The same MHC recombinational hot spots are active in crossing-over between wild/wild and wild/inbred mouse chromosomes

Four recombinational breakpoints were mapped in the K-A interval of the mouse major histocompatibility complex (MHC) by Southern blot analysis. The breakpoint in B10.SBR, containing a b/s recombinant MHC haplotype, is located about 45 kb upstream of the A ₂gene close to the breakpoint in B10.AQR. Crossover in two cas3/cas4 and one cas4/cas3 recombinant haplotypes has taken place in the previously identified K/A ₃and A ₃/A ₂recombinational hot spots. The same hot spots are thus active in crossover between two Mus musculus castaneus MHC haplotypes and in crossover between a laboratory and a M. m. castaneus MHC haplotype.     

No dosage effect of recombinational hotspots in the mouse major histocompatibility complex

The sites of meiotic recombination in the proximal region of the mouse major histocompatibility complex (MHC) are clustered at hotspots. Some MHC haplotypes derived from Asian wild mice increase the frequency of recombination at such hotspots when heterozygous with standard laboratory haplotypes. The wm7 and cas3 haplotypes, have a hotspot close to the Lmp-2 gene (Lmp-2 hotspot), and the cas4 haplotype has a hotspot about 100 kilobase (kb) proximal, close to the Pb gene (Pb hotspot). To examine the effect of a double dose of hotspots, we estimated the rate of recombination and determined the location of the breakpoints in crosses of wm7/cas3 and wm7/cas4. In 3570 backcross progeny we identified 29 new recombinants in the H-2K to Ab interval, at a frequency of 0.81%. This frequency is 40-fold higher than in crosses between laboratory haplotypes and very similar to those previously obtained in crosses between these wild and standard laboratory haplotypes. Thus, a double dose of hotspots has no additive effect on the frequency of meiotic recombination. The site-specificity of recombination was also conserved. Twenty-three breakpoints were confined within 5.4 kb in the Lmp-2 hotspot, and six breakpoints from the cas4 cross were located in the Pb hotspot, which we have now confined to a 15 kb segment. Correspondence to: T. Shiroishi.     

Genetic control of sex-dependent meiotic recombination in the major histocompatibility complex of the mouse.

Meiotic recombination within the proximal region of the major histocompatibility complex (MHC) of the mouse is not random but occurs in clusters at certain restricted sites, so-called recombinational hotspots. The wm7 haplotype of the MHC, derived from the wild mouse, enhances recombination specifically during female meiosis within a fragment of 1.3 kb of DNA located between the A beta 3 and A beta 2 genes in genetic crosses with laboratory haplotypes. Previous studies revealed no significant strain differences in nucleotide sequences around the hotspot, irrespective of the ability of the strain to enhance the recombination. It appeared that a distant genetic element might, therefore, control the rate of recombination. In the present study, original recombinants whose breakpoints were defined by direct sequencing of PCR-amplified DNAs were tested for the rate of secondary recombination in the crosses with laboratory strains in order to determine the location of such a genetic element. The results clearly demonstrated that the chromosomal segment proximal to the hotspot is essential for enhancement of recombination. Moreover, the male recombination is suppressed by a segment distal to the hotspot.     

Multiple sites of crossing over within the Eb recombinational hotspot in the mouse

The Eb gene of the mouse major histocompatibility complex (MHC) contains a well-documented hotspot of recombination. Twelve cases of intra-Eb recombination derived from the b, d, k and s alleles of the Eb gene were sequenced to more precisely position the sites of meiotic recombination. This analysis was based on positioning recombination breakpoints between nucleotide polymorphisms found in the sequences of parental haplotypes. All twelve cases of recombination mapped within the second intron of the Eb gene. Six of these recombinants, involving the k and s haplotypes, mapped to two adjoining DNA segments of 394 and 955 base pairs (bp) in the 3 half of the intron. In an additional two cases derived by crossing over between the d and s alleles, breakpoints were positioned to adjoining segments of 28 and 433 bp, also in the 3 half of the intron. Finally, four b versus k recombinants were mapped to non-contiguous segments of DNA covering 2.9 kb and 1005 bp of the intron. An analysis of the map positions of crossover breakpoints defined in this study suggests that the second intron of the Eb gene contains a recombinational hotspot of approximately 800–1000 bp which contains at least two closely linked recombinationally active sites or segments. Further examination of the sequence data also suggests that the postulated location for the recombinational hotspot corresponds almost precisely to an 812 bp sequence that shows nucleotide sequence similarity to the MT family of middle repetitive DNA.     

