Paz-Artal E et al 1997

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Genetics of MHC

shorter cytoplasmic tail than classical HLA class I genes. In contrast to classical HLA class I genes, the primary transcript of HLA-G gene is alternatively spliced in trophoblasts and other fetal or adult tissues; three different forms of HLA-G mRNA (all lacking exon 7) were observed: the full-length mRNA transcript (HLA-GI) and two transcripts lacking either exon 3 (HLA-GP) or exons 3 and 4 (HLA-G3). Materials and Methods: Human first trimester trophoblasts were obtained from voluntary terminations of pregnancy at 6 to 10 weeks of gestation. Local ethical committee approval was obtained for this study. Total mRNA was isolated from 1 g of frozen tissue and the RT-PCR amplification was performed using HLA-G specific primers. The specificity of PCR products was confirmed by hybridization with exon-specific probes and subsequent sequencing. Resutts: After analysis of splice sites we have postulated the presence of the additional transcript of similar size as G2 but lacking exon 4 (HLA-G4). By using exon 4 specific RT-PGR primers we have demonstrated the presence of 64 transcript. Moreover we and others have demonstrated the presence of an additional alternatively spliced transcript containing intron 4 (HLA-G5). The presence of a stop codon in lntron 4 deletes the transmembrane region (exon 5) of the HLA-G molecule which may produces a soluble antigen. Concluelon: We have demonstrated the existence of HLA-G4 and HLA-G5 new alternatively spliced forms of the HlA-G mRNA. HLA-G5 transcript provide an original processing of soluble antigen since among classical HLA class I genes the alternatively spliced transcript lacking exon 5 is preferred. Moreover the presence of different alternatively spliced mRNAs suggests the possible expression of at least five antigen isofonns.

P.1.03.05

Identification and segregation analysis of woodchuck MHC class I gene products by one dimensional isoelectric focusing (1 D-IEF)

V. Rebmann l, M. PBOler’. J. Maschke*, S. Menne*, M. Roggendotf 2, B. Tennant 3, H. Grosse-Wilde ‘. lDepartment of /mmunology, University Hospital of Essen, GemKmy, *Lkyartment of Vimlo~ University Hospital of Essen, German)! 3Cornell lJnive&~ College of Veterinary Medicine, Gastrointestinal Unit, Ithaca, New York, USA Introduction: Since the woodchuck hepatitis virus (WHV) is closely related to human hepatitis B virus (HBV), woodchucks infected with WHV are the nearest pathobiological model for HBV infections in man. It is considered that T cell mediated immune response to HBWWHV derived peptides presented by MHC molecules may determinate the course of hepatitis. Studies of cytotoxic T-lymphocyte responses, however, are hampered by the lack of informations about organisation and polymorphism of the MHC class I gene products in this animal model. Therefore we started a study on woodchuck MHC class I gene products by biochemical methods (lD-IEF). Materials and Methods: Membrane proteins of peripheral blood lymphocytes from unrelated woodchucks (N = 34) and 6 families (N = 52 members) kept in a breeding colony were extracted by TX114 and analysed by 1D-IEF. After western blot the woodchuck class I molecules were detected by a cross reacting anti-human MHC class I antiserum. The isoelectric points (IP) of MHC class I specific bands were characterised by densitometry. Results: In the unrelated woodchucks 19 distinct banding positions were identified with IP ranging from pH 7.14 to 5.76. At least 2 and in some animals 4 different class I variants were observed. The class I variants appeared with different frequencies but 4 of the 19 variants comprised more than 50% of the variants defined (IP: 6.23, 6.50, 6.63, 6.73). These MHC class I variants were also found in members of the woodchuck families analyzed and a typical codominant segregation was observed. In 3 families we could analyse the segregation pattern over 3 generations. Conclusion: This preliminary study clearly demonstrates, that Woodchuck MHC class I gene products are polymorphic and segregate according to Mendel. As 4 distinct class I vatiants could be detected in most of the unrelated woodchucks, 2 separate MHC class I coding genes can be anticipated. However, more infonative families have to be analysed in this respect.

