A continuous restriction map from HLA-E to HLA-F. Structural comparison between different HLA-A haplotypes

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.     

A new major histocompatibility complex class I b gene expressed in the mouse blastocyst and placenta

 Because of the role major histocompatibility complex (MHC) class I b molecules may play during mouse embryonic development, we thought it would be interesting to search for additional MHC class I b molecules that might be expressed in preimplantation embryos, and in particular in the trophoblastic lineage. We therefore screened a mouse preimplantation blastocyst cDNA library for MHC class I sequences. This search led to the identification and characterization of a new MHC class I b gene, blastocyst MHC. Sequences identical to the exons and 3′ untranslated region of this gene have been found in many laboratory mouse strains, as well as in the related mouse species Mus spreciligus. The presence of this gene in mouse strains of different MHC class I haplotypes argues that blastocyst MHC is a unique, newly-described gene rather than a new allele of a previously described mouse MHC class I gene. Blastocyst MHC has the structure of an MHC class I b gene, with the six exons characteristic of T-region genes. It is linked to H2-D. The amino acid sequence encoded by this gene maintains all the features of a functional antigen-presentation domain. The blastocyst MHC gene, like the human class I b gene HLA-G, is expressed at the blastocyst stage and in the placenta, and may be the mouse analog for HLA-G. Received: 31 May 1996 / Revised: 19 August 1996     

Inter-Locus and intea-allelic polymorphisms of HLA class I antigen gene mRNA

We have constructed cDNA clone libraries from two lymphoblastoid cell lines, JY (HLA-A2, B7, C untypeable) and LB (HLA-A28, B40, Cw3), and isolated clones encoding class I HLA antigens. We have characterized short oligonucleotide probes derived from the coding region of the HLA class I antigens which are specific for the HLA-A and -B loci. These probes have been used to subdivide the class I cDNA clones into subclasses. DNA sequencing of several HLA-A and -B related clones has allowed us to extend the primary structural characterization of these cell-surface antigens. This analysis has also detected a sequence polymorphism at the HLA-A locus, indicating that the previously considered homozygous typing cell line LB expresses two alleles of similar, although not identical, serological specificity.     

The distribution of polymorphic Alu insertions within the MHC class I HLA-B7 and HLA-B57 haplotypes

There are five polymorphic Alu insertion (POALIN) loci within the major histocompatibility complex (MHC) class I region that have been strongly associated with HLA class I alleles, such as HLA-A1, HLA-A2 and HLA-B57. In order to assess the variability and frequency of POALIN distribution within two common HLA-B haplotypes, we detected the presence of the MHC class I POALIN by PCR in a panel of 15 individuals with HLA-B57 and 47 homozygous individuals with 7.1 AH (HLA-B7, -Cw7, -A3) obtained from the Australian Bone Marrow Donor Registry, and also from four families (25 individuals) containing the HLA-B57 allele. Only two of the 47 HLA-B7 genotypes had a detectable POALIN, whereas all of the HLA-B57 genotypes had at least one or more POALINs present, confirming that certain MHC class I haplotypes are relatively POALIN-free and others are POALIN-enriched. Six distinct HLA-B57 haplotypes, based on differences at the HLA-A locus and three of five POALIN loci, were identified that appear to have evolved by different mechanisms, including either by shuffling different combinations of conserved alpha and beta blocks or by recombination events involving two or more previously generated HLA-B57 haplotypes.     

