mapping of h2-m homolog and mog genes in the rat mhc

Immunogenetics (1995) 42:418-421 © Springer-Verlag 1995

Doris Lambracht • Christa Prokop Hans Jiirgen Hedrich • Kirsten Fischer Lindahl Kurt Wonigeit

Mapping of H2-M homolog and MOG genes in the rat MHC

Received: 16 May 1995 / Revised: 3 July 1995

Class I genes of the rat major histocompatibility complex (MHC; RTI system) are encoded by two subregions, which are separated by regions coding for class II (RT1.B/D) and class III molecules. By convention, the RT1.A region is left of the class II region; it codes for one or very few classical class I molecules. The RT1. C/E region, located on the other side of the RT1 complex, contains a large number of class I genes (Jameson et al. 1992). The internal organization of this extended class I region has not been defined in detail. It is therefore of interest that genes similar to H2-M genes, which are the most distal group of class I genes in the mouse, have been demonstrated in the rat (Wang et al. 1991, 1995). The rat genes similar to H2-M genes are now called RT1.M genes, and they are different from the previously defined oligomorphic class I gene RT1.M (Wonigeit and Hfinisch 1991), which has been renamed RT1.R (Gill et al. 1995). A homolog of the H2-M3 gene has been isolated, and the sequence of RT1.M3 cDNA is 88% identical to H2-M3 (Wang et al. 1995). RT1.M3 as well as homologs of H2-M2 and H2-M4 have been mapped with established recombinant haplotypes to the extended class I region on the RT1. C/E side of the class II/III region (Wang et al. 1995). We now report on a new recombinant haplotype, r38, derived from the parental strains LEW (RTIO and BN (RTIn), in which the rat homologs of H2-M2 and H2- M4 are separated from other class I genes that differ

D. Lambrachtl • K. Wonigeit (~) Transplantationslabor, Klinik ftir Abdominal- und Transplantationschirurgie, Medizinische Hochschule Hannover, D-30625 Hannover, Germany

C. Prokop - H. J. Hedrich Zentrales Tierlabor, Medizinische Hochschule Hannover, D-30625 Hannover, Germany

K. Fischer Lindahl Howard Hughes Medical Institute, Departments of Microbiology and Biochemistry, University of Texas Southwestern Medical Center, Dallas TX 75235-9050, USA Present address: 1 Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas TX 75235-9050, USA

between haplotypes l and n. This recombinant defines a new class I subregion beyond RT1.C. It also permitted mapping of the rat gene that codes for the myelin/oligodendrocyte glycoprotein (MOG) to this region of the RT1 complex.

Haplotype r38 was found in a detailed analysis of the LXB family of 12 recombinant inbred (RI) rat strains. The progenitor strains of this RI strain set are LEW and BN (Hedrich 1990). Accordingly, the individual strains are expected to possess either the RT1 l or the RT1 n haplotype. Of the 12 RI strains analyzed, eight had previously been shown to possess the RT1.A and RT1.B/D region of the RT1 l haplotype and four the RT1.A and RT1.B/D region of RTln. Serological typing for the polymorphic RTI.C region gene, RT1.C 1, present in the RT11 but not the RT1 n haplotype, gave concordant results, demonstrating that in all strains the RT1.C gene has been derived from the same parental haplotype as the RT1.A and RT1.B/D regions. This was confirmed by restriction fragment length polymorphism (RFLP) analysis with the rat class I probe pWW6.3 encod- ing the extracellular part of a rat class I molecule (Wurst 1988), which detects the RT1.A gene as well as multiple class I genes mapping to the RT1.C/E region (Fig. 1).

Haplotype-specific patterns for the RT1.M genes, differentiating l and n, could be demonstrated with the H2-M2 probe after digestion of genomic DNA with Hin dII, and with the H2-M4 probe after digestion with Barn HI, Hin dIlI, or Pvu II (Table 1; Fig. 2). One RI strain, LXB17L, showed a deviant result: although typing as 1 in all other assays, it displayed the n pattern with the probes for H2-M2 and H2- M4. This strain possesses a 4.4 kilobase (kb) Barn HI fragment hybridizing to M4 (Fig. 2) and 16 and 10 kb Hin dII fragments that hybridized to M2 (Table 1). LXB 17L thus possesses a recombinant haplotype, termed r38, in which the rat homologs of H2-M2 and H2-M4 are separated from all other class I genes tested in this study. This maps the RT1.M genes to the right of the RT1.C region and establishes a new class I region.

