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A map of the SLA complex, or swine major histocompatibility complex (MHC), class I region was constructed by alignment of yeast artificial chromosomes (YACs) harboring MHC class I genes as well as anchor genes already mapped within the human MHC complex (HLA). Five YACs containing 9 anchor genes built a contig of about 1.0-1.2 Mb between the SLA class III BAT1 locus and the olfactory receptor-like genes OLF42. Ten different SLA class I sequences, including putative allelic forms of published classical and non-classical SLA class I genes, were assigned to the 400-kb enclosing centromeric part of the contig. Three additional YACs comprising the OLF89 genes and two YACs containing the butyrophilin gene were located telomeric to the contig. Comparison between the human and porcine MHC complexes showed a perfect conserved order of anchor genes, whereas no orthologous relationships were found for the class I loci.

Using a positional cloning strategy to identify the hemochromatosis gene (HFE), we isolated seven cDNAs by cDNA selection from a region of 400 kilobases (kb) located near the HLA-A and HLA-F loci. In this paper, we report the study of one of the corresponding genes, referred to as HCG V (hemochromatosis candidate gene), localized 150 kb centromeric to HLA-A. This gene was found to be expressed ubiquitously in the form of a 1.8 kb transcript, and to be apparently well conserved during evolution. The gene spanned 3.1 kb and is organized in three exons and two introns. The cDNA of 1620 base pairs (bp) showed an open reading frame of 378 bp, encoding for a 126 amino acid polypeptide which displayed a strong identity with the predicted product of a mouse Tctex-5 gene (t complex, testis expressed) localized in the t complex on chromosome 17. The HCG V gene was assessed as a potential candidate for hemochromatosis in regard to its localization in the linkage disequilibrium area between HFE and polymorphic markers. The study of deletions and point mutations in hemochromatosis patients revealed a single bp polymorphism within the coding region; however, no associated disease changes were found. Therefore we conclude that HCG V is unlikely to be involved in the pathogenesis of hemochromatosis.

Human families in which recombinant meiotic event(s) are known to have occurred are powerful tools with which to analyze more precisely the structures of defined genomic regions, especially unstable areas. Such families allow the determination of the haplotypes of each member and, taking into account the recombinant event, it is possible to localize very precisely the point of crossover. Using families in which crossovers between the genes HLA-A and -B have occurred, we have constructed a meiotic map localizing the meiotic breakpoint events with respect to both anonymous markers and the principal genes of the region. Such mapping, which depends on the direct analysis of genomic DNA, is essential for fine structural analysis and is a powerful means of verification of the order and the localization of markers: physical mapping alone, using yeast artificial chromosomes, presents some uncertainties due to the numerous chimeras and inversions that can be produced. The establishment of this map will allow us to determine efficiently the precise location for new markers already localized to the map region. Three microsatellites (D6S265, D6S276, and D6S306), localized in the HLA region by linkage analysis, have been precisely located with respect to the points of recombination in the class I region. The sites of meiotic recombination in the MHC class I region seem to be not randomly distributed but in the majority of cases occurred between HLA-C and the microsatellite D6S265. This study also shows two cases of abnormal segregation of alleles. We discuss how these mutations correspond to a spontaneous mutation event at the somatic or germinal level.

We have refined and extended the map of the distal half of the human major histocompatibility complex. The map is continuous from HLA-E to 1000 kb telomeric of HLA-F and includes six new markers and genes. In addition, the corresponding sequences that were not previously mapped in the mouse genome have been located. The human and the mouse organizations have therefore been compared. This comparison allows us to demonstrate that the structure of the distal part of the MHC is similar in the two species. In addition, this comparison shows the presence of a breakpoint of synteny telomeric of the distal part of the H-2 region. Indeed, the region telomeric of HLA in human is found on a chromosome different from that carrying H-2 in mouse. The mapping analysis of paralogous genes (structurally related genes) around the breakpoint shows that the human organization probably represents the putative human/mouse ancestral one. This evolutionary breakpoint was precisely mapped in human, and the surrounding region was cloned into yeast artificial chromosomes. Finally, we show that the region found around the breakpoint was involved several times in chromosome recombinations in the mouse lineage, as it seems to correspond also to the t-complex distal inversion point.

