Meiotic recombination between two polymorphic restriction sites within the beta globin gene cluster.

Analysis of beta globin gene haplotypes for prenatal diagnosis of beta thalassaemia has revealed a recombination event within the beta globin gene cluster. Both a change in the AvaII polymorphic site within the beta globin gene and a change in the phenotype of the beta globin gene were observed. Paternity was established by the pedigree analysis of hypervariable 'minisatellite' DNA polymorphisms and the most probable explanation of the recombination event is a crossover between the psi beta globin gene and the beta globin gene. The data provide direct evidence in support of a DNA region 3' to the beta globin gene with a recombination frequency much higher than expected, and have important implications for the prenatal diagnosis of beta thalassaemia by linked restriction fragment length polymorphisms.     

Meiotic recombination and germ cell aneuploidy

Data on human trisomic conceptuses suggest that the extra chromosome commonly has a maternal origin, and the amount and position of crossing-over on nondisjoined chromosomes is commonly altered. These observations may provide important clues to the etiology of human germ cell aneuploidy, especially in regard to evaluating whether environmental factors play a role. There is concordance of effects of environmental agents on fungi, plants, and animals, which suggests that the overall process of meiosis is well conserved and that chemical and physical agents can affect meiotic recombination, leading to aneuploidy. It seems likely that meiosis in humans will fit the general pattern of meiosis in terms of sensitivity to radiation and chemicals. Thus studies on other organisms provide some insight into the procedures necessary for obtaining useful human data. For example, frequencies of spontaneous meiotic recombination are not uniform per physical length in Drosophila, and different regions of a chromosome respond differently to treatment. Treatments that relieve constraints on the distribution of meiotic exchange, without changing greatly the overall frequency of exchange, may increase the number of univalents and give the impression that there are chromosome- specific responses. Recombination studies that monitor one or a few relatively short genetic regions may also give a false impression of the effects of a treatment on recombination. In addition, meiotic mutants in Saccharomyces and Drosophila highlight a number of processes that are important for production of an exchange event and the utility of that event in the proper segregation of both homologues and sisters. They also suggest that tests for pairing at pachytene, chiasmata at diplotene, and genetic crossing-over may give different results. © 1996 Wiley-Liss, Inc.     

Alignment of recombination sites in Hin-mediated site-specific DNA recombination

The Hin site-specific recombination system normally promotes inversion of DNA between two recombination sites in inverted orientation. We show that the rate of deletion of DNA between two directly repeated recombination sites is 10-300 times slower than inversion between sites in their native configuration as measured in vivo and in vitro, respectively. In vitro studies have shown that the deletion reaction has the same requirement for Fis, a recombinational enhancer, and DNA supercoiling as the inversion reaction. These requirements, together with the finding that the deletion products are interlinked once suggest that the deletion synaptic complex is similar to the invertasome intermediate that generates inversion. The inefficiency of the deletion reaction is not a function of a reduced ability to recognize or synapse recombination sites in direct orientation. Not only do these substrates support an efficient knotting reaction, but directly repeated recombination sites with symmetric core sequences also invert efficiently. These findings demonstrate that the recombination sites are preferentially assembled into the invertasome structure with the sites aligned in the configuration for inversion regardless of their starting orientation. We propose that the dynamics of a supercoiled DNA molecule biases the geometric assembly of specific intermediates. In the case of Hin-mediated recombination, inversion is overwhelmingly preferred over deletion because DNA supercoiling favors a specific alignment of DNA strands in the synaptic complex.     