The E beta hot spot of recombination in wild-derived natural recombinant MHC haplotypes. Cross-over site mapping and the identification of a 1.0-kb E beta deletion in the p and w14 haplotypes

Detailed molecular analysis of three wild-derived MHC haplotypes provided evidence for an important role of the E beta recombinational hot spot in the recent evolution of the mouse I region. Examination of RFLP and restriction maps of cloned DNA permitted the mapping of the natural cross-over events in the haplotypes carried by strains B10.GAA37 (w21) and B10.KPB128 (w19) to a fragment of DNA not exceeding 4.1 kb, which lies almost entirely within the intron separating the beta 1 and beta 2 exons of the E beta gene. In the w14 haplotype (strain B10.STC77), which appears to be a natural recombinant between a p-like parental haplotype and another wild-derived haplotype, the site of crossing over can be mapped to a segment between the beta 2 exon of the E beta gene (left border) and the E beta 2 gene (right border). This segment containing the cross-over site in the w14 haplotype includes the E beta hot spot. In addition, the w14 haplotype as well as the standard p haplotype contain a deletion of approximately 1.0 kb in the second intron of the E beta gene, which may represent the product of an unequal cross-over event in a E beta recombinational hot spot.     

Recombinational hotspot specific to female meiosis in the mouse major histocompatibility complex

Thewm7 haplotype of the major histocompatibility complex (MHC), derived from the Japanese wild mouseMus musculus molossinus, enhances recombination specific to female meiosis in theK/A interval of the MHC. We have mapped crossover points of fifteen independent recombinants from genetic crosses of thewm7 and laboratory haplotypes. Most of them were confined to a short segment of approximately 1 kilobase (kb) of DNA between theA 3 andA 2 genes, indicating the presence of a female-specific recombinational hotspot. Its location overlaps with a sex-independent hotspot previously identified in theMus musculus castaneus CAS3 haplotype. We have cloned and sequenced DNA fragments surrounding the hotspot from thewm7 haplotype and the corresponding regions from the hotspot-negative B10.A and C57BL/10 strains. There is no significant difference between the sequences of these three strains, or between these and the published sequences of the CAS3 and C57BL/6 strains. However, a comparison of this A3/A2 hotspot with a previously characterized hotspot in theE gene revealed that they have a very similar molecular organization. Each hotspot consists of two elements, the consensus sequence of the mouse middle repetitive MT family and the tetrameric repeated sequences, which are separated by 1 kb of DNA.The nucleotide sequence data reported in this paper have been submitted to the DNA Data Bank of Japan nucleotide sequence database and have been assigned the accession numbers d90007-9. Offprint requests to: T. Shiroishi.     

Structural and genetic properties of the Eb recombinational hotspot in the mouse

The Eb gene of the mouse contains a recombinational hotspot which plays a predominant role in meiotic crossing-over within the I region of the mouse major histocompatibility complex (MHC). The nucleotide sequences of five recombinants derived from H-2 ^k /H-2 ^b heterozygotes at the Eb locus placed the sites of recombination in each recombinant haplotype within a 2.9 kilobase (kb) segment located fully within the second intron of the Eb gene. Further resolution of the crossover sites was not possible since the nucleotide sequences of the parental and recombinant haplotypes are identical within this segment. The molecular characterization of these five recombinants considered in conjunction with three previously reported intra-Eb recombinants indicates that there are at least two distinct sites of recombination within the Eb recombinational hotspot. In a related study, an examination of the nucleotide sequence of the H-2 ^p allele of the Eb gene revealed a major genetic rearrangement in the 5' half of the intron in this haplotype. A 597 base pair (bp) nucleotide sequence found in the H-2 ^p haplotype is replaced by a 1634 bp segment found in the H-2 ^b and H-2 ^k haplotypes. Sequence analysis of this 1634 bp segment shows strong nucleotide sequence similarity to retroposon long terminal repeat (LTR), env, and pol genes indicating that this segment of the second intron has evolved through retroposon insertion. The location of these retroposon sequences within the 2.9 kb recombination segment defined by the five H-2 ^k /H-2 ^b recombinant haplotypes suggests a possible relationship between these retroviral elements and site-specific recombination within the second intron of the Eb gene. Offprint requests to: H. C. Passmore     

