The molecular organization of the H-2K region of two t-haplotypes: implications for the evolution of genetic diversity.

The genetic diversity between the t12 and tw5 haplotype chromosomes was studied by analyzing the molecular organization of the H-2K region. Twenty-one cosmid clones spanning over 150 kb of the H-2K region of both t-haplotypes were defined, and high resolution restriction maps were determined. Detailed comparison of the t12 and tw5 restriction maps revealed the following. (i) The H-2K regions of both t-haplotypes retain a very similar molecular organization to that reported for B10, BALB/c and AKR. The nucleotide sequence diversity estimated from restriction site polymorphism is 0.68% between the t12 and tw5 haplotypes; these two t-haplotypes are no more similar to one another than BALB/c is to AKR. (ii) Genetic recombination is strongly implicated in generating H-2 polymorphism. (iii) Genetic polymorphisms, defined as small restriction fragment size differences, are observed at multiple sites along the H-2K region. An Alu-like B2 sequence and BAM5-R homologous sequence were identified as the inserted/deleted DNA segments of two of these sites, suggesting that insertion/deletion of mobile elements is a general mechanism for generating genetic diversity.     

Searching for coding sequences in the mammalian genome: the H-2K region of the mouse MHC is replete with genes expressed in embryos.

We have searched for expressed genes in 170 kb of cosmid cloned DNA from the H-2K region of the mouse MHC. This region is known to contain two genes, H-2K and K2. We identified unique/low copy sequences evenly spaced along the cloned DNA, and used these as probes to search for conserved sequences in Southern blots from a variety of mammalian species. The majority of the unique sequences were found to have homologues and most of these were associated with CpG non-methylated islands. Northern blot analysis and isolation of clones from 5.5 and 10.5-day embryo cDNA libraries showed five additional genes encoded in the H-2K region. Four of these are abundant in embryos; the fifth is exclusively expressed in lymphoid cells. Our data indicate a minimum of seven genes in 170 kb, an unexpectedly high gene density. These results differ from two recent studies where similar lengths of cloned DNA were examined for expressed genes, and only one, or a part of one gene was found. The combined data suggest that the spatial organization of genes in the mammalian genome may not be random.     

Genomic Organization Around the Centromeric End of the HLA Class I Region: Large-Scale Sequence Analysis

We previously sequenced two regions around the centromeric end of HLA class I and the boundary between class I and class III. In this paper we analyze the two regions of about 385 kb and confirm, giving a new line of evidence, that the following two pairs of the genomic segments were duplicated in evolution: (i) a 43-kb genomic segment including the HLA-B gene showing the highest polymorphism among the classical HLA class I loci (class Ia) and a 40-kb segment including the HLA-C locus showing the lowest polymorphism and (ii) a 52-kb segment including the MIC (MHC class I chain related gene) B and a 35-kb segment including MICA. We also found that repetitive elements such as SINEs, LINEs, and LTRs occupy as much as 47% of nucleotides in this 385-kb region. This unusually high content of repetitive elements indicates that repeat-mediated rearrangements have frequently occurred in the evolutionary history of the HLA class Ia region. Analysis of LINE compositions within the two pairs of duplicated segments revealed that (i) LINEs in these regions had been dispersed prior to both the duplication of the HLA-B and -C loci and the duplication of the MICB and MICA loci, and (ii) the divergence of the HLA-B and -C loci occurred prior to the duplication of the MICA and MICB loci. To find novel genes responsible for HLA class I-associated or other diseases, we performed computer analysis applying GenScan and GRAIL to GenBank's dbEST. As a result, at least five as yet uncharacterized genes were newly mapped on the HLA class I centromeric region studied. These novel genes should be analyzed further to determine their relationships to diseases associated with this region. Received: 16 June 1998 / Accepted: 18 August 1998     

