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Download of the Rat MHC Genes of the Telomeric Class I Gene Region
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Physical Map and Expression Profile of Genes of the Telomeric Class I Gene Region of the Rat MHC This information is current as of August 11, 2017. Sofia Ioannidu, Lutz Walter, Ralf Dressel and Eberhard Günther J Immunol 2001; 166:3957-3965; ; doi: 10.4049/jimmunol.166.6.3957 http://www.jimmunol.org/content/166/6/3957 Subscription Permissions Email Alerts This article cites 61 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/166/6/3957.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Downloaded from http://www.jimmunol.org/ by guest on August 11, 2017 References Physical Map and Expression Profile of Genes of the Telomeric Class I Gene Region of the Rat MHC1,2 Sofia Ioannidu,3 Lutz Walter,3 Ralf Dressel, and Eberhard Günther4 T he MHC is of particular relevance for controlling disease susceptibility and graft rejection. In humans, a great variety of diseases are controlled by the HLA complex, most of them being of autoimmune or infectious nature (1, 2). The laboratory rat (Rattus norvegicus) is a well-established and widely used model for certain human diseases and organ transplantation (3–5). MHC control has been shown for spontaneously occurring type I diabetes mellitus in the BB strain (6), and models for a large number of experimentally induced diseases under MHC control have been developed (4). Thus, various types of experimental allergic encephalomyelitis present different courses of multiple sclerosis (7, 8), and several forms of experimental arthritis correspond to rheumatoid arthritis in human (9). Susceptibility control by the MHC is mostly assigned to the class I and class II molecules that control specific immune responsiveness. However, many other genes map into the MHC, some encoding proteins involved in the immune response, such as Ag-processing and peptide-loading proteins (proteasome subunits 8 and 9, TAP1 and 2, tapasin), cytokines (lymphotoxin ␣ and , TNF-␣), complement components (C2, C4, BF), and heat-shock proteins (hsp70 –1, hsp70 –2). Others have no apparent function in the immune response. Their expression and function are mostly not well understood, and their involvement in disease control is unclear (10 –12). To understand the role of the MHC in controlling disease susceptibility and graft rejection, a detailed knowledge of this gene Division of Immunogenetics, University of Göttingen, Göttingen, Germany Received for publication October 18, 2000. Accepted for publication January 4, 2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by the European Union (Contract BIO4CT960562). complex is necessary. The human MHC has been mapped physically and sequenced recently (12). The overall structure of the rat MHC, the RT1 complex, is similar to the homologous systems in mice and humans (Fig. 1). A characteristic difference between mouse and rat MHC on the one hand and the human MHC on the other hand is the presence of an additional region of classical class I (class Ia) genes centromeric to the class II region (13). The telomeric class I region of the rat MHC, RT1-C/E/M, is homologous to the region containing the HLA-A, B, C, E, F, G genes in humans and to the H2-D/Q/T/M region in mice. We have recently established a physical map of the centromeric part of the rat MHC by making use of a P1-derived artificial chromosome (PAC)5 library of BN strain origin (13). We now present a sequence-ready physical map of the telomeric class I region of the rat MHC, RT1-C/E/M, together with expression data, providing a genomic basis for including this part of the MHC in disease and transplantation studies at the molecular level. Materials and Methods Screening of the PAC library and contig construction The PAC library (RPCI-31), encompassing 10 genome equivalents of BN rat (RT1n) origin (14), was supplied as filters by the Resource Center of the German Human Genome Project (Berlin, Germany). The filters were screened under stringent conditions with two probes of the rat class I gene RT1-Au (15), containing exon 2, introns 1 and 2 (␣1 probe), and exon 4 (␣3 probe) (see Table I), respectively. Positive clones were digested with various restriction enzymes, notably BamHI and EcoRI, and hybridized with the screening probe for verification and then with further class I as well as non-class I probes (Table I). The non-class I probes were generated from known sequences of other MHC genes (12), H2 markers (16), or on the basis of sequences obtained from the PAC clones. Clones were ordered according to restriction fragment overlap, hybridization patterns with various probes, and sequence data. Sequencing of PAC clone ends was performed as previously described (13) and analyzed with an ABI310 sequencer. 