Hotspots of homologous recombination in mouse meiosis

The molecular mapping of recombinational breakpoints in the proximal region of the mouse MHC has revealed four hotspots at which breakpoints are clustered. A direct comparison of the nucleotide sequences of two independent hotspots revealed common molecular elements: a consensus sequence of the middle-repetitive MT-family, a repeat of tetramer sequences and a sequence homologous to a solitary LTR of mouse retroviruses. Extremely high frequency of recombination is observed at these hotspots when particular MHC haplotypes are used in genetic crosses. Wild mouse-derived wm7 haplotype instigates recombination at the hotspot located at the 3′-end of the Lmp-2 gene only during female meiosis. Fine genetic analysis demonstrated that the wm7 haplotype carries a genetic factor to instigate recombination and another factor to suppress recombination specifically during male meiosis. In addition, there is no dose effect of the hotspot on frequency of recombination. Finally, we described an attempt to establish an efficient in vitro assay system for monitoring recombination using plasmid DNAs that contain the Lmp-2 hotspot and nuclear extracts prepared from mouse testis.     

The beta -globin recombinational hotspot reduces the effects of strong selection around HbC, a recently arisen mutation providing resistance to malaria

Recombination is expected to reduce the effect of selection on the extent of linkage disequilibrium (LD), but the impact that recombinational hotspots have on sites linked to selected mutations has not been investigated. We empirically determine chromosomal linkage phase for 5.2 kb spanning the beta -globin gene and hotspot. We estimate that the HbC mutation, which is positively selected because of malaria, originated <5,000 years ago and that selection coefficients are 0.04-0.09. Despite strong selection and the recent origin of the HbC allele, recombination (crossing-over or gene conversion) is observed within 1 kb 5' of the selected site on more than one-third of the HbC chromosomes sampled. The rapid decay in LD upstream of the HbC allele demonstrates the large effect the ss-globin hotspot has in mitigating the effects of positive selection on linked variation.     

Recombinational resolution in primate cells of two homologous human DNA segments with a gradient of sequence divergence.

Human alpha-thalassemia-2 genotype -alpha 4.2 is the result of meiotic recombination between two 1.3 kb long, homologous DNA segments, X(alpha 2) and X(alpha 1), located in the adult alpha globin locus. The two segments can also undergo intramolecular recombination on extrachromosomal vectors transfected into mitotically dividing primate cells (COS 7). The existence of a gradient of sequence divergence between X(alpha 2) and X(alpha 1) makes them an interesting system to study the relationship between efficiencies of homologous DNA recombination and the extent of dispersed and localized base mismatches. By partial restriction mapping and DNA sequencing of plasmids recombined in COS 7 cells and rescued from bacteria HB 101, we have determined the distribution of recombinational resolution sites along the two X blocks. Most, if not all, of the homologous recombination events between the two X blocks appear to be single crossing-over without efficient gene correction or repair of base mismatches. The distribution of the sites of recombinational resolution is inversely correlated with that of the gradient of sequence divergence, with only approximately 7% of the X recombinants resolved within the 3' third of the X blocks where two diverged Alu family repeats reside. The Alu sequence within which one of the X recombinants resolved is homologous to a previously characterized alpha thalassemia deletion point.     

Evidence that nucleotide sequence identity is a requirement for meiotic crossing over within the mouse Eb recombinational hot spot.

Meiotic recombination in the mouse is sometimes restricted to specific chromosomal sites. For example, when recombinants within the I region of the mouse major histocompatibility complex (MHC) are examined, the breakpoints between standard alleles can usually be mapped to the Eb gene. DNA sequence analysis of five cases of meiotic crossing over associated with this gene suggests that the recombinational hot spot may be confined to large regions of nucleotide identity located within the second intron of the Eb gene.     

