are done for each BAC clone. Various programs such as Sequencher (Gene Codes Corp., Ann Arbor, MI) or PHRAP (CodonCode Corp., Dedham, MA) are used to align the fragments. Programs such as GENSCAN (Stanford University, Palo Alto, CA) are used to identify genes from the sequence data.
Preliminary analysis of the sequence data indicates that the RP81-321-G24 insert is 150 kb long and contains DNA encoding for DLA-64, DLA-12, DLA-12a, DLA-53, TNF-, and a putative protein (Wagner JL, unpublished data) that has 95% amino acid similarity to the human class III gene HSPA1A (HSP-70-1) (Honore et al. 1992). After the analysis of this BAC clone is completed, the next BAC clone that overlaps the least with this clone will be sequenced using a similar approach. After sequencing multiple overlapping BAC clones not only will a physical map of the MHC be generated, but several new genes will be identified. Further understanding of the DLA region will require functional studies of the gene products.
|
|
|
Acknowledgments |
|---|
This work was supported by grant nos. RR12558 and CA78902 from the National Institutes of Health, DHHS, Bethesda, MD. Yaniv Palti and Donna DiDario provided technical support. This paper was delivered at the Advances in Canine and Feline Genomics symposium, St. Louis, MO, May 16–19, 2002.
|
Footnotes |
|---|
Corresponding Editor: Naoya Yuhki
Received July 15, 2002
Accepted September 18, 2002
|
References |
|---|
|
TopAbstractReferences |
|---|
-
Beck S, Geraghty D, Inoko H, Rowen L, 1999. Complete sequence and gene map of a human major histocompatibility complex. Nature. 401:921-923.[CrossRef][Medline]
Breen M, Jouquand S, Renier C, Mellersh CS, Hitte C, Holmes NG, Cheron A, Suter N, Vignaux F, Bristow AE, Priat C, McCann E, Andre C, Boundy S, Gitsham P, Thomas R, Bridge WL, Spriggs HF, Ryder EJ, Curson A, Sampson J, Ostrander EA, Binns MM, Galibert F, 2001. Chromosome-specific single-locus FISH probes allow anchorage of an 1800-marker integrated radiation-hybrid/linkage map of the domestic dog genome to all chromosomes. Genome Res. 11:1784-1795.
Burnett RC, DeRose SA, Wagner JL, Storb R, 1997. Molecular analysis of six dog leukocyte antigen class I sequences, including three complete genes, two truncated genes, and one full-length processed gene. Tissue Antigens. 49:484-495.[Web of Science][Medline]
Burnett RC, Geraghty DE, 1995. Structure and expression of a divergent canine class I gene. J Immunol. 155:4278-4285.[Abstract]
Cornell KK, Bostwick DG, Cooley DM, Hall G, Harvey HJ, Hendrick MJ, Pauli BU, Render JA, Stoica G, Sweet DC, Waters DJ, 2000. Clinical and pathological aspects of spntaneous canine prostate carcinoma: a retrospective analysis of 76 cases. Prostate. 45:173-183.[CrossRef][Web of Science][Medline]
Dewey CW, Bailey CS, Shelton GD, Kass PH, Cardinet GH, 1997. Clinical forms of acquired myasthenia gravis in dogs: 25 cases (1988–1995). J Vet Intern Med. 11:50-57.[Web of Science][Medline]
Doxiadis G, Rebmann V, Doxiadis I, Krumbacher K, Vriensdorp HM, Grosse-Wilde H, 1986. Polymorphism of the fourth complement component in the dog. Immunobiology. 169:563-569.[Web of Science]
Dutra AS, Mignot E, Puck JM, 1996. Gene localization and syntenic mapping by FISH in the dog. Cytogenet Cell Genet. 74:113-117.[Web of Science][Medline]
Fournel C, Chabanne L, Caux C, Faure JR, Rigal D, Magnol JP, Monier JC, 1992. Canine systemic lupus erythematosus I: a study of 75 cases. Lupus. 1:133-139.
Gordon EN, 1967. The clinical management of diabetes mellitus in dogs and cats. Aust Vet J. 43:568-574.[Medline]
Green ED, 2001. Strategies for the systematic sequencing of complex genomes. Nat Rev Genet. 2:573-583.[CrossRef][Web of Science][Medline]
Gruen JR, Nalabolu SR, Chan TW, Bowlus C, Fan WF, Goei VL, Wei H, Sivakamasundari R, Liu Y, Xu HX, Parimoo S, Nallur G, Ajioka R, Shukla H, Bray-Ward P, Pan J, Weissman SM, 1996. A transcription map of the major histocompatibility complex (MHC) class I region. Genomics. 36:70-85.[CrossRef][Web of Science][Medline]
Halliwell RE, Lavelle RB, Butt KM, 1983. Canine rheumatoid arthritis—a review and a case report. J Small Anim Pract. 13:239-248.[CrossRef]
Happ GM, 1995. Thyroiditis—a model of canine autoimmune disease. Adv Vet Sci Comp Med. 39:97-139.[Web of Science][Medline]
Honore B, Leffers H, Madsen P, Rasmussen HH, Vandekerckhove J, Celis JE, 1992. Molecular cloning and expression of a transformation-sensitive human protein containing the TPR motif and sharing identity to stress-inducible yeast protein ST11. J Biol Chem. 267:8485-8491.