2 The nucleotide data reported in this work have been assigned the database accession numbers AJ294759, AJ294760, and AJ294761 (see Table I). 3 S.I. and L.W. contributed equally. 4 Address correspondence and reprint requests to Dr. Eberhard Günther, Division of Immunogenetics, University of Göttingen, Heinrich-Düker-Weg 12, 37073 Göttingen, Germany. E-mail address: [email protected] Copyright © 2001 by The American Association of Immunologists 5 Abbreviations used in this paper: PAC, P1-derived artificial chromosome; Grc, growth and reproduction complex; STS, sequence-tagged site; utr, untranslated region. 0022-1767/01/$02.00 Downloaded from http://www.jimmunol.org/ by guest on August 11, 2017 The rat is an important model for studying organ graft rejection and susceptibility to certain complex diseases. The MHC, the RT1 complex, plays a decisive role in controlling these traits. We have cloned the telomeric class I region of the RT1 complex, RT1-C/E/M, of the BN inbred rat strain in a contig of overlapping P1-derived artificial chromosome clones encompassing ⬃2 Mb, and present a physical map of this MHC region. Forty-five class I exon 4-hybridizing BamHI fragments were detected, including the previously known rat class I genes RT1-E, RT-BM1, RT1-N, RT1-M2, RT1-M3, and RT1-M4. Twenty-six non-class I genes known to map to the corresponding part of the human and mouse MHC were tested and could be fine mapped in the RT1-C/E/M region at orthologous position. Four previously known microsatellite markers were fine mapped in the RT1-C/E/M region and found to occur in multiple copies. In addition, a new, single-copy polymorphic microsatellite has been defined. The expression profiles of several class I genes and the 26 non-class I genes were determined in 13 different tissues and exhibited restricted patterns in most cases. The data provide further molecular information on the MHC for analyzing disease susceptibility and underline the usefulness of the rat model. The Journal of Immunology, 2001, 166: 3957–3965. 3958 GENOMIC AND EXPRESSION ANALYSIS OF THE RAT MHC Table I. List of RT1-C/E/M region genes, markers, and probes Gene Product, Characteristics Bat1 Pou5f1, Oct3, Otf3 Tcf19, Sc-1 Spr1, Pcg Cdsn, S Gtf2h4, TfIIh M75168 (rat) NM_013633 (mouse) NM_007109 (human) AF159091 (mouse) AI600060 (rat) AA875300 (rat) Ddr1, Cak, Nep Ier3, Prg1, Dif2, Iex1 Flot1 Tubb AW144724 (rat) X96437 (rat) AA955780 (rat) AA859924 (rat) RNA helicase Transcription factor Transactivating factor Psoriasis candidate gene Corneodesmosin Subunit of transcription factor IIH Cell adhesion kinase Early response protein Flotillin, cell adhesion Tubulin -polypeptide Kiaa0170 BE120346 (rat) Ddx16, Dbp2 Ptd017 Ppp1r10, Pnuts, Fb19 AA851883 (rat) AA901244 (rat) AF040954 (rat) Abcf1, Abc50 AA900151 (rat) Cat56 Gnl1, Gna-rs1, Hsr1 Znf173, Zfp173 Znfb7 Rnf9, Rfb30 Tctex5 Tctex4 Mog BE120346 (rat) AI501298 (rat) NM_003449 (human) AI577576 (rat) AI324236 (mouse) Gabbr1 Olfr42, Tu42 Ubd D20lmg2 Grc D205T I162T P084T 255D16T 573K1S Leh525 RT1-M2 RT1-M3 RT1-M4 RT-BM1 RT1-N1 AV318173 (mouse) L21995 (rat), M99485 (rat) AA817879 (rat) AI030354 (rat) L81133 (rat) AJ294759 (rat) AJ294760 (rat) AJ294761 (rat) B88392 (mouse) AB004434 (mouse) AB004423 (mouse) M26156 (mouse) U16025 (rat) AF024712 (rat) X16979 (rat) NM_012646.1 (rat) Homologous to Drosophila photoreceptor protein calphotin RNA helicase Unknown function Protein phosphatase 1 regulatory subunit 10 TNF-␣-inducible ATPbinding protein Proline-rich protein GTP-binding protein Zinc finger protein Zinc finger protein Ring finger protein Testis-expressed gene Testis-expressed gene Myelin oligodendrocyte glycoprotein ␥-Aminobutyric acid B receptor Olfactory receptor Diubiquitin, ubiquitin D Microsatellite marker STS marker STS marker STS marker STS marker STS marker STS marker STS marker Class Ib molecule Class Ib molecule Class Ib pseudogene Class Ib molecule Class Ib molecule Primers Used Source TGACGTGCAGGACCGTTTCG CGAGAAGAGTATGAGGCTAC GACTTTGCTGCCATTACCATC CCACTGGAAGCACCATCCTG CAACGTTTATTGAGCCCTGC GAAATGTCCCGATGTGTAAAC CTGATAAATCAGGACTAGGT ACCCCAAAGCTCCAGGTTCT GCTCACTCTCATCATCCAGT GCTTCTCTCGATCAGCAGAA AATCGTTGGGTTCCCACCAT GTGGACTTTGAATTGCTGCTG Ref. 54, GenBank GenBank GenBank GenBank