Evidence for the location of RT1.M4 close to rat homologs of H2-M5 (RT1.M5) and H2-M6 (RT1.M6) was obtained by analysis of twenty cosmid clones that encode

D. Lambracht et al.: Location of rat RT1.M region

kb

---

23----*

9.4

6.5- ' -*

4,3

Fig. 2 RT1.M4 pattern of Barn HI-digested genomic rat DNA after hybridization to the locus-specific mouse probe for H2-M4 (described in Figure 3). The hybridizing fragment is 4.4 kb in strains carrying the k b

haplotype n or r37, which possess the RT1.Cregion ofn. It is 4 kb in

23 ---* strains LEW (RTP) and in strains LEW.1R14 and LEW.1LM1, 9.4 ---- which share part or the entire RT1.C region with LEW. 6.5 ----* LXB17L possesses the n pattern, 4.3 ----* which indicates that it is a recombinant haplotype

419

z z × × I ~ W

2.3 2 "-"

0.5

Fig. 1 Class I pattern of Bam HI-digested genornic rat DNA hybridized to the cDNA probe pWW6.3. The strains used are indicated above the lanes. Strain LXB17L was indistinguishable from the standard RT1 l haplotype of the LEW rat strain and is clearly different from the strains BN and LXB17N possessing the RTI~ haplotype. BN.1B and LEW.1R14 carry recombinant haplotypes with the subregion structures: AbBbDbC n for r37 (Wonigeit et ai. 1993) and AuBuDuC 1 for r14 (Hedrich 1990). LEW.ILM1 possesses a mutant RT1 haplotype that is characterized by a deletion of about 100 kb nonclassical class I genes from the RT1.C region (Wurst et al. 1989). Genomic DNA was isolated from the different strains by standard procedures, digested with restriction enzymes, separated in horizontal agarose gels, and blotted onto nitrocellulose filters (Maniatis et al. 1982). Hybridizations were carried out overnight in roller tubes at 68 °C in a buffer containing 6 x standard sodium citrate (SSC)/ 5 x Denhardt's solution/0.5% sodium dodecyl sulfate (SDS) 10% (w/v) dextran sulfate. The filters were washed in decreasing salt concentrations at 68 °C. For the M genes and Mog, the final wash was 1 x SSC/0.1% SDS at 68 °C (Figs. 2, 3, 4)

class I genes and were isolated from a LEW genomic library (Lambraeht 1994). One clone, cosmid 3, hybridized with H2-M4, M5, and M6 probes. Because each probe hybridized to a different fragment, this cosmid must contain separate genes (Fig. 3). Thus it is likely that all three genes are located distal to the crossover in r38. In the mouse, M4, M6, and M5 are also next to each other (Wang and Fischer Lindahl 1993). H2-M4 and H2-M6 are pseudogenes due to in frame stop codons; the status of the rat homologs is not known yet. The most telomeric class I gene in the MHC of the mouse is H2-M3 (Jones et al. 1995). Its rat homolog RT1.M3 has already been mapped to the RT1. C side of the class IUIII region using a different set of recombinants (Wang et al. 1995), and it is likely that RT1.M3 also maps in the RT1.M region. Because the H2-T region is located proximal-to the H2-M region in the mouse, it was of interest to see whether the rat homologs of H-2T genes, RT1.N1, N2, and N3 (Kirisits et al. 1994), are separated

2.3

from the RT1.M genes by the crossover in r38. Therefore, we analyzed rat H2-T homologous genes with the rat probe 5' pMK (Kirisits et al. 1994). However, we found no potymorphism differentiating haplotypes l and n, and it has not yet been possible to determine their location relative to the recombination point in r38.

A limited screening of various rat strains with probes for H2-M2 and H2-M4 showed three distinct restriction fragment length polymorphism (RFLP) patterns of two fragments each for RT1.M2 and three patterns for RT1.M4. When RT1.M2 and RT1.M4 were considered together, five different RFLP combinations emerged (Table 1). Interest- ingly, strains AS and LEW, which share the RTF haplotype,

Fig. 3 Cosmid clone 3 was digested with Eco RI and hybridized to (panel a) H2-M4, (panel b) H2-M5, and (panel c) H2-M6 probes. The lanes shown were run in parallel in the same gel and hybridized separately to the H2-M probes. The probes were a 1.1 kb Kpn I fragment of H2-M4 and a 0.15 kb Kpn I/Sma I fragment of H2-M5 (Wang and Fischer Lindahl 1993). Testing of the 1.1 kb Barn HUKpn I fragment of H2-M6 (Wang and Fischer Lindahl 1993) showed multiple hybridizing bands with rat genomic DNA. To obtain a single copy probe for this gene, the fragment was shortened by Msp I digestion and a 0.2 kb fragment was identified as a locus-specific probe (Lambracht 1994)

a b c

kb

23

9.4 ---,.

6 , 5 4 - - .

4 . 3 . ,

2 . 3

2

420

Table 1 RFLP analysis of RT1 haplotypes with H2-M probes


Fragment sizes (kb)

Rat strain Source 1 RT1 haplotype H2-M4 probe and Pvu II H2-M24 probe and Hin dII