Evolutionary dynamics of duplicated microsatellites shared by sex chromosomes

Segmental duplications on sex chromosomes constitute an important proportion of recent duplications (approximately 30%). Among those, the evolution of duplicated noncoding DNA is still poorly investigated. We focus our work on repeated DNA sequences extensively used in population genetics and evolution: microsatellites. Six duplicated (CA), microsatellite loci, located on the homologous region of human sex chromosomes, were studied at the intraspecific level in Homo sapiens and by an orthologous comparison in eight primate species. At the intraspecific level, we evaluated the congruence in paralogous divergence between the flanking sequences of the six microsatellites and the approximately 2.2-kb surrounding sequences and observed that both phylogenies are congruent. At the interspecific level (8 species of primates: 54 individuals), we analyzed the sequence polymorphism and divergence of each orthologous locus for both the flanking sequence and the microsatellite. The results showed a lower divergence of flanking sequences than expected in noncoding DNA and a relative stability of the first nucleotides close to the microsatellite. The location of each CAIII locus in a Low Copy Repeated element containing duplicated VCX/Y genes (approximately 1 kb) suggested that direct or indirect selection could explain these results. Moreover, the substitution rates in the flanking sequences and in the microsatellites were correlated. Thus, the evolutionary dynamics of microsatellites seems closely linked to the variation of spontaneous mutations in the surrounding regions.     

Evidence of gene orthology and trans‐species polymorphism,but not of parallel evolution,despite high levels of concerted evolution in the major histocompatibility complex of flamingo species

The major histocompatibility complex (MHC) is a cornerstone in the study of adaptive genetic diversity. Intriguingly, highly polymorphic MHC sequences are often not more similar within species than between closely related species. Divergent selection of gene duplicates, balancing selection maintaining trans‐species polymorphism (TSP) that predate speciation and parallel evolution of species sharing similar selection pressures can all lead to higher sequence similarity between species. In contrast, high rates of concerted evolution increase sequence similarity of duplicated loci within species. Assessing these evolutionary models remains difficult as relatedness and ecological similarities are often confounded. As sympatric species of flamingos are more distantly related than allopatric species, flamingos represent an ideal model to disentangle these evolutionary models. We characterized MHC Class I exon 3, Class IIB exon 2 and exon 3 of the six extant flamingo species. We found up to six MHC Class I loci and two MHC Class IIB loci. As all six species shared the same number of MHC Class IIB loci, duplication appears to predate flamingo speciation. However, the high rate of concerted evolution has prevented the divergence of duplicated loci. We found high sequence similarity between all species regardless of codon position. The latter is consistent with balancing selection maintaining TSP, as under this mechanism amino acid sites under pathogen‐mediated selection should be characterized by fewer synonymous codons (due to their common ancestry) than under parallel evolution. Overall, balancing selection maintaining TSP appears to result in high MHC similarity between species regardless of species relatedness and geographical distribution.     

Nucleotide polymorphism at the Atmyb2 locus of the wild plant Arabidopsis thaliana

DNA variation was studied in a 2.2 kb region of the regulatory gene Atmyb2 using 20 ecotypes of Arabidopsis thaliana and one accession each of Arabis gemmifera and Arabidopsis himalaica. Nucleotide diversity (pi) in the region was 0.0027, which was lower than for other loci in A. thaliana. The MYB domain of the Atmyb2 gene (pi = 0.0036) had a larger variation than the non-MYB region (pi = 0.0013). Tajima's test and Fu and Li's test did not give a significant result. In contrast to the low level of polymorphism, the degree of divergence of the Atmyb2 region was higher between A. thaliana and A. gemmifera (K = 0.0730) than for other loci. The MYB domain (K = 0.0436) had smaller divergence than the non-MYB region (K = 0.0939). The HKA test detected significant discordance in the ratio of polymorphism to divergence in some comparisons. The pattern of low polymorphism and high divergence, which is mainly observed in the non-MYB region of the gene, is inconsistent with the neutral mutation theory. Strong purifying selection after establishment of A. thaliana and a species-specific adaptive process could be invoked to account for this pattern of polymorphism and divergence of Atmyb2.     

Structural relationships among the H-2 D-regions of murine MHC haplotypes

The number of genes encoding functional Ag-presenting molecules in the D region of the murine MHC differs among haplotypes. For example, the H-2b D region contains a single "D/L" gene, H-2Db, whereas the d-haplotype encodes two, H-2Dd and Ld. Using D/L specific oligonucleotide probes, we have found that, as with H-2d, the q- and v-haplotypes contain two D/L genes, whereas the other haplotype examined have one. Hybridization analysis using cloned probes that map between H-2Dd and Ld revealed similar structures in each of the three haplotypes (d, q, and v) which have "duplicated" D regions. Two approaches were used to examine allelic relationships among the D/L genes. First, the 5' region of the H-2Db gene was sequenced, and found to be more similar to H-2Ld than to H-2Dd. Second, oligonucleotide probes that distinguish H-2Ld from H-2Dd revealed H-2Ld-related genes in several haplotypes, including the duplicated haplotypes H-2q and H-2v. Analogous probes specific for H-2Dd, however, did not detect similar sequences in the other haplotypes. We interpret these results to mean that the three duplicated D regions arose from a common duplication event, and share the five gene structure of the D region cluster defined in H-2d. However, subsequent events have generated sequence divergence at the D-locus.     