Polymorphic SVA retrotransposons at four loci and their association with classical HLA class I alleles in Japanese, Caucasians and African Americans

Polymorphic insertion frequencies of the retrotransposons known as the “SVA” elements were investigated at four loci in the MHC class I genomic region to determine their allele and haplotype frequencies and associations with the HLA-A, -B or -C genes for 100 Japanese, 100 African Americans, 174 Australian Caucasians and 66 reference cell lines obtained from different ethnic groups. The SVA insertions representing different subfamily members varied in frequency between none for SVA-HF in Japanese and 65% for SVA-HB in Caucasians or African Americans with significant differences in frequencies between the three populations at least at three loci. The SVA loci were in Hardy–Weinberg equilibrium except for the SVA-HA locus which deviated significantly in African Americans and Caucasians possibly because of a genomic deletion of this locus in individuals with the HLA-A*24 allele. Strong linkage disequilibria and high percentage associations between the human leucocyte antigen (HLA) class I gene alleles and some of the SVA insertions were detected in all three populations in spite of significant frequency differences for the SVA and HLA class I alleles between the three populations. The highest percentage associations (>86%) were between SVA-HB and HLA-B*08, -B*27, -B*37 to -B*41, -B*52 and -B*53; SVA-HC and HLA-B*07; SVA-HA and HLA-A*03, -A*11 and -A*30; and SVA-HF and HLA-A*03 and HLA-B*47. From pairwise associations in the three populations and the homozygous cell line results, it was possible to deduce the SVA and HLA class I allelic combinations (haplotypes), population differences and the identity by descent of several common HLA-A allelic lineages.     

Allelic repertoire of the humanMHC class IMICA gene

 The hallmark of the classical major histocompatibility complex (MHC) class I molecules is their astonishing level of polymorphism, a characteristic not shared by the nonclassical MHC class I genes. A distinct family of MHC class I genes has been recently identified within the human MHC class I region. The MICA (MHC class I chain-related A) gene in this family is a highly divergent member of the MHC class I family and has a unique pattern of tissue expression. We have sequenced exons encoding the extracellular α1, α2, and α3 domains of the MICA gene from twenty HLA homozygous typing cell lines and four unrelated individuals. We report the identification of eleven new alleles defined by a total of twenty-two amino acid substitutions. Thus, the total number of MICA alleles is sixteen. Interestingly, a tentative superimposition of MICA variable residues on the HLA-A2 structure reveals a unique pattern of distribution, concentrated primarily on the outer edge of the MICA putative antigen binding cleft, apparently bordering an invariant ligand binding site. Received: 13 May 1996 / Revised: 29 May 1996     

Molecular mapping of a new public HLA class I epitope shared by all HLA-B and HLA-C antigens and defined by a monoclonal antibody

It has previously been shown that a mouse monoclonal antibody, designated 4E, reacts with an epitope common to all HLA-B and -C antigens and those of the HLA-Aw19 cross-reactive group, namely, HLA-A29,-A30, -A31, -A32, -Aw33, and -Aw74. In order to pinpoint the amino acid residues which comprise the public specificity recognized by 4E, an HLA-A29 cDNA clone was isolated and its predicted amino acid sequence compared with those of other clonedHLA class I genes. The isolated HLA-A29 cDNA corresponded to the rarer of the twoA29 variant alleles,A29.1. Two amino acid residues of HLA-A29.1, gln-144 and arg-151, were found in all 24HLA-B andHLA-C alleles examined but were present in only one of 15HLA-A alleles for which sequence data are available. Importantly, this exceptional allele wasHLA-A32, another member of the HLA-Aw19 cross-reactive group. Gln-144 and arg-151 should be capable of jointly contributing to the binding site for 4E, as they are situated in successive alpha-helical subregions and are predicted to be juxtaposed in the three-dimensional HLA molecule. Four other residues in the first or second external domains of HLA-A29.1 (thr-9, leu-62, gln-63, and his-102) were unique among theHLA-A alleles, but none of these was found in corresponding positions ofHLA-B or-C alleles and thus failed to correlate with presence or absence of the 4E determinant. These observations are consistent with the notion that gln-144 and arg-151 define a determinant common to HLA-B, HLA-C, and the HLA-Awl9 cross-reactive group and the binding site of the monoclonal antibody 4E.     