Meiotic recombination favors the spreading of deleterious mutations in human populations

Although mutations that are detrimental to the fitness of organisms are expected to be rapidly purged from populations by natural selection, some disease-causing mutations are present at high frequencies in human populations. Several nonexclusive hypotheses have been proposed to account for this apparent paradox (high new mutation rate, genetic drift, overdominance, or recent changes in selective pressure). However, the factors ultimately responsible for the presence at high frequency of disease-causing mutations are still contentious. Here we establish the existence of an additional process that contributes to the spreading of deleterious mutations: GC-biased gene conversion (gBGC), a process associated with recombination that tends to favor the transmission of GC-alleles over AT-alleles. We show that the spectrum of amino acid-altering polymorphisms in human populations exhibits the footprints of gBGC. This pattern cannot be explained in terms of selection and is evident with all nonsynonymous mutations, including those predicted to be detrimental to protein structure and function, and those implicated in human genetic disease. We present simulations to illustrate the conditions under which gBGC can extend the persistence time of deleterious mutations in a finite population. These results indicate that gBGC meiotic drive contributes to the spreading of deleterious mutations in human populations.     

The evolutionary turnover of recombination hot spots contributes to speciation in mice

Meiotic recombination is required for the segregation of homologous chromosomes and is essential for fertility. In most mammals, the DNA double-strand breaks (DSBs) that initiate meiotic recombination are directed to a subset of genomic loci (hot spots) by sequence-specific binding of the PRDM9 protein. Rapid evolution of the DNA-binding specificity of PRDM9 and gradual erosion of PRDM9-binding sites by gene conversion will alter the recombination landscape over time. To better understand the evolutionary turnover of recombination hot spots and its consequences, we mapped DSB hot spots in four major subspecies of Mus musculus with different Prdm9 alleles and in their F1 hybrids. We found that hot spot erosion governs the preferential usage of some Prdm9 alleles over others in hybrid mice and increases sequence diversity specifically at hot spots that become active in the hybrids. As crossovers are disfavored at such hot spots, we propose that sequence divergence generated by hot spot turnover may create an impediment for recombination in hybrids, potentially leading to reduced fertility and, eventually, speciation.     

Structural basis for human PRDM9 action at recombination hot spots

The multidomain zinc finger (ZnF) protein PRDM9 (PRD1–BF1–RIZ1 homologous domain-containing 9) is thought to influence the locations of recombination hot spots during meiosis by sequence-specific DNA binding and trimethylation of histone H3 Lys4. The most common variant of human PRDM9, allele A (hPRDM9_A), recognizes the consensus sequence 5′-NCCNCCNTNNCCNCN-3′. We cocrystallized ZnF8–12 of hPRDM9_A with an oligonucleotide representing a known hot spot sequence and report the structure here. ZnF12 was not visible, but ZnF8–11, like other ZnF arrays, follows the right-handed twist of the DNA, with the α helices occupying the major groove. Each α helix makes hydrogen-bond (H-bond) contacts with up to four adjacent bases, most of which are purines of the complementary DNA strand. The consensus C:G base pairs H-bond with conserved His or Arg residues in ZnF8, ZnF9, and ZnF11, and the consensus T:A base pair H-bonds with an Asn that replaces His in ZnF10. Most of the variable base pairs (N) also engage in H bonds with the protein. These interactions appear to compensate to some extent for changes from the consensus sequence, implying an adaptability of PRDM9 to sequence variations. We investigated the binding of various alleles of hPRDM9 to different hot spot sequences. Allele C was found to bind a C-specific hot spot with higher affinity than allele A bound A-specific hot spots, perhaps explaining why the former is dominant in A/C heterozygotes. Allele L13 displayed higher affinity for several A-specific sequences, allele L9/L24 displayed lower affinity, and allele L20 displayed an altered sequence preference. These differences can be rationalized structurally and might contribute to the variation observed in the locations and activities of meiotic recombination hot spots.     