New Molecular Markers for the Distal End of the T-Complex and Their Relationships to Mutations Affecting Mouse Development

Many mutations affecting mouse development have been mapped to the t-complex of mouse chromosome 17. We have obtained 17 cosmid clones as molecular markers for this region by screening a hamster-mouse chromosome 17 and 18 cell hybrid cosmid library with mouse-specific repetitive elements and mapping positive clones via t-haplotype vs. C3H restriction fragment length polymorphism (RFLP) analysis. Twelve of the clones mapping distal to Leh66B in t-haplotypes are described here. Using standard RFLP analysis or simple sequence length polymorphism between t-haplotypes, exceptional partial t-haplotypes and nested sets of inter-t-haplotype recombinants, five cosmids have been mapped in or around In(17)3 and seven in the most distal inversion In17(4). More precise mapping of four of the cosmids from In(17)4 shows that they will be useful in the molecular identification of some of the recessive lethals mapped to the t-complex: two cosmids map between H-2K and Crya-1, setting a distal limit in t-haplotypes for the position of the tw5 lethal, one is inseparable from the tw12 lethal, and one maps distal to tf near the t0(t6) lethal and cld.     

Comparative analysis of the bovine MHC class IIb sequence identifies inversion breakpoints and three unexpected genes

The bovine major histocompatibility complex (MHC) or BoLA is organized differently from typical mammalian MHCs in that a large portion of the class II region, called class IIb, has been transposed to a position near the centromere on bovine chromosome 23. Gene mapping indicated that the rearrangement resulted from a single inversion, but the boundaries and gene content of the inverted segment have not been fully determined. Here, we report the genomic sequence of BoLA IIb. Comparative sequence analysis with the human MHC revealed that the proximal inversion breakpoint occurred approximately 2.5 kb from the 3' end of the glutamate-cysteine ligase, catalytic subunit (GCLC) locus and that the distal breakpoint occurred about 2 kb from the 5' end from a divergent class IIDRbeta-like sequence designated DSB. Gene content, order and orientation of BoLA IIb are consistent with the single inversion hypothesis when compared with the corresponding region of the human class II MHC (HLA class II). Differences with HLA include the presence of a single histone H2B gene located between the proteasome subunit, beta type, 9 (PSMB9) and DMB loci and a duplicated TAP2 with a variant splice site. BoLA IIb spans approximately 450 kb DNA, with 20 apparently intact genes and no obvious pseudogenes. The region contains 227 simple sequence repeats (SSRs) and approximately 167 kb of retroviral-related repetitive DNA. Nineteen of the 20 genes identified in silico are supported by bovine EST data indicating that the functional gene content of BoLA IIb has not been diminished because it has been transposed from the remainder of BoLA genes.     

Adaptive evolution at the molecular level of the duplicatedAmy gene system inDrosophila

Analyses of the nucleotide sequences of the duplicatedAmy genes in the eight species of theDrosophila melanogaster species subgroup have revealed concerted evolution of the coding regions and divergent evolution between the duplicated genes of the 5’-flanking regions. Homogenization between the duplicated genes in the coding region is maintained by frequent genetic exchange in various portions of the coding region. On the other hand, such genetic exchange seems to produce a large amount of DNA sequence variation and protein polymorphism at the two loci within a species. The puzzling observation that concerted evolution is restricted to the coding regions seems to be explained by not only adaptive evolution of the AMY proteins in speciation but also adaptive fixation of selectively advantageous mutations in the intergenic region that differentiate expression of the twoAmy genes. We review molecular work on theAmy gene system inDrosophila, including evidence from biochemical characterization of the AMY proteins and molecular characterization of the cis regulatory elements.     