Dominant Sequences of Human Major Histocompatibility Complex Conserved Extended Haplotypes from HLA-DQA2 to DAXX

We resequenced and phased 27 kb of DNA within 580 kb of the MHC class II region in 158 population chromosomes, most of which were conserved extended haplotypes (CEHs) of European descent or contained their centromeric fragments. We determined the single nucleotide polymorphism and deletion-insertion polymorphism alleles of the dominant sequences from HLA-DQA2 to DAXX for these CEHs. Nine of 13 CEHs remained sufficiently intact to possess a dominant sequence extending at least to DAXX, 230 kb centromeric to HLA-DPB1. We identified the regions centromeric to HLA-DQB1 within which single instances of eight “common” European MHC haplotypes previously sequenced by the MHC Haplotype Project (MHP) were representative of those dominant CEH sequences. Only two MHP haplotypes had a dominant CEH sequence throughout the centromeric and extended class II region and one MHP haplotype did not represent a known European CEH anywhere in the region. We identified the centromeric recombination transition points of other MHP sequences from CEH representation to non-representation. Several CEH pairs or groups shared sequence identity in small blocks but had significantly different (although still conserved for each separate CEH) sequences in surrounding regions. These patterns partly explain strong calculated linkage disequilibrium over only short (tens to hundreds of kilobases) distances in the context of a finite number of observed megabase-length CEHs comprising half a population''s haplotypes. Our results provide a clearer picture of European CEH class II allelic structure and population haplotype architecture, improved regional CEH markers, and raise questions concerning regional recombination hotspots.     

The nucleotide sequence of the murine I-E beta b immune response gene: evidence for gene conversion events in class II genes of the major histocompatibility complex.

We have determined the DNA sequence of the murine I-E beta b immune response gene of the major histocompatibility complex (MHC) of the C57BL/10 mouse and compared it with the sequence of allelic I-E and non-allelic I-A genes from the d and k haplotypes. The polymorphic exon sequences which encode the first extracellular globular domain of the E beta domain show approximately 8% nucleotide substitutions between the E beta b and E beta d alleles compared with only approximately 2% substitutions for the intron sequences. This suggests that an active mechanism such as micro gene conversion events drive the accumulation of these mutations in the polymorphic exons. The fact that several of the nucleotide changes are clustered supports this hypothesis. The E beta b and E beta k genes show approximately 2-fold fewer nucleotide substitutions than the E beta d/E beta b pair. The A beta bm12, a mutant I-A beta b gene from the C57BL/6 mouse, has been shown to result from three nucleotide changes clustered in a short region of the beta 1 domain, which suggests that a micro gene conversion event caused this mutation. We show here that the E beta b gene is identical to the non-allelic A beta bm12 DNA sequence in the mutated region and suggest, therefore, that the E beta b gene was the donor sequence for this intergenic transfer of genetic information. Diversity in class II MHC genes appears therefore to be generated, at least in part, by the same mechanism proposed for class I genes: intergenic transfer of short DNA regions between non-allelic genes.     

High-resolution sperm typing of meiotic recombination in the mouse MHC Ebeta gene

Meiotic crossovers detected by pedigree analysis in the mouse MHC cluster into hotspots. To explore the properties of hotspots, we subjected the class II E(beta) gene to high-resolution sperm crossover analysis. We confirm the presence of a highly localized hotspot 1.0-1.6 kb wide in the second intron of E(beta) and show that it is flanked by DNA which is almost completely recombinationally inert. Mice heterozygous for haplotype s and another MHC haplotype show major haplotype-dependant variation in crossover rate but always the same hotspot, even in crosses including the highly diverged p haplotype. Crossovers in reciprocal orientations occur at similar rates but show different distributions across the hotspot, with the position of centre points in the two orientations shifted on average by 400 bp. This asymmetry results in crossover products showing biased gene conversion in favour of hotspot markers from the non-initiating haplotype, and supports the double-strand break repair model of recombination, with haplotype s as the most efficient crossover initiator. The detailed behaviour of the E(beta) hotspot, including evidence for highly localized recombination initiation, is strikingly similar to human hotspots.     