Potential regulatory sequences in the untranslated regions of the baboon MHC class Ib gene, <Emphasis Type="Italic">Paan-AG</Emphasis>, more closely resemble those in the human MHC class Ia genes than those in the class Ib gene, <Emphasis Type="Italic">HLA-G</Emphasis>

The baboon major histocompatibility complex (MHC) class Ib gene, Paan-AG, is structurally similar to the human MHC class Ia gene, HLA-A, but exhibits characteristics similar to those of the class Ib gene HLA-G. These include limited polymorphism, alternative splicing of a single message, and restricted tissue distribution, with high expression in the placenta. In order to determine whether regulatory elements controlling expression of Paan-AG resemble those of HLA-A or HLA-G, we cloned the 5 and 3 untranslated regions of Paan-AG. Unexpectedly, sequence comparisons showed that potential regulatory elements in Paan-AG strikingly resembled those in HLA-A and differed in major respects from those in HLA-G. Unlike HLA-G, Paan-AG contained an intact interferon- stimulated response element (ISRE) in the promoter. Studies using luciferase reporter assays showed that the Paan-AG ISRE was functional. The basal activity of the Paan-AG ISRE and its response to interferon- was similar to that of class Ia MHC genes. Further, we identified an ISRE in the 3 untranslated region of Paan-AG that is known to be functional in HLA-A2 but is deleted in HLA-G. These experiments predict that functional studies may demonstrate differences in regulation of expression of Paan-AG and HLA-G genes, which could restrict the use of the baboon as a primate model for studying HLA-G expression and function.The nucleotide sequence data reported in this paper are available in the DDBJ/EMBL/GenBank databases under accession numbers AY434095, AY434096, AY434097 and AY434103.     

Class I gene contraction within the HLA-A subregion of the human MHC.

Individuals expressing either the HLA-A24 or the HLA-A23 histocompatibility antigens have been found to possess an HLA-A class I subregion approximately 50 kb smaller in size than those studied from individuals expressing other HLA-A haplotypes. This originally manifested itself as a haplotype-associated size variation in the NotI and MluI megabase fragments observed on pulsed-field electrophoresis gels after blotting and probing with HLA-A subregion-specific genomic probes. The contracted region falls between the HLA-A and the HLA-G class I genes and specifically includes the novel HLA-A-related pseudogene, HLA-H, as well as the adjacent deteriorated class I pseudogene, 7.0 p. The intactness of locus D6S128, defined by probe pMC6.7 located telomeric to the HLA-H gene, demonstrates that the distal rearrangement point falls within a 20-kb stretch of DNA separating HLA-H from pMC6.7. This extends a previous report regarding variation in class I gene number within the human major histocompatibility complex and precisely localizes the genomic residence of sequences that may define a recombination hot spot. Because the size variation maps to a recombinogenic area, its characterization may ultimately reveal important biological information relevant to the events that shaped the organization of the human HLA class I multigene family.     

Novel detection of restriction fragment length polymorphisms in the human major histocompatibility complex