GenBank EST UI-R-E0-cn-e-12-0-UI.s1 GCAGGATGACCCTCTCTGC GCTCTACCCTCGAGTGGTGA GCCCTTCCAAGTTCCCAATG GATACACTCCCAGGCCATC GTAATGGGCGCAGAGATGGG AGGGCTCAGTCTCGTTTAAT GenBank Ref 53, PAC end sequencing EST UI-R-E1-fg-b-06-0-UI.s1 GACTATGGACTCCGTTCGCT AGTCCCGAAAGGCTGGTT GACAGAGTCAACCAGCTCAG GACCGAGGCATGCTGGG EST UI-R-E0-cg-c-06-0-UI.s1 EST UI-R-CA0-baq-d-03-0-UI AGGTATTGGAGATCGAAAGC GAATACCAGAACCGCTATGG GCAGCATCGCCTGGAGCA ATTAAATAAAGTATGGAAGG CCTAAAGTCAACATGCAACT GCCTGTGAAGAAGAACTCCA GenBank EST UI-R-E0-bq-d-05-0-UI-s1 Ref. 55, PAC end sequencing GAGACTTCAGTTCAGGAGCT CGAGACTTTTGCCACTCAGA EST UI-R-0-dm-c-10-0-UI.r1 GGTTTCCACTGTGTTTATTAC GGCAGGTGATGAAGAGGAGG CACGGAAGAAGTTCTGGTTT TGATTTCCAACTTGTCTGCA GTGAGGCTGGCTTGGGGCTT DNA probe GGTGGTGTGCATACAACAGC GGACAGTTCAGAGTGATAGG TGTGAAAACAGCAGTGAACA CACAATCAGGGTGGCACAGG ACTTCCACTTCCCAGTAGAC AAGGTGGAGACAGGTGGAAG CAGGGAAAAACTGGACCCTC EST UI-R-C1-kv-b-11-0-UI.s1 EST UI-R-C2p-ru-d-05-0-UK.s1 GenBank EST UI-R-Y0-vk-3-03-0-UI-s1 GenBank TCCCTCCCTCGAATCATGCG GAACCTCACGTTCTGGATCCT GenBank Refs. 56 and 57, GenBank GTCTATAAGGAAAGGCTCTT ATGTTGGAAATGCTTCGGGT EST UI-R-A0-af-b-05-0-UI.s2 DNA probe CCACTCTTGATGTTGTAGTC CTGAGCTCCCTAGGACCTACAT GCTGCATCTAATCAGTCTGTG CGCGACCAAAAGCAACATGG GTACAGGAGACAACTGTGAC GGATCAAGGACTAAGTACCAC CTGCAGTGTGCTTTCAAATTC GCTTCTGTTCTGTGTGTGCC GTCAGTCAATACCACAGTTC GATCCCACTCCTTGCGGTAC GTTCTCACATCCTGCAGTGG GCTGTGATTACCCTCACTGG GCAAGGCTTTCCAGATCTCT ACTTCCACACACTACAGTGG TTATGGGATTGACAAGGAAA TCTCTTGTGTCAGGCTAATTAC GCAGAATGCAGTAGTGGGCAT GAGCAAACCACCCGGAGCAA GGCTTGCAACAGTCCACAAC GGAACACGTGGCGCTCAGAC CCAAGTTCTTCCCTGCTGCT GACTGTATCGGTTGCTGGAA GTTCTAGCAGGTATCTGTAT GTTCTGGCTCTGGTTGTAGT GCAGGAATCTATGGAGCAGC AGCTCCCGTACTAGAGTGTG GACCATAAGAGGAATGGTGGA GAACGCAGCAGCCTCTCCTT EST UI-R-C0-iu-h-01-0-UI.s1 PAC end sequencing Ref. 37, GenBank PAC end sequencing PAC end sequencing PAC end sequencing Ref. 31, GenBank Ref. 58, GenBank Ref. 58, GenBank GenBank Ref. 32, GenBank Ref. 21, GenBank Ref. 26, GenBank Ref. 30, GenBank 1 kb flanking to position 217 (X82669) Positions 218–677 (X82669) Positions 2019–2308 (X82669) Positions 2954–3688 (X82669) Ref. 15, GenBank Probes for Class I genes Promoter Exon 2 Exon 4 Exon 7, 8, 3⬘-utr Ref. 15, GenBank Ref. 15, GenBank Ref. 15, GenBank a Where no designation for the rat ortholog existed or was different from human, the designation of the human gene is chosen or listed first. The official designations in mouse and human are different for Cak/DDR1, Zfp173/ZNF173, and Gna-rs1/GNL1, respectively. No approved designations exist thus far for Bat1, Spr1, Kiaa0170, Ptd017, Cat56, Znfb7, Rnf9, Olfr242, or Ubd, and they are used according to Refs. 10 –12, 16, and http://www.sanger.ac. HGP/Chr6/current_MHC_gene_list.shtml. The EST sequences were obtained from the EST database at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) or from the University of Iowa EST Project http://ratest.eng.UIowa.edu/cgi-bin/map-info?chr⫽20. Probes for the mouse Tctex5 and Olfr42 genes are a gift of Dr. Pierre Pontarotti. Expression analysis Total RNA was prepared (17) from various organs of 3-mo-old male BN/ Gun rats and day 12 embryos (LEW.1W/Gun strain) bred in our own colony and from BN lymphocytes after 3 days of stimulation with Con A. RNA from three BN rats were pooled and analyzed by Northern blot (washing conditions 2⫻ SSC, 60°C for 2 ⫻ 10 min). In most cases, probes were derived by PCR from the 3⬘-untranslated region (utr) using DNA from the corresponding rat PAC clones, and -actin served as loading and hybridization control. The primers were designed according to rat gene or EST (expressed sequence tag) sequences when available, or on the basis of mouse and human sequence information (Table I). Hybridization signals were scanned (Epson Scanner, software ScanPack; Biometra, Göttingen, Germany), and the ratio of gene to -actin signal was determined. Microsatellite analysis The D20Arb2 (18, 19), D20Mgh3 (18), D20Wox4 (18), D20Wox5 (20), RT1-M4 (21), and D20Img2 (this study; Table I) microsatellites were analyzed using primers and PCR conditions according to the respective references. The PCR profile for D20Img2 was 30 cycles of 94°C, 30 s; 55°C, 45 s; 72°C, 60 s. Genomic DNA of inbred rat strains BN/Gun, LEW/Ztm, Downloaded from http://www.jimmunol.org/ by guest on August 11, 2017 Accession No. of Sequence (Species) Gene Designation(s)a The Journal of Immunology LEW.1A/Gun, LEW.1W/Gun, bred in our colony, was isolated according to standard methods. Amplificates were analyzed on 2% agarose gels. Additionally, the 5⬘ primer of D20Arb2 was 6-FAM labeled, and analysis was conducted on an ABI310 with GeneScan software. 