Polymorphism and Linkage of the αa-Crystallin Gene in t-Haplotypes of the Mouse

Restriction fragment polymorphisms were used to order the alpha A-crystallin locus (Crya-1) relative to other genes in mouse t-chromatin and to investigate the relatedness of alpha-A-crystallin sequences among different t-haplotypes. Analysis of DNA from t-recombinant mice mapped Crya-1 to the K end of the H-2 complex and within the distal inverted region characteristic of t-haplotypes. Hybridization with Crya-1 cDNA revealed three distinct phenotypic groups among the 17 different t-haplotypes studied. A majority (9 of 17) of the t-haplotypes were classified into a novel group (Crya-1t) characterized by restriction fragments apparently unique to t-chromosomes and therefore thought to contain alpha A-crystallin sequences descended from the original t-chromosome. A second group of t-haplotypes had restriction fragment patterns indistinguishable from those observed among many common inbred strains of mice of the Crya-1a type, and a third restriction fragment pattern, observed only in the tw121 haplotype, was indistinguishable from the fragment pattern for C3H/DiSn (Crya-1b) and several other inbred strains of mice. Thus, with respect to sequences around the Crya-1 locus, different t-haplotypes show restriction fragment polymorphisms, some of which are comparable to those found in wild-type chromosomes and provide further evidence for genetic heterogeneity in DNA from the distal region of t-haplotypes.     

Contrasting patterns of selection acting on MHC class I and class II DRB genes in the Alpine marmot (Marmota marmota)

The major histocompatibility complex (MHC) genes code for proteins that play a critical role in the immune system response. The MHC genes are among the most polymorphic genes in vertebrates, presumably due to balancing selection. The two MHC classes appear to differ in the rate of evolution, but the reasons for this variation are not well understood. Here, we investigate the level of polymorphism and the evolution of sequences that code for the peptide-binding regions of MHC class I and class II DRB genes in the Alpine marmot (Marmota marmota). We found evidence for four expressed MHC class I loci and two expressed MHC class II loci. MHC genes in marmots were characterized by low polymorphism, as one to eight alleles per putative locus were detected in 38 individuals from three French Alps populations. The generally limited degree of polymorphism, which was more pronounced in class I genes, is likely due to bottleneck the populations undergone. Additionally, gene duplication within each class might have compensated for the loss of polymorphism at particular loci. The two gene classes showed different patterns of evolution. The most polymorphic of the putative loci, Mama-DRB1, showed clear evidence of historical positive selection for amino acid replacements. However, no signal of positive selection was evident in the MHC class I genes. These contrasting patterns of sequence evolution may reflect differences in selection pressures acting on class I and class II genes.     

Colony Specificity in the Colonial Tunicate Botryllus and the Origins of Vertebrate Immunity

SYNOPSIS. Colonies of the compound tunicate Botryllus show thecapacity for self—nonself discrimination by fusion betweenseparated pieces of the same colony and rejection between piecesof unrelated colonies. We have found that genes controllingthis colony specificity are similar to those which cause transplantrejection in the vertebrates. Like the loci within the vertebratemajor histocompatibility complex (MHC), Botryllus fusibility(or histocompatibility) genes are highly polymorphic. In Botryllus,the histocompatibility complex also controls self—sterility,and limits cross—fertilization between colonies sharinghistocompatibility alleles. The mouse MHC, the H-2 region, islinked to loci which also affect the frequencies of allelesat H-2 loci in mouse populations. Thus both systems containcharacters which could act to promote the heterozygous conditionat the linked histocompatibility loci. We suggest that suchlinked characters are responsible for the evolution of allogeneicpolymorphism in vertebrates (however currently maintained),and that tunicate fusibility loci may be the evolutionary precursorsof vertebrate MHC genes.     