Cosmid genomic DNA clones have been used as hybridization probes in genomic Southern blot analysis to define restriction fragment length polymorphisms (RFLPs) in the major histocompatibility complex (MHC). Using 14 different enzymes and three overlapping cosmid clones we have detected six RFLPs in a 100 kilobase (kb) segment of DNA in the class III region extending centromeric of theTNFA gene towardHLA-DR. Four of the five RFLPs, defined using the enzymesTaqI,Rsa I,Hinc II, andHind III, and detected by the cosmid clone cosM7B, map to a 29 kb segment of DNA that includes all of the recently described G2 (BAT2) gene and a large portion of the 3 end of the G3 (BAT3) gene. The different RFLP variants were established by analyzing the DNA from three informative families and a panel of 51HLA-homozygous typing cell lines. CosM7B detectsTaq I variants of 4.3 kb, and 2.9 kb or 2.8 kb, Rsa I variants of 2.9 kb or 2.4 kb,Hinc II variants of 5.8 kb or 3.8 kb and 1.4 kb, and aHind III variant of 4.8 kb, while cosOT2 detects Taq I variants of 4.5 kb or 4 kb. The distribution of theRsa 1, Hinc II and Taq I RFLPs detected by cosM7B, and theTaq I RFLP detected with cosOT2, within the panel of cell line DNAs was assessed by Southern blotting. The 4.3 kbTaq I variant was observed in only one cell line with the extended haplotypeHLA-A29, C-, B44, SC30, DR4. The other RFLPs, however, occurred much more frequently. The 2.8 kb Taq I variant was observed in 20 % of haplotypes, the 2.9 kbRsa I variant was observed in 42% of haplotypes, and the 5.8 kbHinc I variant was observed in 12 % of haplotypes analyzed. The 4.5 kbTaq I variant detected by the overlapping cosmid cosOT2 was present in 21 % of haplotypes. Analysis of the RFLP variants with each other revealed seven different haplotypic combinations. Three of the haplotypic combinations were each subdivided into two subsets on the basis of the Nco I RFLP variant they carried at theTNF-B locus. These haplotypic combinations potentially allow differentiation among different extended haplotypes such asHLA-B8, SC01, DR3, HLA-B18, F1 C30, DR3, andHLA-B44, FC31, DR7. The RFLPs detected by the cosmid clones thus provide new tools which will be useful in the further genetic analysis of the MHC class III region.     

Fine localization of a major disease-susceptibility locus for diffuse panbronchiolitis

Diffuse panbronchiolitis affecting East Asians is strongly associated with the class I human leukocyte antigen (HLA) alleles. Recent observations suggest that a major disease-susceptibility gene may be located between the HLA-B and HLA-A loci in the class I region of the major histocompatibility complex on chromosome 6. To test this possibility, we analyzed 14 polymorphic markers in 92 Japanese patients and 93 healthy controls. Of these, seven marker alleles, including HLA-B54 and HLA-A11, were significantly associated with the disease. Maximum-likelihood haplotype analysis and subsequent direct determination of individual haplotypes identified a group of disease-associated haplotypes, one of which contained all seven disease-associated marker alleles. Another haplotype, containing HLA-B*5504, was also associated with the disease. All these haplotypes seem to have diverged from a common ancestral haplotype in East Asians and share a specific segment containing three consecutive markers between the S and TFIIH loci in the class I region. Furthermore, one of the markers within the candidate region showed the highest delta value, indicating the strongest association. Of 20 Korean patients with diffuse panbronchiolitis, 17 also shared the combination of the disease-associated marker alleles within the candidate region. These results indicate that an HLA-associated major susceptibility gene for diffuse panbronchiolitis is probably located within the 200 kb in the class I region 300 kb telomeric of the HLA-B locus on the chromosome 6p21.3.     

<Emphasis Type="Italic">HLA-A</Emphasis> allele associations with viral <Emphasis Type="Italic">MER9-LTR</Emphasis> nucleotide sequences at two distinct loci within the <Emphasis Type="Italic">MHC alpha</Emphasis> block

The study of the association of the Human Leukocyte Antigen (HLA) alleles and polymorphic retrotransposons such as Alu, HERV, and LTR at various loci within the Major Histocompatibility Complex allows for a better identification and stratification of disease associations and the origins of HLA haplotypes in different populations. This paper provides sequence and association data on two structurally polymorphic MER9-LTR retrotransposons that are located 54 kb apart and in close proximity to the multiallelic HLA-A gene involved in the regulation of the human immune system. Direct DNA sequencing and analysis of the PCR products identified DNA nucleotide variations between the MER9-LTR sequences at the two loci and their associations with HLA-A alleles as potential haplotype and evolutionary markers. All MER9-LTR sequences were haplotypic when associated with common HLA-A alleles. The number of SNP loci was 2.5 times greater for the solo LTR at the AK locus, which is located closer to the HLA-A gene than the solo or 3′ LTR at the HG locus. Our study shows that the nucleotide variations of the MER9-LTR DNA sequences are additional informative markers in fine mapping HLA-A genomic haplotypes for future population, evolutionary, and disease studies.     