PVG Ola c -, 3.9, - 1.5 - 16, 10 LEW. 1C (WIST) Han c - 3.9, 2.11 231 - ' 10 LEW Won l - , 3.9, 2.1, - 23, -, -, 10 AS Ztm I -, 3.9, 2.1, - -, 16, -, 10 PVG.1L (LEW) Iap l -, 3.9, 2.1, - 23, -, -, 10 PVG.1L (AGUS) 2 Ola 1 -, 3.9, 2.1, - -, 16, -, 10 BH Ztm lv33 -, 3.9, 2.1, - -, 16, -, 10 LEW.1LV3 Won lv33 -, 3.9, 2.1, - -, 16, -, 10 LXB17L Han r383 -, 3.9, -, 1.5 -, 16, -, i0 LEW.1N (BN) Won n -, 3.9, -, 1.5 -, 16, -, 10 BN Han n -, 3.9, - 1.5 16, -, 10 LEW.IU (WP) Won u -, 3.9, 2.11 - -,-' 12, 10 PVG.1U (AO) Ola u 6, -, -, - 23, -', -, 10 AO Ola u 6, -, -, - 23, - , -, 10 LOU/C Ota u 6, -, -, - 23, -, -, 10 BDE Han u 6, -, -, - 23, -, -, 10

1 The breeder symbols are: Han (Zentralinstitut ftir Versuchstierzucht, Hannover, Dr. Hedrich), Iap (Institute of Animal Physiology, Cambridge, Dr. Butcher and Dr. Howard), Ola (Harlan Olac, Bicester), Won (Transplantationslabor, Medizinische Hochschule Hannover, Dr. Wonigeit), Ztm (Zentrales Tierlaboratorium, Medizinische Hochschule Hannover) 2 Differences from the standard RT11 haplotype were detected by RFLP analysis and described in Naper and co-workers (1995) 3 The RT1 subregion structure for haplotype lv3 is AIB1DIClv3M iv3 and for haplotype r38 A1BID1OM ~ 4 The mouse H2-M2 probe is a 1.3 kb Eco RI fragment of 3/12 (Brorson et al. 1989)

differed in RTI.M2. Strain LEW.1C shared the pattern for M2 and M4 with L E W but not with the reference RT1 c strain, PVG. It is not clear whether this difference is caused by a recombinat ion of the RT1.M region during backcross- ing to LEW, or whether the donor of the RTlc haplotype differed in the RT1.M genes from PVG. A surprising finding was the identity of patterns obtained for the strains PVG and BN, which differ in all other RT1 regions. Several strains carrying the haplotype u displayed identical patterns when hybridized with the class I probe pWW6.3 (data not shown); however, the reference strain, LEW. 1U, differed in RT1.M2 and RT1.M4 from the other RT1, strains. Because it differed also from the background strain L E W in RT1.M2, it is l ikely that the RT1.M genes of L E W . I U are derived from the extinct strain, W R from which the RTI~ haplotype was transferred. Similarly, the polymorphism that maps M3 to the RT1.C/E region (Wang et al. 1995) distinguishes LEW. 1U from PVG.1U. Analysis of other congenic strains and the respective parental strains could lead to the detec- tion of additional recombinants in this part of the MHC.

The recombinant haplotype r38 permitted mapping of the MOG gene in the RT1.M region. The MOG gene belongs to a subset of the immunoglobul in superfamily with a 9 5 % - 8 8 % conserved sequence in cattle, mouse, and rat, and 41% similari ty to B-G antigens of the chicken MHC region in its extracellular domain (Pham-Dinh et al. 1993). M O G is a pr imary target antigen in experimental autoimmune encephalit is (EAE; Gardinier et al. 1992). The human MOG gene maps distal to HLA-F (Pham-Dinh et al. 1995), and in the mouse Mog is located in the H2-M region 25 kb distal of H2-M5 (Jones et al. 1995; Pham-Dinh et al. 1995). To map MOG in the rat, the RFLP patterns of r38 and its parental haplotypes 1 and n were analyzed. After Hin dII digestion a clearcut po lymorphism was found

(Fig. 4). Haplotype r38 showed the n pattern, locating the rat MOG gene within the RT1.M region. As rat strains carrying the RT1 haplotypes l and n display MHC-depen- dent differences in susceptibili ty to EAE (Gasser et al. 1983) and other neurological disorders, the recombinant r38 might be helpful in differentiating effects of other MHC genes from those of different MOG alleles.

The recombinant haplotype r38 separates the RT1.M region from all other serological ly defined RTI regions and locates the RT1.M genes outside the RT1.C region. It is very l ikely that the RT1.M region represents one end of the RT1 complex, and the RT1.M genes and MOG will be important marker genes for estimation of the physical size of the MHC and the organization of rat class I regions. The

Fig. 4 A polymorphism for rat MOG was found after Hin dII digestion of DNA from the indicated strains. The probe is a polymerase chain reaction-gen- erated exou 2 sequence of the mouse Mog gene (Daubas et al. 1994). Haplotype r38 showed the pattern of n, mapping MOG within the RT1.M region

kb

23 -...*

9 . 4 ~

6 . 5 ~

4.3

co

2.3 2 --~


location of RT1.M4, M5, and M6 on a single cosmid clone suggests that, in addition to the map position, the internal organization of the M region in rat and mouse may be very similar, providing a further example of conservation of gene organization in related species.

Acknowledgments We thank Dr. J. T. Vaage for sending tissue ma- terial for DNA isolation of the LOU/C, AO and all PVG rat strains and Dr. A. Dantigny for Mog probes. This work was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 244).

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mapping of h2-m homolog and mog genes in the rat mhc

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