3959 from Fig. 2, four subregions with class I genes are separated by three subregions of non-class I genes. Class I genes Results Construction of a PAC contig of the RT1-C/E/M region FIGURE 1. Scheme of the MHC structure in human, mouse, and rat. A, The class I, class II, and class III regions are indicated by roman numbers and marked by different symbols. The centromere is shown to the left. The position of some genes is included for orientation. The region designated here as RT1-C/E is also called RT1-C or RT1-E/C/Grc in the literature. B, The bars below the rat MHC indicate the status of its physical cloning in cosmid, PAC, or yeast artificial chromosome (YAC) vectors (references in brackets) described for RT1-A (13), RT1-H/DO (59, 60), RT1-B/D (61), and RT1-C/E/M (Refs. 42, 43, 62, and 63 and this study). The cosmid clones described in Ref. 42 cannot be assigned to particular parts of the RT1-C/E/M region and are, therefore, not shown by bars. Downloaded from http://www.jimmunol.org/ by guest on August 11, 2017 The structure of the rat MHC is schematically shown in Fig. 1. The analysis focuses on the RT1-C/E/M class I region. Probes ␣1 and ␣3, representing class I exons 2 and 4, respectively, were used for initial screening of the PAC filters. Ninety-eight clones were obtained and verified to carry class I sequences. These PAC clones were analyzed by Southern blot with various class I and non-class I gene probes. In a first step, 31 PAC clones could be ordered into four separate contigs (22) on the basis of overlapping restriction fragments and hybridization patterns. Class I gene-carrying clones that mapped to the RT1-A region have been reported elsewhere (13). The four initial contigs were then assembled into a single contig by screening the PAC library with either non-class I probes known to map to the respective part of the MHC or probes derived from information of PAC end sequencing (sequence-tagged site (STS) markers). A single PAC contig of ⬃2 Mb could thus be constructed for the RT1-C/E/M region, based on a usually deep coverage with overlapping clones (Fig. 2). The contig starts with the Tnf and Bat1 genes and is thereby anchored in the RT1 class III region. The contig ends beyond the RT1-M3b gene. As is evident The individual BamHI restriction fragments that hybridized with the ␣1 and/or ␣3 class I probes (symbolized by rectangles in Fig. 2) will be provisionally designated according to their size. In total, 45 fragments hybridized with the ␣3 probe, which represents the most conserved part of a class I gene. In addition, in the second class I subregion, 24 consecutive BamHI fragments were detected that hybridized only with the ␣1, but not the ␣3 probe. In the first class I cluster, between Bat1 and Pou5f1, 15 class I genes may be present, if one assumes that a single BamHI fragment that hybridizes with both the ␣1 and ␣3 probes, or two neighboring fragments hybridizing with ␣1 and ␣3 probes, respectively, represent a complete class I gene. Most of the class I genes in the first subregion (but none in the other class I subregions) also hybridized with promoter and 3⬘-utr probes (Table I) derived from the class Ia RT1-Au gene, indicating that indeed complete class I genes are present and that they are very similar to class Ia genes. Several class I fragments have been partially sequenced. By comparison with published data, the class I exon 2 sequence of BamHI fragment 1 is nearly identical with the RT1-Cc clone cc1 (23), and fragment 4 was identified as the RT1-E gene (24, 25). Exon 2 sequences of fragments 2, 19, 23, and 10 revealed highest similarity (above 90%) to class Ia genes of the RT1-A region, but not 3960 GENOMIC AND EXPRESSION ANALYSIS OF THE RAT MHC Downloaded from http://www.jimmunol.org/ by guest on August 11, 2017 FIGURE 2. Physical map of the RT1-C/E/M region based on the PAC contig. The contig is constructed from 101 PAC clones. Locations of microsatellite markers are shown by bars above the gene designations. Positions of class I gene cross-hybridization (squares) are indicated by the size of BamHI restriction fragments and the cross-hybridizing probes, pr (promoter), ␣1 (exon 2, introns 1 and 2), ␣3 (exon 4), 3⬘ (exons 7, 8, intron 7, and 3⬘-utr). Already known class I genes that have been mapped on the contig are indicated. Framework genes (triangles) are indicated by their designation. STS markers are shown by open circles (mouse origin) or by closed circles (constructed