Hurvitz AI, Feldman E, 1975. A disease in dogs resembling human pemphigus vulgaris: case reports. J Am Vet Med Assoc. 166:585-590.[Web of Science][Medline]
Inoue H, Ishi H, Alder H, Snyder E, Druck T, Huebner K, Croce CM, 1997. Sequence of the FRA3B common fragile region: implications for the mechanism of FHIT deletion. Proc Natl Acad Sci USA. 94:14584-14589.
Kennedy LJ, Angles JM, Barnes A, Carter SD, Francino O, Gerlach JA, Happ GM, Ollier WE, Thomson W, Wagner JL, 2001. Nomenclature for factors of the dog major histocompatibility system (DLA), 2000: second report of the ISAG DLA Nomenclature Committee. Anim Genet. 32:193-199.[CrossRef][Web of Science][Medline]
Kennedy LJ, Barnes A, Happ GM, Quinnelt RJ, Bennett D, Angles JM, Day MJ, Carmichael N, Innes JF, Isherwood D, Carter SD, Thomson W, Ollier WER, 2002. Extensive interbred, but minimal intrabreed, variation of DLA class II alleles and haplotypes in dogs. Tissue Antigens. 59:194-204.[CrossRef][Web of Science][Medline]
Li R, Mignot E, Faraco J, Kadotani H, Cantanese J, Zhao B, Lin X, Hinton L, Ostrander EA, Patterson DF, de Jong PJ, 1999. Construction and characterization of an eightfold redundant dog genomic bacterial artificial chromosome library. Genomics. 58:9-17.[CrossRef][Web of Science][Medline]
MacEwen EG, 1990. Spontaneous tumors in dogs and cats: models for the study of cancer biology and treatment. Cancer Metastasis Rev. 9:125-136.[CrossRef][Web of Science][Medline]
Ollier WE, Kennedy LJ, Thomson W, Barnes AN, Bell SC, Bennett D, Angles JM, Innes JF, Carter SD, 2001. Dog MHC alleles containing the human RA shared epitope confer susceptibility to canine rheumatoid arthritis. Immunogenetics. 53:699-673.
Sarmiento UM, Storb RF, 1988a. Characterization of the class II alpha genes and DLA-D region allelic associations in the dog. Tissue Antigens. 32:224-234.[Web of Science][Medline]
Sarmiento UM, Storb RF, 1988b. Restriction fragment length polymorphism of the major histocompatibility complex of the dog. Immunogenetics. 28:117-124.[CrossRef][Web of Science][Medline]
Sarmiento UM, Storb RF, 1990. Nucleotide sequence of a dog class I cDNA clone. Immunogenetics. 31:400-404.[CrossRef][Web of Science][Medline]
Soderlund C, Humphrey S, Dunham A, French L, 2000. Contigs built with fingerprints, markers and FPC V4.7. Genome Res. 10:1772-1787.
Teichner M, Krumbacher K, Doxiadis I, Doxiadis G, Fournel C, Rigal D, Monier JC, Grosse-Wilde H, 1990. Systemic lupus erythematosus in dogs: association to the major histocompatibility complex class I antigen DLA-A7. Clin Immunol Immunopathol. 55:255-262.[CrossRef][Web of Science][Medline]
Wagner JL, Burnett RC, DeRose SA, Storb R, 1996a. Molecular analysis and polymorphism of the DLA-DQA gene. Tissue Antigens. 48:199-204.[Web of Science][Medline]
Wagner JL, Burnett RC, Storb R, 1996b. Molecular analysis of the DLA-DR region. Tissue Antigens. 48:549-553.[Web of Science][Medline]
Wagner JL, Burnett RC, Storb R, 1999. Organization of the canine major histocompatibility complex: current perspectives. J Hered. 90:35-38.
Wagner JL, DeRose SA, Burnett RC, Storb R, 1995. Nucleotide sequence and polymorphism analysis of canine DRA cDNA clones. Tissue Antigens. 45:284-287.[Web of Science][Medline]
Wagner JL, Hayes-Lattin B, Works JD, Storb R, 1998. Molecular analysis and polymorphism of the DLA-DQB genes. Tissue Antigens. 52:242-250.[Web of Science][Medline]
Wagner JL, Sarmiento UM, Storb R, 2002. Cellular, serological, and molecular polymorphism of the class I and class II loci of the canine major histocompatibility complex. Tissue Antigens. 59:205-210.[CrossRef][Web of Science][Medline]
Willard MD, Schall WD, McCaw DE, Nachreiner RF, 1982. Canine hypoadrenocorticism: report of 37 cases and review of 39 previously reported cases. J Am Vet Med Assoc. 180:59-62.[Web of Science][Medline]
Zucker K, Lu P, Fuller L, Asthana D, Esquenazi V, Miller J, 1994. Cloning and expression of the cDNA for canine tumor necrosis factor-alpha in E. coli. Lymphokine Cytokine Res. 13:191-196.[Web of Science][Medline]
Zur G, Ihrke PJ, White SD, Kass PH, 2002. Canine atopic dermatitis: a retrospective study of 266 cases examined at the University of California, Davis, 1992–1998. Part I. Clinical features and allergy testing results. Vet Dermatol. 13:89-102.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  N. Yuhki, T. Beck, R. Stephens, B. Neelam, and S. J. O'Brien Comparative Genomic Structure of Human, Dog, and Cat MHC: HLA, DLA, and FLA J. Hered., August 3, 2007; (2007) esm056v1. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Aguilar, S. V. Edwards, T. B. Smith, and R. K. Wayne Patterns of Variation in MHC Class II {beta} Loci of the Little Greenbul (Andropadus virens) with Comments on MHC Evolution in Birds J. Hered., March 1, 2006; 97(2): 133 - 142. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||