Eur. J. Immunol. 1990. zyxwvutsrqp 20: 1337-1343 zyxwvutsrqp Mus suxicolu wild mouse class zyxw I1 [3 genes 1337 Philippe Camv, Evelyne Jouvin-Marche, Christian LeGuernO and Patrice N. Marche Institut Pasteur, Paris and National Institutes of Healtho, Bethesda Structure of class I1 genes in wild mouse Mus saxicola: functional and evolutionary implications* Class I1 zyxwvutsr p genes have been characterized from Mus suxicofu, a wild mouse which diverged five millions years ago from the house mouse. The nucleotide sequences of the exons 2 and 3 of Epl, Ap2 and Ep2 genes have been determined in order to define the molecular mechanisms of interspecies variability. The Epl and Apl (J. X. She, personal communication) genes from Mus zyxw suxicolu display extensive sequence variability in exon 2 when compared to those of the house mouse, in association with the variability of residues probably involved in antigen binding. On the other hand, most putativeT cell contact residues from the I-Ep chain, and to a lesserextent from the I-Ap chain,were found conserved in mice.T cell contact residues from Mus suxicofu I-Ap and I-Ep chains are identical to those of the b and q, and of the k haplotypes of the house mouse, respectively. Comparisons performed with Ap2 and Ep2 genes revealed that both are highly conserved and, in contrast to A61 and Epl genes, that a strong selective pressure for conservation occurs in the Ap2 second exon. The different aspects of Ap2 and Ep2 genes evolution may correlate with the original function of their products. Comparisons of the intervening sequences revealed that the repeated motifs of the hotspot region present in the Eplb gene are also conserved in Mus suxicolu. Recombina- tions may have occurred, in some cases in association with X-like sequences. 1 Introduction The MHC is a genetic region composed of class I and class I1 genes which control the regulation of the immune response. Class I1 molecules are heterodimeric transmem- brane proteins which are encoded by an a and a p gene [l, 21. In the house mouse, the class zyxwvut I1 gene products, I-A and I-E, are mainly expressed on B cells, monocytes, M@ and dendritic cells [2-4].The I-A and I-E molecules display allelic polymorphism [5], especially in the aminoterminal portion of the chains [6].Transfection studies have shown that the NH? terminal variability of both the a and p chains is related to antigen recognition by Tcells and to MHC- restriction phenomena [7-91. In addition to I-A- and I-E-encoding genes, Ap2 and Ep2 class I1 genes, whose function remains unknown, were found in the H-2 I region [lo, 111. Ep2, Ap2 and its human equivalent DOp [12] are non-polymorphic and show an unusual pattern of transcrip- tion [13, 141. Using RFLP studies, comparisons of class I1 genes among distantly related members of the genus Mus have been reported ([15], Cam et al., in preparation). Evidence has also been obtained that a large part of the MHC polymor- phims pre-dates speciation and that alleles may be main- [I 81551 ~ ~ _______ * This work was supported by INSERM Grant No. 873. Recipient of an allocation from Ministkre de la Recherche et de I’Enseignement SupCrieur. Correspondence: Philippe Cam, Unite d’Immunochimie Analy- tique, DCpartement d’Immunologie, Institut Pasteur, 25, rue du Dr. Roux, F-75724 Paris, Cedex 15, France Abbreviations: ARS: Antigen recognition site sax: M. saxicola haplotype tained from species to species [16, 171. Most of the house mouse Apl gene alleles can be organized into two evolu- tionary groups on the basis of restriction site polymorphism and by the presence of a short interspersed nucleotide element [ 181. However, little data is available concerning the variability among the class I1 genes between distant Mus species, in particular the Ap2 and Ep2 genes. The present work extends the study of these genes to Mus suxicolu.The structure of Epl, Ap2 and Ep2 genes has been determined in this species, for which class I1 genes restric- tion endonuclease fragments (REF) had been found to display a high degree of variability compared to those of the house mouse. The Epl, Ap2 and Ep2 genes, as well as the Apl gene from M. zyxwv suxicofu have been analyzed in the light of the structural model of class I1 antigens [19]. 2 Materials and methods 2.1 Probes The probes used for hybridizations were purified fragments corresponding to exons 3 to 4 of an Apl genomic clone [20], exon 2 of an Epl genomic clone [21] and exons 3 of DOg and DPp cDNA clones [12]. These probes were labeled by the random priming procedure [22]. 2.2 Cloning and nucleotide sequence determination An M. saxicofu genomic library, constructed in vector phage hJ1, was screened by colony hybridization [23] with DPp, DOp and Epl class I1 probes, and 17 clones were selected. Filters were washed at low stringency in 0.5 X SSC, 30 min at 50°C. Hybridizing clones were further characterized by hybridization with a panel of probes followed by increasingly stringent washes. High and 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990 0014-2980/90/0606-1337$02.50/0