from rat PAC sequences). The order of genes under brackets has not yet been determined in the rat. PAC lengths, although determined in most cases, are not to scale. The intervals between the genes have not been examined and are shown schematically with equal size and, therefore, not to scale. PAC coverage varies between 15- and 2-fold, except the Tcf19/Gtf2 h4, D205T/Ier1, P084T/Rnf9, and Tu42/Ubd intervals for which only a single PAC clone was found, respectively (indicated by ⬃). The region around STS marker D205T appears to be unstable in bacteria, especially clones E15393 and A24506 (shaded in gray). The gap between the complete contig and the RT1-M3c/M2 cluster has not been closed yet, but the latter has been mapped to the MHC chromosomal region by fluorescence in situ hybridization (K. Helou and L. Walter, unpublished results). In some cases, transcriptional orientation could be determined by partial sequencing and is indicated by an arrow below the respective gene designation. nd, Not determined. The Journal of Immunology FIGURE 3. PCR patterns of the D20Mgh3 (A), D20Wox4 (B), D20Wox5 (C), D20Arb2 (D, E), and D20Img2 (F) microsatellite markers. In A, B, C, D, and F, PCR amplified products were separated in 2% agarose gels. In E, the 6-FAM-labeled PCR product was amplified from DNA of several inbred rat strains plus one representative PAC clone and analyzed with an ABI310 automated sequencer. Amplification products are marked by arrowheads (A–D, F); in E, the exact sizes (bp) are indicated. Non-class I genes In the RT1-C/E/M-corresponding parts of the human and mouse MHC, represented by the HLA class I and the H2-D/Q/T/M regions, respectively, clusters of conserved non-class I genes (also designated framework genes; 16) have been described extending from Bat1 to Ubd (10 –12, 16). Of the ⬃34 non-class I genes (excluding known pseudogenes) reported (10 –12), 26 have been included in the PAC analysis. These genes could be assigned to the RT1-C/E/M region (Fig. 2, genes symbolized by triangles), and their order, as far as determined, is the same as in the HLA (10 – 12) and H2 complex (16, 35). The first three class I subregions are interspersed at orthologous positions in the three species, as defined by framework genes Bat1, Pou5f1, Gnl1 (Gna-rs1), Znf173, Tctex4, and Mog (Fig. 2). A syndrome of decreased male fertility, dwarfism, and increased susceptibility to chemical carcinogenesis has been mapped to the RT1-C region of the RT1r16 haplotype (36). The respective genes ft, dw3, and rcc are assumed to be part of a growth and reproduction complex (Grc) in the MHC. The Grc, defined at the molecular level as a region hybridizing multiply with the grc1.4 probe (37), could now be assigned with this probe by PAC hybridization to the second class I subregion (Fig. 2). Microsatellite markers Besides the RT1-M4 microsatellite in the RT1-M region (21), four known microsatellite markers can be localized in the RT1-C/E/M region. The D20Mgh3 and D20Wox4 markers (18) are both derived from the class I gene RT1A-4 (38) and are identical, except for an incomplete overlap of the 3⬘-primer (see http://www.nih.gov/niams/scientific/ratgbase/data/ARBPR20.htm). Both markers have now been mapped to the first class I subregion (Fig. 2). They do not occur in single copy, but are repeated at least five times, as is documented by their presence on three nonoverlapping PAC clones from which one or two amplification products can be obtained (Fig. 3, A and B). A composite of these amplificates is seen in BN genomic DNA (Fig. 3, A and B). The D20Wox5 microsatellite (20), derived from the RT1-O gene (39), is present in at least Downloaded from http://www.jimmunol.org/ by guest on August 11, 2017 to H2-D or H2-Q genes, which reside in the corresponding part of the mouse MHC. In the second class I cluster, extending between Gln1 and Znf173 (Fig. 2), the RT-BM1 gene (26 –28), which is identical to RT1-S3 (29), could be assigned to fragment 4.4 on the basis of specific hybridization and partial sequence information obtained from the 3⬘-utr of fragment 4.4. Similarly, the RT1-N1 gene (30) was identified according to specific hybridization and partial sequence data and assigned to BamHI fragment 4.6. Sequence analysis of the ␣1-cross-hybridizing fragment 2.1 (PAC clone H12587) revealed the presence of 81 nucleotides from the 3⬘ half of a class I exon 2 without any neighboring class I sequences. Most likely, the 24 consecutive ␣1-cross-hybridizing fragments (starting with ␣1-fragment 8 and ending with ␣1-fragment 