Meiotic recombination generates rich diversity in NK cell receptor genes,alleles, and haplotypes

Natural killer (NK) cells contribute to the essential functions of innate immunity and reproduction. Various genes encode NK cell receptors that recognize the major histocompatibility complex (MHC) Class I molecules expressed by other cells. For primate NK cells, the killer-cell immunoglobulin-like receptors (KIR) are a variable and rapidly evolving family of MHC Class I receptors. Studied here is KIR3DL1/S1, which encodes receptors for highly polymorphic human HLA-A and -B and comprises three ancient allelic lineages that have been preserved by balancing selection throughout human evolution. While the 3DS1 lineage of activating receptors has been conserved, the two 3DL1 lineages of inhibitory receptors were diversified through inter-lineage recombination with each other and with 3DS1. Prominent targets for recombination were D0-domain polymorphisms, which modulate enhancer function, and dimorphism at position 283 in the D2 domain, which influences inhibitory function. In African populations, unequal crossing over between the 3DL1 and 3DL2 genes produced a deleted KIR haplotype in which the telomeric “half” was reduced to a single fusion gene with functional properties distinct from its 3DL1 and 3DL2 parents. Conversely, in Eurasian populations, duplication of the KIR3DL1/S1 locus by unequal crossing over has enabled individuals to carry and express alleles of all three KIR3DL1/S1 lineages. These results demonstrate how meiotic recombination combines with an ancient, preserved diversity to create new KIR phenotypes upon which natural selection acts. A consequence of such recombination is to blur the distinction between alleles and loci in the rapidly evolving human KIR gene family.Among the most polymorphic and structurally diverse human loci are genes related to immune function (Redon et al. 2006; Frazer et al. 2007; Korbel et al. 2007). A principle example is the KIR locus, which displays both polymorphic and structural diversity throughout all human populations (Parham 2005; Bashirova et al. 2006). The protein products, the killer cell immunoglobulin-like receptors (KIR), recognize determinants of conserved and polymorphic major histocompatibility complex (MHC) Class I molecules (Boyington et al. 2001). Interaction of KIR on immune-system cells with MHC Class I on other cell types allows the health of tissues to be monitored and responded to when compromised by infection or malignant transformation. In the human MHC, the HLA complex, each of the highly polymorphic Class I genes—HLA-A, HLA-B, and HLA-C—has some alleles that encode KIR ligands. KIR are principally found on the surface of natural killer (NK) cells, lymphocytes that function in the early, or innate, immune response to virus infection (Lanier 2008), but they also contribute to an early stage of reproduction, when they remodel the maternal blood vessels that will supply the placenta and nourish the fetus (Moffett and Loke 2006). KIR are also expressed on some T-lymphocytes, cells that are central to the adaptive immune response to infection (Snyder et al. 2004; Moretta et al. 2006).In humans and other primates, the KIR are encoded by a diverse and rapidly evolving gene family that exhibits considerable species specificity due to continual gene turnover (Parham 2005; Bashirova et al. 2006). In contrast, in mice, the widely used animal model in immunology, the KIR genes are few in number (two) and do not encode NK cell receptors for MHC Class I, those functions having been assumed by the independently evolved KLRA1 (also known as Ly49) receptors (Kelley et al. 2005). This lability and plasticity in genes encoding NK cell receptors likely reflects the strengths of the different and sometimes conflicting selections imposed by the needs of immune defense and placental reproduction, but also by the functional and genetic complexity of matching polymorphic ligands and receptors encoded by unlinked genes (Parham 2005; Moffett and Loke 2006; Lanier 2008).The KIR locus is part of the leukocyte receptor complex (LRC) on human chromosome 19, which comprises several families of cell-surface receptors expressed by cells of the immune system (Wilson et al. 2000). The KIR genes are flanked on the centromeric side by the leukocyte immunoglobulin-like receptor (LILR) gene family and on the telomeric side by FCAR, the gene encoding the receptor for immunoglobulin A that is expressed on phagocytic cells. Human KIR haplotypes vary in gene content, having between seven and 15 genes (Uhrberg et al. 1997). Each KIR haplotype is divided into two parts by three conserved framework regions. The centromeric part contains KIR2D genes encoding HLA-C receptors, and the telomeric part contains KIR3D genes encoding HLA-A and -B receptors (Bashirova et al. 2006). The latter two genes, comprising KIR3DL1/S1 and KIR3DL2, are the subject of this study. KIR3DL1/S1 recognizes sequence motifs at residues 77–83 of HLA-A and HLA-B allotypes that form the Bw4 antigen, or epitope, defined by HLA serology (Cella et al. 1994; Gumperz et al. 1995; Thananchai et al. 2007), and KIR3DL2 recognizes HLA-A3 and –A11 (Dohring et al. 1996; Pende et al. 1996).Like HLA-A and -B, KIR3DL1/S1 is a highly polymorphic protein with more than 60 allotypes defined (Robinson et al. 2006). The natural variation affects receptor function by altering the frequency of cellular expression, abundance at the cell surface (Pando et al. 2003; Thomas et al. 2008), avidity and specificity for ligand (O''Connor et al. 2007; Thananchai et al. 2007), and the nature—either inhibitory or activating—of the intracellular signals generated upon ligand engagement (Carr et al. 2007). Furthermore, a person''s combination of HLA-A, HLA-B, and KIR3DL1/S1 allotypes influences the development and function of the NK cell repertoire (Foley et al. 2008; Yawata et al. 2008), and in the population, such combinations are associated with disease susceptibility and progression, notably for HIV infection (Martin et al. 2007). The foundation for 3DL1/S1 variety is three ancient lineages of alleles—3DS1 lineage encoding activating receptors and 3DL1-005 and 015 lineages encoding inhibitory receptors—maintained by balancing selection for >3 million years and present in all modern human populations (Norman et al. 2007). Of the three lineages, 3DS1 is essentially homogeneous, whereas both 3DL1 lineages have been extensively diversified by point mutation and recombination. Because recombination with other KIR genes and between KIR3DL1/S1 lineages has the potential to erode the lineage distinctions, we examined the impact that meiotic recombination has had on the KIR3DL1/S1 locus and on human NK cell functional diversity.     