P.1.03.06

identification of a pz-microgiobuiin dependent impaired breeding phenotype in mice: A role in MHC- dependent fecundity

N. Femandez. Department of Biologica and Chemical Sciences, Central Campus, University of Essax, Wiwnhoe Park, Cotchester, England /Jz-microglobulin plays a pivotal role in the biology of mammals, including its association with MHC Class I products. The latter molecules have been shown to affect reproduction in both mice and humans (I), although the exact mechanism is still unknown. Here we report the results of a longitudinal study of the reproductive performance of the transgenic ,92-m deficient mouse (also as a consequence, MHC Class I deficient). Our data show that this mouse strain has an impaired, &-m-associated, reproductive performance (2). However, the mice superovulate well and show a normal oestrus cycle. Breeding studies from

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crosses between the transgenic mice and wild type mice show that &m deficient mice have a significantly lower frequency of mating than the control &m +/+ C57BU6 mice (p < 0.0001). In addition, the litter size and weaning success of Be-m deficient mice were lower than the control. Petinatal lethalii of the &-m deficient offspring was also inflicted by cannibalism of the young pups by the Be-m deficient female. This impaired breeding phenotype (IBP) can be reversed by re-introducing the f12-m gene in FI heterozygous B2-m +/- animals; thus the presence of fin-m confers a normal breeding pattern. The acquisition of an impaired breeding phenotype (IBP) as a result of the knock out of fin-m directly implicates @z-min the reproductive cycle of mice and raises the possibility of an effect of 67-m in the reoroduction of other mammals. [l] Sprinks, M.T., Sellens, M.H., Dealtry, G.B. and Femandez, N., Immunogenetics 1993. 36: 35. [2] Cooper, J.C., Femandez, N., and Dealtry, G.B. lmmunotogy 1995. 63 (1): 51.

P.l.03.07

Aiieiic diversity at the primate MC-G locus: Exon 3 bears stop codons in ail Cercophltecinee sequences

M.J. Castro, P. Morales, V. FemBndez-Soda, B. Suarez, M.J. Recio, M. Alvarez, J.M. Martin-Villa, A. Amaiz-Villena. Deparfamento de Inmunologia, Hospital 12 de Octubre, Universidad Complutense, Madrid, Spain Twenty seven Ma@ his&compatibility complex (Mhc)-G exon 2, exon 3 and exon 2+3 allelic sequences were obtained together with 12 different intron 2 sequences. Homo sapiens, Pan tmgl~es, Panpaniscus, Gorilla gorilla, Rmgo pygmaeus, Macaca fasciculatfs, Macaca mulatta and Cercopithecus aetfMps individuals were studied. Polymorphism does not follow the classical pattern of three hypervartable regions perdomain and is found in all species studied; exon 3 (equivalent to ~2 protein domain) shows stop codons in the Cercopithecinae group but not in the Pongidae and human groups. Dendrograms show that cotton top tamarin (Saguinus Oedipus) f&c-G sequences are closer to Homo sapiens and Rxgidae than to Cercopithecinae, probably due to the stop codons existing at exon 3 of the latter. There is a clear bans-species evolution of allelism in Cercopithecihae and also in exon 2 of all the other apes studied, but a generation of allelism within each species may be present on exon 3 sequences; this discrepancy may be due to the preferential use of exon 2 over exon 3 at the mRNA splicing level within each species in order to obtain the appropriate functional G product. Mhc-G intron 2 shows conserved motifs in all species studied, particulaty a 23 bp deletion between positions 161 and 163 which is locus specific, and some of the invariant residues, important for peptide presentation, conserved in classical class I molecules from fish and reptiles to humans were not found in Mhc-G alleles; intron 2 dendrogram also shows a particular pattern of allelism within each species. In summary, Mhc-G has substantial differences with other classical class I genes: polymorphism patterns, tissue distribution, gene structure, splicing variability, and probably an allelism variability within each species at exon 3. The G proteins may also be different. This indicates that Mhc-G function may not be peptide presentation to the clonotypic T-cell receptor.

P.1.03.08

Primate DRB6 gene expression and evolution: A study In Macaca mulatta and Cemoplthecus aethiops

E. Paz-Artal, A. Corell, P. Varela, J. Martinez-Laso, E. Gomez-Casado, VM. Femandez-Soda, M.A. Moreno. A. Amaiz-Villena. Departamento de Inmunologia, Hospital 12 de &tubre, Universidad Compltiens%. Madrid, Spain DRB6 has been found to be transcribed in human and apes. Promoter region and exon 1come from a 5’ LTRfrom a mammary tumour retrovirus. However, the putative protein structure would be very different to other DR molecules and it is doubtful that it may function as an antigen presenting molecule. Primate DRB6 alleles previously published together with the two new macaque sequences reported here support the existence of a strong selective pressure working on exon 2 to generate stop codons at the end of the exon (between codons 74 and 94) during at least 23 million years. The topology of dendrograms constructed with different primate DRB6 alleles supports the ‘trans-species’ evdution proposed for MHC class I, class II and possibly C4 genes. Finally, DRBG, which is one of the oldest DRB genes, has been lost in the HLA-DRB3 (or DR52) group of haplotypes (DR3, DR5, DR6 and DR6) and a small DRB6 sequence is present at the exon 2 first hypervartable region of DRB4 (or DR53) gene, which is present in DR4. DR7 and DR9 haplotypes.