5 in the second class I subregion, Fig. 2) are duplications of the same module, since they hybridize with the 3⬘ half of exon 2, but not with the 5⬘ half (not shown). The presence of the rat homologue of the mouse STS marker 255D16T (31) is of interest because it maps to the beginning of the H2-M region in the mouse (16). In the third class I cluster between Tctex4/Tctex5 and Mog, the RT1-M4 gene was identified by a RT1-M4-specific microsatellite (21). RT1-M3 (32) was assigned to a fourth class I cluster telomeric to Ubd by hybridization of the specific probe and by partial sequence data. Different from the mouse, three RT1-M3 genes, designated provisionally RT1-M3a, M3b, and M3c, are found (Fig. 2), as well as three copies of the Leh525 homologous marker. The RT1-M3c gene, together with the RT1-M2 gene (33), is localized on a contig that could not yet be linked to the main contig directly, but has been assigned to the RT1 region by fluorescence in situ hybridization (K. Helou and L. Walter, unpublished data). This finding is in accord with the identification of three H2-M3 homologous genes on a single clone of a rat yeast artificial chromosome (YAC) library (34). No cross-hybridization signal with MHC class I-related probes could be found for any PAC clone of the contig, confirming negative genomic Southern blot results (not shown). 3961 3962 GENOMIC AND EXPRESSION ANALYSIS OF THE RAT MHC Table II. Expression profiles of RT1-C/E/M genes based on Northern blot analysisa Bat1 Skin Liver Kidney Small intestine Lung Heart 0 0.03 s 0.10 l 0.18 s 0.07 l 0.11 s 0.11 l 0.09 s 0.11 l 0.08 s 0.05 l 0.08 s Muscle Brain Testis Lymphoblasts Spleen 0.04 s 0.17 l 0.18 s 0.11 l 0.10 s 0.37 l 0.36 s 0.16 l 0.08 s 0.20 l 0.10 s Embryo Tcf19 Spr1 Cdsn 0 0 0.29 0 0 0 0 0 0.11 0 0 0 0.14 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Gtf2h4 0.07 s 0.51 l 0.52 s 0.36 l 0.27 s 0.29 l 0.19 s 0.29 l 0.22 s 0.25 l 0.21 s Ddr1 Ier3 0.20 0.15 0.69 l 1.17 m 0.32 0.16 l 0.77 m 0.08 0.19 l 0.47 m 0.03 0.14 l 0.58 m 0.08 0.2l l 0.80 m 0.02 0.31 m 0.02 0.20 l 0.53 m 0.02 0.13 l 0.99 m 0.01 0.34 m 0.01 0.59 l 0.73 m 0.38 0.37 l 0.64 m 0.91 s 0.06 0.28 l 0.33 m 0.65 s 0.02 0.21 m 0.17 0.40 0.24 0.23 0.17 0.03 0.06 s 0.41 l 0.28 s 0.32 l 0.31 s 0.19 l 0.19 s 0.19 l 0.32 s 0 0 0 0 0 0 0 0 0 0.18 0.02 0 0 0.15 0 0 0 0.14 0 0 0.19 l 0.11 s 0.39 0 0.06 0 0 0.30 l 0.16 s 0.50 0.23 s 0.20 0.42 0.04 0.04 Flot1 Tubb Kiaa0170 Ddx16 Ptd017 Ppp1r10 0 0 0 0.02 0 0 0.09 0.65 0 0 0.08 0.58 0 0 0.03 0.55 0 0 0.02 0.48 0 0 0.06 0.29 0 0 0.03 0.06 0 0.03 0.12 0.35 0.04 0.04 0.07 0.90 0.01 0.02 0.02 0.40 0 0.12 0.01 0.32 1.10 l 0.95 s 0 0 0.01 0.21 1.35 l 1.51 s 0 0.03 0.03 0.20 0.76 s 0.69 l 1.12 s 0.94 l 0.96 s 0.63 l 0.64 s 1.06 l 1.03 s 0.57 l 0.88 s 0.42 s 0.86 l 3.29 s 0.25 l 1.88 s 0.82 l 1.20 s 1.35 l 1.92 s a The ratio of gene to -actin signal is shown (mean of two to four independent experiments). Where several transcripts were detected, l indicates longest, m medium, and s shortest transcript. (Table continues.) three copies in the second class I subregion (Fig. 2). One and two amplificates are detectable with two nonoverlapping PAC clones, respectively (Fig. 3C). The BN genomic DNA shows both PCR product lengths (Fig. 3C). The D20Arb2 microsatellite (18, 19), derived from the RT1-N1 gene (30), also maps to the second class I subregion at multiple positions (Fig. 2). An amplification product of the same length can be obtained with three nonoverlapping PAC clones (Fig. 3, D and E). Up to five D20Arb2 amplification products of different length can be obtained in other RT1 haplotypes (Fig. 3E). By end sequencing of PAC N0751 a CA dinucleotide repeat and corresponding flanking primers (Table I) could be established (Fig. 2). This microsatellite, 288 bp long (RT1n haplotype), designated D20Img2, occurs only once in the RT1-C/E/M region and is polymorphic in different strains (Fig. 3F). Expression profiles of RT1-C/E/M genes Northern blot analysis was conducted with total RNA from 11 organs, lymphoblasts, and day 12 embryo using 3⬘-utr probes (Table I) from each of the 26 framework and four class I genes of the RT1-C/E/M region (Table II). Only few genes such as Tubb are ubiquitously expressed. The class I genes tested and most framework genes exhibit differential expression profiles, often in an organ-specific manner, such as Mog and Gabbr1 showing brain-specific expression and Tctex4 being testis specific. The expression of genes such as Ubd is induced in mitogen-stimulated lymphocytes, in accord with stimulation of this