Spectrum of MHC class II variability in Darwin's finches and their close relatives

The study describes >400 major histocompatibility complex (MHC) class II B exon 2 and 114 intron 2 sequences of 36 passerine bird species, 13 of which belong to the group of Darwin's finches (DFs) and the remaining 23 to close or more distant relatives of DFs in Central and South America. The data set is analyzed by a combination of judiciously selected statistical methods. The analysis reveals that reliable information concerning MHC organization, including the assignment of sequences to loci, and evolution, as well as the process of species divergence, can be obtained in the absence of genomic sequence data, if the analysis is taken several steps beyond the standard phylogenetic tree construction approach. The main findings of the present study are these: The MHC class II B region of the passerine birds is as elaborate in its organization, divergence, and genetic diversity as the MHC of the eutherian mammals, specifically the primates. Hence, the reported simplicity of the fowl MHC is an oddity. With the help of appropriate markers, the divergence of the MHC genes can be traced deep in the phylogeny of the bird taxa. Transspecies polymorphism is rampant at many of the bird MHC loci. In this respect, the DFs behave as if they were a single, genetically undifferentiated population. There is thus far no indication of alleles that could be considered species, genus, or even DF group specific. The implication of these findings is that DFs are in the midst of adaptive radiations, in which morphological differentiation into species is running ahead of genetic differentiation in genetic systems such as the MHC or the mitochondrial DNA. The radiations are so young that there has not been enough time to sort out polymorphisms at most of the loci among the morphologically differentiating species. These findings parallel those on Lake Victoria haplochromine fishes. Several of the DF MHC allelic lineages can be traced back to the MHC genes of the species Tiaris obscura, which we identified previously as the closest extant relative of DFs in continental America.     

Gene Conversion in the Evolution of Both the H-2 and Qa Class I Genes of the Murine Major Histocompatibility Complex

In order to better understand the role of gene conversion in the evolution of the class I gene family of the major histocompatibility complex (MHC), we have used a computer algorithm to detect clustered sequence similarities among 24 class I DNA sequences from the H-2, Qa, and Tla regions of the murine MHC. Thirty-four statistically significant clusters were detected; individual analysis of the clusters suggested at least 25 past gene conversion or recombination events. These clusters are comparable in size to the conversions observed in the spontaneously occurring H-2K(bm) and H-2K(km2) mutations, and are distributed throughout all exons of the class I gene. Thus, gene conversion does not appear to be restricted to the regions of the class I gene encoding their antigen-presentation function. Moreover, both the highly polymorphic H-2 loci and the relatively monomorphic Qa and Tla loci appear to have participated as donors and recipients in conversion events. If gene conversion is not limited to the highly polymorphic loci of the MHC, then another factor, presumably natural selection, must be responsible for maintaining the observed differences in level of variation.     

Distinct evolutionary trajectories of MHC class I and class II genes in Old World finches and buntings

Major histocompatibility complex (MHC) genes code for key proteins of the adaptive immune system, which present antigens from intra-cellular (MHC class I) and extra-cellular (MHC class II) pathogens. Because of their unprecedented diversity, MHC genes have long been an object of scientific interest, but due to methodological difficulties in genotyping of duplicated loci, our knowledge on the evolution of the MHC across different vertebrate lineages is still limited. Here, we compared the evolution of MHC class I and class II genes in three sister clades of common passerine birds, finches (Fringillinae and Carduelinae) and buntings (Emberizidae) using a uniform methodological (genotyping and data processing) approach and uniform sample sizes. Our analyses revealed contrasting evolutionary trajectories of the two MHC classes. We found a stronger signature of pervasive positive selection and higher allele diversity (allele numbers) at the MHC class I than class II. In contrast, MHC class II genes showed greater allele divergence (in terms of nucleotide diversity) and a much stronger recombination (gene conversion) signal. Gene copy numbers at both MHC class I and class II evolved via fluctuating selection and drift (Brownian Motion evolution), but the evolutionary rate was higher at class I. Our study constitutes one of few existing examples, where evolution of MHC class I and class II genes was directly compared using a multi-species approach. We recommend that re-focusing MHC research from single-species and single-class approaches towards multi-species analyses of both MHC classes can substantially increase our understanding MHC evolution in a broad phylogenetic context.Subject terms: Molecular evolution, Immunogenetics     

Mapping and characterization of the DQ subregion of the ovine MHC

A map of the ovine MHC class II DQ subregion has been constructed from overlapping cosmid clones. This region consists of two loci linked on a linear tract of 130 kb DNA. Each locus consists of a DQA and a DQB gene in a tail-to-tail orientation. The genes in each locus are transcribed but only those designated DQ1 express class II molecules at the surface of mouse L cells following DNA-mediated gene transfection. The DQA1 and DQB1 genes are separated by 11kb while the DQA2 and B2 genes are 25 kb apart. The loci are separated by 22 kb.