Download of the Rat MHC Genes of the Telomeric Class I Gene Region

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
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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
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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,
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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.
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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
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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
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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.
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and D20Wox5, known to localize to the RT1-C/E/M region, were fine
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could be identified in the second framework gene region.
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E/M gene probes are being produced to speed up this analysis.
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graft rejection, and disease susceptibility. Furthermore, the contig
provides the basis for sequencing this part of the rat MHC.
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