Protein-protein interaction sites are hot spots for disease-associated nonsynonymous SNPs

Many nonsynonymous single nucleotide polymorphisms (nsSNPs) are disease causing due to effects at protein-protein interfaces. We have integrated a database of the three-dimensional (3D) structures of human protein/protein complexes and the humsavar database of nsSNPs. We analyzed the location of nsSNPS in terms of their location in the protein core, at protein-protein interfaces, and on the surface when not at an interface. Disease-causing nsSNPs that do not occur in the protein core are preferentially located at protein-protein interfaces rather than surface noninterface regions when compared to random segregation. The disruption of the protein-protein interaction can be explained by a range of structural effects including the loss of an electrostatic salt bridge, the destabilization due to reduction of the hydrophobic effect, the formation of a steric clash, and the introduction of a proline altering the main-chain conformation.     

The cis-configuration effects of rare chromosomal fragile sites

Rare chromosomal fragile sites may adversely affect specific alleles of closely linked genes when in cis-configuration. This is illustrated by data published in Sutherland & Hecht (1985) on chromosome 16 at band q22.1.     

Mapping recombination sites in the HLA class II region.

Studies of linkage disequilibrium across the HLA class II region have been useful in predicting where recombination is most likely to occur. Strong associations exist within the 100kb region from DQB1 to DRB1, suggesting a low frequency of recombination in this region. Conversely, a lack of association between alleles of TAP1 and TAP2 (about 15kb) has been observed, suggesting that recombination occurs here with relative frequency. Analysis of familial-derived recombinant chromosomes is likely to enrich the current knowledge of recombination across the class II region which has been derived from disequilibrium studies of known class II haplotypes. Families containing recombinant chromosomes within the 700kb interval between the DPB1 and DRB1 genes have been identified for such purposes. Using single-strand conformation polymorphism analysis, 122 novel polymorphic markers distributed throughout the class II region were identified and used to delineate the site of crossover events in 26 class II recombinant chromosomes. The three regions where recombination occurred most frequently are as follows: the 45kb interval between DN-A and RING3 in 10 cases, the 40kb interval between DQB3 and DQB1 in 6 cases, and an 8.8kb segment of the TAP2 gene in 3 cases. The final seven recombinant chromosomes require further resolution, but all of these potentially fall within one of the three intervals mentioned. Two of the three TAP2 recombinant chromosomes have been mapped to overlapping 139bp and 850bp segments of intron 2. Associations between polymorphic markers immediately flanking each of the 3 defined regions using data from unrelated individuals will be compared with the familial recombination data observed in these regions.     