Heterogeneity of HLA-G Genes Identified by Polymerase Chain Reaction/Single Strand Conformational Polymorphism (PCR/SSCP)

A genomic HLA-G clone named 7.0E was isolated from a Japanese placenta. The deduced amino acid sequence of the 7.0E was identical to two HLA-G genomic clones and two cDNA clones previously described. The DNA sequences of α1 and α2 domains of the HLA-G gene from 5 cell lines also encoded the same amino acids. However, a 14 bp insertion, ATTTGTTCATGCCT, was present in the 3′ untranslated region of 7.0E compared with the originally described HLA-G clone (HLA 6.0). Polymerase chain reaction (PCR)/single strand conformational polymorphism (SSCP) analysis of exon 8 allowed the HLA-G gene to be classified into two alternative types, G6.0 and 7.0 E, those correlated to the absence or the presence of the 14 bp stretch. Each group had minor sequence variant(s), and the alleles of the 7.0E-type were more heterogeneous than those of the G6.0- type. The 14 bp deletion is present only in the G6.0-type of HLA-G alleles among HLA class I genes. Thus it was suggested that G6.0 alleles were generated after diversification of the HLA-G.     

Nucleotide sequencing analysis of the swine 433-kb genomic segment located between the non-classical and classical<Emphasis Type="Italic"> SLA</Emphasis> class I gene clusters

Genome analysis of the swine leukocyte antigen (SLA) region is needed to obtain information on the MHC genomic sequence similarities and differences between the swine and human, given the possible use of swine organs for xenotransplantation. Here, the genomic sequences of a 433-kb segment located between the non-classical and classical SLA class I gene clusters were determined and analyzed for gene organization and contents of repetitive sequences. The genomic organization and diversity of this swine non-class I gene region was compared with the orthologous region of the human leukocyte antigen (HLA) complex. The length of the fully sequenced SLA genomic segment was 433 kb compared with 595 kb in the corresponding HLA class I region. This 162-kb difference in size between the swine and human genomic segments can be explained by indel activity, and the greater variety and density of repetitive sequences within the human MHC. Twenty-one swine genes with strong sequence similarity to the corresponding human genes were identified, with the gene order from the centromere to telomere of HCR - SPR1 - SEEK1 - CDSN - STG - DPCR1 - KIAA1885 - TFIIH - DDR - IER3 - FLOT1 - TUBB - KIAA0170 - NRM - KIAA1949 - DDX16 - FLJ13158 - MRPS18B - FB19 - ABCFI - CAT56. The human SEEK1 and DPCR1 genes are pseudogenes in swine. We conclude that the swine non-class I gene region that we have sequenced is highly conserved and therefore homologous to the corresponding region located between the HLA-C and HLA-E genes in the human.The nucleotide sequence data reported in this paper have been submitted to DDBJ, EMBL and GenBank databases under accession numbers AB113354, AB113355, AB113356, AB113357     

Molecular characterization of major histocompatibility complex class 1 (MHC-I) from squirrel monkeys (<Emphasis Type="Italic">Saimiri sciureus</Emphasis>)

Little is known about the major histocompatibility complex (MHC) class 1 in squirrel monkeys (Saimiri sciureus). We cloned, sequenced and characterized two alleles and the cDNA of the coding region of MHC class 1 in these New World monkeys. Phylogenetic analyses showed that these sequences are related to HLA class 1 genes (HLA-A and HLA-G). The structure and organization of one of the two identified clones was similar to that of a class 1 MHC gene (HLA-A2). All the exon/intron splice acceptor/donor sites are conserved and their locations correspond to the HLA-A2 gene. The sequences of the newly described cDNAs reveal that they code for the characteristic class 1 MHC proteins, with all the features thought necessary for cell surface expression. Typical sequences for the leader peptide, ₁, ₂, ₃, transmembrane and cytoplasmic domains were found.The nucleotide sequence data reported in this paper have been submitted to the GenBank database and have been assigned the accession numbers AJ438576 (Sasc-G*31), AJ438577 (Sasc-G*25), AY282760 (Sasc-G*03), AY282761 (Sasc-G*04) and AY282762 (Sasc-G*05). Sequences were named as recommended by Klein and co-workers (1990)     