gene by IFN-␥ (40). For some genes, no positive Northern blot signal could be detected, because expression might be too weak or restricted to tissues not included in the panel. In the case of Spr1 and Rnf9, expression was checked by RT-PCR with RNA from liver, small intestine, lung, lymphoblasts, spleen, and embryo, but no amplification products could be obtained. In general, the expression patterns obtained are in accord with the relative tissue representation of corresponding ESTs (expressed sequence tags; see http://www.ncbi.nlm.nih.gov/ UniGene/query.cgi and http://ratest.eng.uiowa.edu/cgi-bin/ map-info?chr⫽20). Discussion The RT1-C/E/M region shows a particular genomic organization. Class I genes occur in clusters that are positioned between regions of non-class I framework genes. Most of these framework genes had not yet been mapped in the rat, and reports (34, 41) on the presence of Pou5f1, Cdsn (S), Gnl1 (Gna-rs1), Znf173, Tctex5, and Mog in the RT1-C/E/M region are confirmed. In total, four class I clusters have been found to alternate with three non-class I gene clusters in between (Fig. 2). This organization is very similar to that described for the H2-D/Q/T/M region in mice (16) and the class I region in humans (10 –12, 16). The boundaries of the first three class I gene clusters are marked by orthologous framework genes in the three species. An exception is the fourth class I gene cluster containing the RT1-M3 genes. It is found telomeric to the Mog gene, similar to the mouse, whereas no class I gene has been described in the corresponding part of the HLA complex. Each of the 26 framework genes tested in the rat has been found in the RT1-C/E/M region at an orthologous position, and none of them mapped into a class I subregion. In this context, the RANBP1 (TC4) sequence found in the second class I cluster of the HLA complex (10 –12) is of interest, because it is not present in the rat RT1-C/E/M region (own unpublished data). Inspection of the sequence indicates that it might be a processed pseudogene, and as such would not be expected to be conserved between rat and human in contrast to expressed framework genes. The number of class I genes in the RT1-C/E/M region of the RT1n haplotype will only be known when the complete sequence of this region is available. On the basis of hybridization with the conserved exon 4 probe, ⬃45 class I genes could be assumed. This Downloaded from http://www.jimmunol.org/ by guest on August 11, 2017 Thymus Pou5f1 The Journal of Immunology 3963 Table II. Continued Abcf1 Cat56 Gnl1 RT-BM1 RT1-N1 Znf173 Znfb7 Rnf9 Tctex5 Tctex4 Mog Gabbr1 Olfr42 Ubd RT1-M3 RT1-M2 0.04 0 0 0.09 0 0 0 0 0 0 0 0 0 0 0 0 0.27 0 0 1.94 0 0.15 l 0.26 s 0 0 0.15 l 0 0 0 0 0 0.09 0 0.21 0.02 0 0.30 0.03 0 0 0.06 l 0 0 0 0 0 0.06 0 0.29 0 0.06 l 0 0 0 0 0 0.03 0 0 0 0.06 l 0 0 0 0 0 0.11 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.03 l 0 0.09 2.12 0 0 0.08 0 0 0 2.22 0 0 0 0.05 0 0 0 0 0.51 l 0.46 s 0 0 0 0.03 0 0.12 0.24 0.07 0 0 0 0 0 0 0 1.55 0.49 0 0 0 0.02 l 0 0 0 0 0 0.12 0.03 0 0 0.04 l 0 0 0.03 0 0 0.01 0.04 0.11 0.01 0 0.19 0 0.12 0.01 0 0.39 0.03 0.13 s 0.06 s 0.03 s 0.31 0 0 0.28 0 0.07 s 0.13 0 0 0.21 0 0.03 s 0.42 0.09 0 0.26 0 0.12 s 1.08 0.04 0 0.16 0 0.14 s 0.04 0 0.62 1.01 0.11 s 0.35 0 0 0.19 0.11 0.18 s 0.17 0.02 0 0.49 0.27 0.10 s 0.13 0.03 0 0.08 0.15 0.10 s number is lower than the 61 (42) and 62 class I genes (43) extrapolated from screening cosmid libraries of the RT1av1 and RT1r21 haplotypes, respectively. Haplotype differences in the number of class I genes are a familiar phenomenon of the MHC, also in the rat (13). It is unknown at present how many of the class I genes are indeed expressed. Among the class I genes described in the RT1C/E/M region before, RT1-E (24, 25), RT-BM1 (26, 27, 29), RT1-N1 (30), RT1-M3 (32), and RT1-M4 (21) have been fine mapped now, and for some of them restricted tissue distribution could be shown (Table II). Genes of the first class I cluster, such as RT1-E, as well as class I sequences obtained from this region, are more similar to rat class Ia genes than to other rat class I genes or mouse H2-D, Q, T, M genes. Thus, whereas the localization of this subregion immediately telomeric to Bat1 corresponds to the H2-D/L/Q region of the mouse, the individual genes are not orthologous. The RT-BM1 (RT1-S3) gene in the second class I gene cluster is assumed to be orthologous to the mouse H2-T23 gene (27, 28), which encodes the Qa-1 molecule, and the RT1-N1 gene is reported to be orthologous to H2-T10 and T22 (30). Furthermore, the Grc probe, defining the Grc region in the second cluster, has been shown to cross-hybridize to sequences flanking the H2T10d/11d and H2-T22d/23d genes in the mouse (44). Consequently, the second class I subregion appears to correspond to the mouse H2-T region. Since in the mouse the 255D16T marker maps to the beginning of the H2-M region, the position of the homologous sequence in the second class I subregion (Fig. 2) could mark the beginning of the homologous region, RT1-M, in the rat, and the ␣3-cross-hybridizing fragments telomeric to this marker therefore might represent homologues of mouse H2-M genes. The H2-M4 orthologous gene RT1-M4 (21) maps to the third class I subregion, and the H2-M3 orthologous gene RT1-M3 (32) to the fourth subregion. Thus, the telomeric part of the whole contig corresponds to the mouse H2-M region, confirming and extending previously published genetic data (33). No human/rat orthology can be established for the class I genes in the various class I clusters. This is in accord with the general lack of interorder class I orthology. It is of note that HLA-MHC class I-related-like genes are missing in the rat MHC (as in the mouse; 45), whereas H2-T-like as well as H2-M-like genes, which are missing in the human MHC, are present in the rat. Therefore, with respect to class I genes, the rat RT1-C/E/M region cannot serve as a model for the HLA complex, whereas the non-class I genes are clearly orthologous. The function of the RT1-C/E/M class I genes is not well defined. It is known that they can act as targets of CTL (23) and as stimulatory targets of alloreactive NK cells (46). Notably, the RT1-E gene product has been shown to stimulate alloreactive NK cells (47). RT1-C/E/M incompatibility has been shown to induce skin and pancreas graft rejection (48) and to modulate the fate of MHC class II-mismatched heart grafts (49). The individual RT1-C/E/M genes that are responsible for the histoincompatibility reaction have not yet been identified. Ag presentation by RT1-C/E/M-encoded class I molecules could not be shown (50) except one report (51). Nevertheless, since H2-Qa1 and H2-M3, for example, are able to present particular peptides in the mouse, a similar function could be assumed for RT-BM1 and RT1-M3, respectively. Also, the class Ia similarity of genes in the first class I subregion could indicate a peptide presentation function. The RT1-C/E/M region is involved in controlling disease susceptibility. Analysis of RT1 recombinants has shown that severity of collagen-induced arthritis is associated with the RT1-C/E/M genotype (52). Similarly, the course of anti-myelin/oligodendrocyte glycoprotein (MOG)-induced encephalomyelitis is modulated by genes in the RT1-C/E/M region (7, 8). The function of the non-class I genes, as far as known, is diverse (Table I). Some genes appear to encode transcription factors controlling cell proliferation or DNA repair, and have been suggested to be involved in cancer development (11). An example is the Downloaded from http://www.jimmunol.org/ by guest on August 11, 2017 0.30 3964 GENOMIC AND EXPRESSION ANALYSIS OF THE RAT MHC 21. Acknowledgments 28. The technical assistance of Petra Kiesel and Diana Otto is gratefully acknowledged. We also thank the Resource Center of the German Human Genome Project for providing PAC pools, filters, and clones. References 1. Thorsby, E. 1997. HLA associated diseases. Hum. Immunol. 53:1. 2. Hill, A. V. S. 1998. 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Downloaded from http://www.jimmunol.org/ by guest on August 11, 2017 Ppp1r10 gene (alias designations Fb19 and Pnuts) that has been detected because of its expression in pancreatic cancer (53). In this context it is noteworthy that the Grc controls susceptibility to diethylnitrosamine-induced hepatocellular carcinomas (36). Unexpectedly, the Grc, as far as defined by the grc1.4 probe (37), maps into the second class I subregion without including known framework genes. To implement RT1-C/E/M genes into whole genome analyses of physiological or pathological traits, microsatellite markers are extremely useful. The microsatellites D20Arb2, D20Mgh3, D20Wox4, and D20Wox5, known to localize to the RT1-C/E/M region, were fine mapped. Surprisingly, PAC analysis revealed that these markers occur in several copies, which are not always distinguishable by length polymorphism. This multicopy nature must be taken into account in genetic analyses. 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Transplant. Proc. 31:1594. 3965