Haplotypes histories as pathways of recombinations

MOTIVATION: The diversity of a haplotype, represented as a string of polymorphic sites along a DNA sequence, increases exponentially with the number of sites if recombinations are taking place. Reconstructing the history of recombinations compared with that of the polymorphic sites is thus extremely difficult. However, in the human genome, because of the relatively simple pattern of haplotype diversity dominated by a few ancestral haplotypes, the complexity of the recombinational network can be reduced, thus making its reconstruction feasible. We focus on the problem of inferring the recombination pathways starting with putative ancestral haplotypes and leading to new rare recombinant haplotypes. RESULTS: We describe classes of recombinations that represent the whole set of minimal recombination pathways leading to a new haplotype. We present an O(n(2)) algorithm that outputs such representative recombination pathways. We apply it to haplotypes of the 8 kb dystrophin gene segment dys44. AVAILABILITY: A software implementing the algorithm and some other extentions has been developed on a Java platform (JDK 1.3.1). It is freely available at http://www.iro.umontreal.ca/~mabrouk/     

Evidence that nucleotide sequence identity is a requirement for meiotic crossing over within the mouse Eb recombinational hot spot

Meiotic recombination in the mouse is sometimes restricted to specific chromosomal sites. For example, when recombinants within the I region of the mouse major histocompatibility complex (MHC) are examined, the breakpoints between standard alleles can usually be mapped to the Eb gene. DNA sequence analysis of five cases of meiotic crossing over associated with this gene suggests that the recombinational hot spot may be confined to large regions of nucleotide identity located within the second intron of the Eb gene.     

High-resolution mapping and recombination interval analysis of mouse Chromosome 17

Analysis of homologous recombination in eukaryotes has shown that some meiotic crossing-over occurs preferentially at specific genomic sites of limited physical distance called recombinational hotspots. In the mouse, recombinational hotspots have only been defined in the major histocompatibility complex (MHC) on chromosome (Chr) 17. In an attempt to examine whether hotspots are unique to the MHC or are present throughout the genome, high-resolution linkage maps of Chr 17 based on five backcrosses involving different inbred strains have been generated. These maps separate many markers that were previously shown at the same map position and allow a detailed analysis of recombination patterns across Chr 17. Corresponding recombination intervals in these maps have been compared for the identification of intervals with very little or no recombination in certain genetic crosses and considerable recombination in other genetic crosses. This approach has been termed Recombination Interval Analysis. Possible haplotype-dependent non-MHC hotspots, as well as previously identified MHC hotspots, have been detected by interval analysis. Received: 1 December 1997/ Accepted: 27 February 1998     

Hotspots of meiotic recombination in the mouse major histocompatibility complex

Meiotic recombination is not random in the proximal region of the mouse major histocompatibility complex (MHC). It is clustered at four restricted positions, so-called hotspots. Some of the MHC haplotypes derived from Asian wild mice enhance recombination at the hotspots in genetic crosses with standard MHC haplotypes of laboratory mouse strains. In particular, the wm7 haplotype derived from Japanese wild mouse indicated an approximately 2% recombination frequency within a 1.2 kb fragment of DNA in the interval between the Pb and Ob genes. Interestingly, this enhancement of recombination was observed only in female meiosis but not in male meiosis. Mating experiments demonstrated that the wm7 haplotype carries a genetic factor in the region proximal to the hotspot, which instigates recombination. In addition, the wm7 haplotype has a genetic factor located in the region distal to the hotspot, which suppresses recombination. From the molecular characterization of the two hotspots located in the Eb gene and the Pb-Ob interval, it appeared that there are several common molecular elements, the consensus of the middle repetitive MT-family, TCTG or CCTG tetramer repeats, and the solitary long terminal repeat (LTR) of mouse retrovirus.     

Familial clustering of IGHC deletions and duplications: functional and molecular analysis

The human immunoglobulin heavy chain constant region locus (IGHC) comprises nine genes and two pseudogenes clustered in a 350 kilobase (kb) region on chromosome 14q32. Several IGHC haplotypes with single or multiple gene deletions and duplications have been characterized. The most likely mechanism accounting for these unusual haplotypes is the unequal crossing-over between homologous regions within the locus. Here we report the analysis of an unusual case of familial clustering of deletions/duplications. In the two branches of the BON family, three duplicated and two deleted haplotypes, all probably independent in origin, have been characterized. The structure of the haplotypes, one of which is described here for the first time, supports the hypothesis of homologous unequal crossing-over as the origin of recombinant haplotypes. The analysis of serological markers in a subject carrying one deleted and one duplicated haplotype allowed us the first direct inferences concerning the functions of the duplicated IGHC haplotypes.