Anonymous Marker Loci within 400 kb of HLA-A Generate Haplotypes in Linkage Disequilibrium with the Hemochromatosis Gene (HFE)

The hemochromatosis gene (HFE) maps to 6p21.3 and is less than 1 cM from the HLA class I genes; however, the precise physical location of the gene has remained elusive and controversial. The unambiguous identification of a crossover event within hemochromatosis families is very difficult; it is particularly hampered by the variability of the phenotypic expression as well as by the sex- and age-related penetrance of the disease. For these practical considerations, traditional linkage analysis could prove of limited value in further refining the extrapolated physical position of HFE. We therefore embarked upon a linkage-disequilibrium analysis of HFE and normal chromosomes from the Brittany population. In the present report, 66 hemochromatosis families yielding 151 hemochromatosis chromosomes and 182 normal chromosomes were RFLP-typed with a battery of probes, including two newly derived polymorphic markers from the 6.7 and HLA-F loci located 150 and 250 kb telomeric to HLA-A, respectively. The results suggest a strong peak of existing linkage disequilibrium focused within the i82-to-6.7 interval (approximately 250 kb). The zone of linkage disequilibrium is flanked by the i97 locus, positioned 30 kb proximal to i82, and the HLA-F gene, found 250 kb distal to HLA-A, markers of which display no significant association with HFE. These data support the possibility that HFE resides within the 400-kb expanse of DNA between i97 and HLA-F. Alternatively, the very tight association of HLA-A3 and allele 1 of the 6.7 locus, both of which are comprised by the major ancestral or founder HFE haplotype in Brittany, supports the possibility that the disease gene may reside immediately telomeric to the 6.7 locus within the linkage-disequilibrium zone. Additionally, hemochromatosis haplotypes possessing HLA-A11 and the low-frequency HLA-F polymorphism (allele 2) are supportive of a separate founder chromosome containing a second, independently arising mutant allele. Overall, the establishment of a likely “hemochromatosis critical region” centromeric boundary and the identification of a linkage-disequilibrium zone both significantly contribute to a reduction in the amount of DNA required to be searched for novel coding sequences constituting the HFE defect.     

Functional characterization of a chicken major histocompatibility complex class II B gene promoter

 A 0.7 kilobase (kb) DNA fragment from the 5′ flanking region of a chicken major histocompatibility complex (MHC) class II B gene was cloned into chloramphenicol acetyltransferase (CAT) reporter vectors and was transfected into a chicken macrophage cell line that expresses a low level of MHC class II antigens. Positive orientation-dependent promoter activity of the chicken DNA was evident in a reporter construct containing an SV40 enhancer. Deletion analysis of this 0.7 kb DNA fragment revealed a short fragment in the 3′ end that was crucial for the promoter function and negative regulatory elements (NRE) located further upstream. The conserved MHC class II X and Y boxes did not have a significant effect on promoter activity. Sequence analysis of the 0.7 kb class II B gene upstream region suggests possible involvement of interferon (IFN), E twenty-six specific (ETS)-related proteins, and other factors in regulating this promoter. A chicken T-cell line culture supernatant increased surface expression of MHC class II antigens, as well as class II promoter activity, in this macrophage cell line. This first functional characterization of a chicken MHC class II B gene promoter will aid in understanding the regulatory mechanisms that control the expression of these genes. Received: 9 July 1996 / Revised: 7 October 1996     