The P5-1 cDNA clone maps to the human MHC class I region (Vernet et al. 1993a). In this paper, we show that the P5-1 cDNA represents a chimeric transcript in which the first exon of an MHC class I gene has been spliced to an unrelated sequence. The corresponding gene P5-1 is composed of the 5' sequence of an MHC class I gene including the promoter region, the first exon, and the half of the first intron fused to an unrelated intron, followed by a large exon. Furthermore, the non-class I part of P5-1 is present within the MHC class I region in multiple copies, defining the P5 family. Another member of the P5 family is fused to a class I gene, although by a type of rearrangement different from P5-1. These two fusion events between members of HLA class I and P5 families reflect the existence of a duplication unit including two class I genes and a P5 sequence. These data shed light on the MHC class I evolution and on the creation and evolution of new genes.

A gene coding for a putative GTP-binding protein, MMR1, has been localized on band C of the murine Chr 17 within or close to the MHC (Denizot et al. 1992). Its human homolog, HSR1, localized to the human MHC class I region, is described in this paper. Its sequence, compared with MMR1, shows that the conceptual proteins encoded by these genes are highly homologous and have thus been subjected to high constraints during evolution. Furthermore, a detailed databank search with HSR1 leads to the characterization of a new subfamily of GTP-binding proteins, of which HSR1 and MMR1 are the only eukaryotic members. The precise localization of HSR1 within the human MHC class I region is also presented.

Problem: We investigated the possible negative regulatory mechanisms that repress classical human leukocyte antigen (HLA) class I gene expression in human spermatozoa and searched for novel testis-specific coding sequences that might be present in MHC class I chromosomal region., Method: We performed a comparative DNA methylation analysis of this genomic region in both purified human spermatozoa and mononuclear blood cells from the same donors, using methylation-sensitive restriction enzymes followed by classical or pulsed field gel electrophoresis and hybridization with HLA class I locus-specific probes., Results: Unmethylated CpG sites were detected in the 3' part of HpaII tiny fragments of the HLA-F and HLA-G genes in spermatozoal DNA. In contrast, no difference was observed in the methylation status of the HLA-B, HLA-C, and HLA-E genes between germ and somatic cells. CpG unmethylation events were also detected in several parts of this chromosomal region (outside the known loci) in spermatozoal DNA., Conclusions: These results suggest that this genomic region undergoes changes in its DNA methylation pattern during the developmental process. We hypothesize that these dynamic changes have functional importance, including a possible transcriptional activity of nonclassical class I genes and/or as yet undescribed testis-specific coding sequences.

During a search for novel coding sequences within the human MHC class I region (chromosome 6p21.3), we found an exon (named B30-2) coding for a 166-amino-acid peptide which is very similar to the C-terminal domain of several coding sequences: human 52-kD Sjögren's syndrome nuclear antigen A/Ro (SS-A/Ro) and ret finger protein (RFP), Xenopus nuclear factor 7 (XNF7), and bovine butyrophilin. The first three of these proteins share similarities over the whole length of the molecule whereas butyrophilin is similar in the C-terminal domain. The N-terminal domain of butyrophilin is similar to rat myelin/oligodendrocyte glycoprotein (MOG) and chicken B blood group system (B-G) protein. These domains are components of a new subfamily of the immunoglobulin superfamily (IgSF). Butyrophilin is thus a mosaic protein composed of the MOG/B-G Ig-like domain and the C-terminal domain of 52-kD SS-A/Ro, RFP, and XNF7 (B30-2-like domain). Moreover, in situ hybridization shows that RFP, butyrophilin, and MOG map to the human chromosome 6p21.3-6p22 region and are thus close to the MHC class I genes. It is therefore possible that the butyrophilin gene is the product of an exon shuffling event which occurred between ancestors of the RFP and MOG genes. To our knowledge, this is the first example of the colocalization of a chimeric gene and its putative progenitors. Finally, regulatory protein T-lymphocyte 1 (Rpt-1) shares similarities with the N-terminal halves of RFP, 52-kD SS-A/Ro, and XNF7, but not with the B30-2-like domain. We show that the ancestral Rpt-1 gene evolved by overprinting.