Double-strand break repair by interchromosomal recombination: suppression of chromosomal translocations

To directly determine whether recombinational repair of double-strand breaks (DSBs) can occur between heterologous chromosomes and lead to chromosomal rearrangements in mammalian cells, we employed an ES cell system to analyze recombination between repeats on heterologous chromosomes. We found that recombination is induced at least 1000-fold following the introduction of a DSB in one repeat. Most (98%) recombinants repaired the DSB by gene conversion in which a small amount of sequence information was transferred from the unbroken chromosome onto the broken chromosome. The remaining recombinants transferred a larger amount of information, but still no chromosomal aberrations were apparent. Thus, mammalian cells are capable of searching genome-wide for sequences that are suitable for DSB repair. The lack of crossover events that would have led to translocations supports a model in which recombination is coupled to replication.     

Spontaneous and induced homologous recombination betweenlacZ chromosomal direct repeats in CV-1 cells

AlacZ substrate for intrachromosomal homologous recombination was generated at a specific site within the genome of CV-1 cells by FLP recombinase-mediated gene targeting. A histochemical stain was used to detect cells that contained recombinedlacZ genes. The spontaneous rate of homologous recombination was approximately 1×10^–5 events per cell generation. Recombi nation was induced 30-fold in cells following exposure to mitomycin C (MMC) and by serum starvation. These results demonstrate the utility of the FLP recombinase in modifying the genome of mammalian cells in a predetermined manner and show that homologous recombination between direct repeats is increased in cells as a result of the withdrawal of serum growth factors.     

Meiotic chromosome synapsis and recombination in Arabidopsis thaliana: new ways of integrating cytological and molecular approaches

Meiosis is an evolutionary conserved mechanism that produces haploid gametes and is essential for the sexual reproduction of higher eukaryotes. Since the late nineteenth century, meiosis has been studied in plants due their large chromosomes compared with other organisms and due to advances in microscopy and cytological approaches. On the other hand, non-plant model organisms like budding yeast have been widely used recently in order to characterise the molecular and functional aspects of meiosis. Arabidopsis arose as a new meiotic model for plants during the last decade of the twentieth century. This emergence was sustained by different molecular and genetic advances, mainly by completing the full genome sequence in 2000. Since then, further development of molecular technologies and the cytological methodologies to analyse the meiotic dynamics in Arabidopsis have permitted researchers to establish plant meiosis at the forefront of international research. Some key plant meiotic recombination events have been established in Arabidopsis. These advances have placed researchers into the position to transfer their knowledge from this plant meiotic model to crops and are likely to have an impact on plant breeding and the development of agriculture in future years.     

Meiotic recombination in the beta globin gene cluster causing an error in prenatal diagnosis of beta thalassaemia.

In the course of a prenatal diagnosis for beta thalassaemia by linkage analysis of restriction fragment length polymorphisms, a homozygous beta thalassaemia fetus was misdiagnosed as beta thalassaemia trait. Extensive studies of the polymorphic sites within the beta globin gene cluster in all the members of the family resulted in the conclusion that the paternal chromosome 11 of the newborn was different from that expected. Paternity was confirmed by HLA typing and blood group studies. The analysis of another polymorphic locus on chromosome 11 within the family was in agreement with the possibility of a crossing over between the two paternal chromosomes in a region 5' to the beta gene, previously indicated to contain a 'hot spot' area for recombination. This report underlines the risk of performing prenatal diagnosis using restriction polymorphisms 5' to the beta gene.