中国壮族和裕固族群体HLA Ⅰ类区域Alu插入多态性及其与HLA-A位点的相关性

近年来研究发现:位于HLAⅠ类基因区域的Alu插入是研究不同群体HLAⅠ类基因区域祖先单倍型和HLAⅠ类基因多样性产生、进化和重组的理想工具。文章对中国壮族和裕固族群体HLAⅠ类基因区域5个Alu插入多态性(AluMICB、AluTF、AluHJ、AluHG和AluHF)进行研究,结合HLA基因分型数据,分析壮族、裕固族、哈尼族、布朗族和傣族5个民族群体中Alu插入与HLA-A等位基因的关系。研究结果显示:(1)壮族和裕固族人群中5个Alu插入频率范围分别为1.5%~35.8%和9.2~34.8%,AluMICB、AluTF和AluHF插入频率在这两个群体中有统计学差异(P<0.05);(2)在5个研究的群体中,AluHG插入与HLA-A*02的不同亚型关联;AluHJ插入与HLA-A*2402在5个群体中都关联,但AluHJ与HLA-A*1101和HLA-A*2407只在布朗族中关联。表明不同群体HLAⅠ类基因区域内Alu插入具有各自的特征,且Alu插入与不同的HLA-A等位基因相关联。这种Alu插入及其与HLA-A的关联特征可作为研究群体中HLAⅠ类基因和单倍型系谱变化的重要遗传标记。     

The HLA-AW24 gene: Sequence,surroundings and comparison with the HLA-A2 and HLA-A3 genes

A cosmid clone containing two class I sequences was found to cause expression of the HLA-AW24 protein after transfection into mouse L cells. The restriction map of this cosmid shows extensive homology over 26 kb with the map of the HLA-A3 region obtained from cosmids of the same library, constructed with DNA from an HLA-A3/HLA-AW24 heterozygote, but diverges over the remaining 14 kb. The HLA-AW24 gene was subcloned from this cosmid and its nucleotide sequence was determined. Amino acid and, more strikingly, nucleotide sequence comparisons with other HLA alleles indicate that the A locus alleles are more closely related to each other than to alleles from other HLA loci. A very skewed distribution of silent substitutions is apparent, and the occurrence of clustered multiple substitutions hints at gene-conversion-like events.     

Frequent segmental sequence exchanges and rapid gene duplication characterize the<Emphasis Type="Italic"> MHC</Emphasis> class I genes in lemurs

Major histocompatibility complex (MHC) class I genes have complicated and profound evolutionary histories. To reconstruct and better understand their histories, partial class I genes (exon 2–intron 2–exon 3) were sequenced in a sampling of prosimians (Strepsirhini, Primates). In total, we detected 117 different sequences from 36 Malagasy prosimians (lemurs) and 1 non-Malagasy prosimian (galago) representing 4 families, 7 genera, and 13 species. Unlike the MHC class II genes (MHC-DRB), MHC class I genes show a generally genus-specific mode of evolution in lemurs. Additionally, no prosimian class I loci were found to be orthologous to HLA genes, even at highly conserved loci (such as HLA-E, HLA-F). Phylogenetic analysis indicates that nucleotide diversity among loci was very small and the persistence time of the polymorphisms was short, suggesting that the origin of the lemur MHC class I genes detected in this study was relatively recent. The evolutionary mode of these genes is similar to that of classic HLA genes, HLA-A, HLA-B, and HLA-C, in terms of their recent origin and rarity of pseudogenes, and differs from them with respect to the degree of gene duplications. From the viewpoint of MHC genes evolution, some interlocus sequence exchanges were apparently observed in the lemur lineage upon phylogenetic and amino acid motif analyses. This is also in contrast to the evolutionary mode of HLA genes, where intralocus exchanges have certainly occurred but few interlocus exchanges have taken place. Consequently, the gene conversion model for explaining the generation of the MHC diversity among different loci can be thought to play more important roles in the evolution of lemur MHC class I genes than in that of HLA genes.Electronic Supplementary Material Supplementary material is available in the online version of this article at