Polymorphic as well as HLA-F and -G genes are repressed in the human cell line JAR, derived from a tumor of trophoblast origin. By contrast, the HLA-E gene as well as the non-HLA novel coding-sequence, R1, located 5' to HLA-E, both remain transcriptionally active. We first demonstrated the role of DNA methylation in the repression of class I genes (except HLA-E) in JAR by the use of the 5-Azacytidine demethylating agent. Following treatment, JAR clones reexpressed polymorphic class I transcripts and cell surface alpha chains. Using methylation-sensitive rare cutter enzymes on JAR genomic DNA, followed by classical or pulse field gel electrophoresis and hybridization with HLA locus-specific probes, we found methylated CpG islands in the 5' region of all class I genes, except for HLA-E. These results, establishing an inverse relationship between states of methylation and transcriptional activity within the MHC class I chromosomal region in JAR, and the observations that the HLA-E and R1 genes were ubiquitously expressed, suggest that the HLA-E chromosomal domain might have functional importance including the presence of housekeeping genes.

We report the successful use of whole yeast artificial chromosomes (YACs) as probes for direct positional cloning of novel expressed sequences in a given genomic fragment. The class I region of the human major histocompatibility complex, in particular the chromosomal fragment spanning the HLA-E locus, was investigated. The screening of a cDNA library with a 210-kb-long YAC clone led to the identification of a new gene, positionally conserved in the major histocompatibility complex of the mouse genome and encoding a putative GTP binding protein. Although its precise function remains unknown, the interspecies conservation of both sequence and map position suggests a regulatory or functional link with the histocompatibility cluster.

The class I region of the human major histocompatibility complex contains genes encoding the classical transplantation antigens (HLA-A, B, and C), at least three new class I genes (HLA-E, F, and G) and many class I pseudogenes (including HLA-H). By pulse field gel electrophoresis and using five rare cutter enzymes, we have constructed a precise and continuous map of 1200 kilobases (kb) around HLA-A. The blots were hybridized with HLA-A, E, and F-specific probes and with new probes derived from yeast artificial chromosomes and cosmids of the class I region. We have compared the genomic organization of the same 1200 kb in three homozygous lymphoblastoid cell lines corresponding to three different HLA haplotypes (A3, A24, and A31). The differences in size observed may have been caused by insertions and deletions and may prove valuable in understanding the evolution of the HLA chromosomal region.

The cloning of large genomic fragments corresponding to the major histocompatibility complex (MHC) class I region provides the necessary framework for a better understanding of its organization and for the localization of new genes involved in MHC-associated disease. Two human genomic libraries constructed in yeast artificial chromosomes (YACs) have been prepared using complete Not I or Mlu I digestion of source DNA. From these libraries three YAC clones with inserts belonging to the MHC class I region have been isolated. They correspond to exact copies of three genomic fragments of 210, 145, and 50 kilobases (kb), respectively and have been precisely located in the restriction map of the region. Detailed rare-cutter restriction maps of the inserts have been generated. Within these clones we have demonstrated the presence of two class I genes, one of which is HLA-E, and of at least three Hpa II tiny fragment (HTF) islands, corresponding to three putative new transcribed sequences. End clones, which are of particular interest in the extension and refinement of the regional map, have been rescued by systematic subcloning of purified YACs.

We have investigated the accessibility of the 5' CpG rich sequences (CpG islands) present in the 5' region of most if not all HLA class I genes to methylation sensitive rare cutter enzymes. We show that for HLA-A, -B, -C genes and a few other (but not all) class I sequences these CpG islands are unmethylated and therefore constitute HTF islands (CpG rich, unmethylated regions of DNA, usually associated with expressed genes). We then map precisely the HTF islands of the HLA-B and HLA-C genes and determine that they are separated by 130 Kb (in agreement with genetic data) and that these two genes are in the same transcriptional orientation on the chromosome.

The large-scale organization and polymorphism of the HLA class I region was investigated by pulsed field gel (PFG) fractionation of DNA from various HLA-typed cell lines cleaved by different 'rare cutter' restriction enzymes, followed by hybridization with 'general' and locus-specific HLA probes. Results indicate that (i) most HLA class I sequences are contained in a 340 kb MluI DNA fragment which also carries the HLA-A gene; (ii) HLA-A, -B and -C genes are present on different fragments bounded by 'HTF islands' (CpG-rich, unmethylated DNA regions containing multiple sites for 'rare cutter' enzymes) which generally coincide with the 5' regions of expressed genes; and (iii) very little fragment size polymorphism is seen, implying that expansion/contraction events in the HLA class I region due to unequal crossing over (as documented in the mouse class I system) are infrequently found in the human population.