通过染色体步查建立水稻G200座位的重叠群

分别以G200及其旁侧区的Y2587L、Y4073L和L688片段为探针筛选水稻基因组YAC文库,共得到4个阳性克隆,均与G200、Y2587L和Y4073L座位重叠,插入片段的大小为240～650kb。用反向PCR法分离YAC克隆插入片段的末端序列,并利用这些末端序列确定克隆的方向以及进行染色体步查,共筛选到7个YAC克隆,建立了一个8厘摩(cM)的G200 YAC重叠群。     

Analysis of the occurrence and nature of repeated DNA in an 850 kb region of Arabidopsis thaliana chromosome 4

The occurrence and nature of repeated DNA sequences has been analysed within an 850 kb YAC contig on Arabidopsis thaliana chromosome 4. Hybridization analysis with seven RFLP markers, six cosmid contigs, 29 YAC end probes and eight YAC clones showed that a least 585 kb of the 850 kb contained only low-copy sequences. One YAC end probe, EG15C8LE, hybridized to multiple genomic fragments and contained a sequence with predicted protein homology to cytochrome P450 monooxygenases. Another one, EG11B7RE, was found to be non-contiguous with the other YAC clones and contained a dispersed repetitive sequence associated with centromeric regions     

Detailed description of four YAC contigs representing 17 Mb of chromosome 4 of Arabidopsis thaliana ecotype Columbia

The detailed arrangement of 563 YAC clones comprising four contigs covering ～17 Mbp of chromosome 4 is presented. YAC clones were positioned relative to each other and to markers by taking into account marker and end fragment hybridization data and the sizes of all YAC clones. This analysis made it possible to estimate physical distances between the majority of chromosome 4 markers. It also identified a relatively large number of YAC clones containing chimaeric inserts. The YAC contig map of the Columbia ecotype presents an important resource for map-based cloning experiments, rapid mapping of DNA sequences and large-scale genomic sequencing programs.     

Characterization of a YAC Contig Spanning the Pseudoautosomal Region

Due to its unique biology of partial sex linkage and high recombination rates, the pseudoautosomal region (PAR1) on both X and Y chromosomes has attracted considerable interest. In addition, an extremely high level of YAC instability has been observed in this region. We have derived 82 YAC clones from six different YAC libraries mapping to this 2.6-Mb region. Of these a subset of 22 YACs was analyzed in detail. YAC contigs were assembled using 67 pseudoautosomal probes, of which 64 were unambiguously ordered. All markers are well distributed over the entire region, including the middle part of the region, which has previously been found difficult to contig. Two gaps of less than 50 kb within the genomic locus of CSF2RA and around XE7 remain, which could not be covered with YACs, cosmids, or phages. This YAC contig anchored on the physical map of PAR1 represents one of the best characterized large regions of the human genome with a map completion greater than 90% at 100-kb resolution and has permitted the accurate localization of all known genes within this region.     

Description of 31 YAC contigs spanning the majority of Arabidopsis thaliana chromosome 5

In order to generate a physical map of Arabidopsis thaliana chromosome 5, 142 molecular markers mapping to chromosome 5 have been used in colony hybridization experiments with four Arabidopsis, ecotype Columbia, yeast artificial chromosome (YAC) libraries. This resulted in 634 YAC clones being anchored on chromosome 5. Southern blot analysis confirmed their positioning and provided data, which along with knowledge of the sizes of all the YAC clones, enabled the clones to be arranged into 31 contigs. Genetic mapping of markers located within 29 of these contigs on the Landsberg erecta/Columbia recombinant inbred lines allowed positioning of the contigs along the chromosome. A high proportion of the YAC clones were found to contain chimaeric inserts. The availability of this YAC contig map will accelerate chromosome-walking experiments, provide substrates for large-scale genomic sequencing projects and facilitate the mapping of new probes to this chromosome.     

Molecular linkage of the HLA-DR, HLA-DQ, and HLA-DO genes in yeast artificial chromosomes.

Eight major histocompatibility complex (MHC) class II loci and the newly defined Y3/Ring 4 locus were isolated in overlapping yeast artificial chromosome (YAC) clones defining a 420-kb segment of human chromosome 6p21.3. YAC B1D12 spanning 320 kb contained seven of these loci from HLA-DRA to HLA-DQB2. A 330-kb YAC, A148A7, spanned from the HLA-DQA1 locus through the Y3/Ring 4 locus and extended at least 130 kb centromeric of YAC B1D12. Southern blotting demonstrated that YAC B1D12 derived from the HLA-DR3 haplotype and that YAC A148A7 derived from the HLA-DR7 haplotype of the heterozygous library donor. A third 150-kb YAC, A95C5, lay within this contig and contained only the HLA-DRA locus. A fourth 300-kb YAC, A76F11, was isolated by chromosome walking from the telomeric end of YAC B1D12. Probes isolated from the ends of the YAC genomic inserts have been used to confirm overlaps between the clones. These analyses demonstrated that the centromeric end of YAC A76F11 used the same genomic EcoRI cloning site as the telomeric end of YAC A95C5. YAC B1D12 used an EcoRI site only 2.1 kb telomeric of the aforementioned EcoRI site. These data suggest that certain EcoRI sites are used preferentially during construction of the library. These YACs complete the linkage of the DR and DQ subregions of the HLA complex in cloned DNA and provide the substrate for precise analysis of this portion of the class II region.     

Localization on a physical map of the NKC-linked Cmv1 locus between Ly49b and the Prp gene cluster on mouse chromosome 6.

The Cmv1 locus controls NK cell-mediated resistance to infection with murine CMV. Our recent genetic analysis of backcross mice demonstrated that the NK gene complex (NKC)-linked Cmv1 locus should reside between the Ly49 and Prp gene clusters on distal mouse chromosome 6. We have aligned yeast artificial chromosome (YAC) inserts in a contig spanning the interval between the Ly49 and Prp gene clusters. This YAC contig includes 13 overlapping YAC inserts that span more than 2 megabases (Mb) in C57BL/6 (B6) mice. Since we have identified genomic clones that span the Ly49-Prp gene region, we hypothesize that at least one should contain the Cmv1 locus. To narrow the Cmv1 critical region, we developed novel NKC genetic markers and used these to genotype informative backcross and intra-NKC recombinant congenic mouse DNA samples. These data suggest that Cmv1 resides on a single YAC insert within an interval that corresponds to a physical distance of approximately 390 kb. This high resolution, integrated physical and genetic NKC map will facilitate identification of Cmv1 and other NKC-linked loci that regulate NK cell-mediated immunity.     

A yeast artificial chromosome contig containing the complete Duchenne muscular dystrophy gene.

A contig of 36 overlapping yeast artificial chromosome (YAC) clones has been constructed for the complete Duchenne muscular dystrophy (DMD) gene in Xp21. The YACs were isolated from a human 48,XXXX YAC library using the DMD cDNA and brain promoter fragments as hybridization probes. The YAC clones were characterized for exon content using HindIII or EcoRI digests, hybridization of individual DMD cDNA probes, and polymerase chain reaction (PCR) amplification of specific exons near the 5' end of the gene. For comparison to the known long-range restriction map of the DMD gene, YAC clones were digested with SfiI and hybridized with DMD cDNA probes. The combined analysis of the exon content and the SfiI map allowed an approximately 3.2-Mb YAC contig to be constructed. The complete 2.4-Mb DMD gene could be represented in a minimum set of 7 overlapping YAC clones.     

High-resolution, human–bovine comparative mapping based on a closed YAC contig spanning the bovine mh locus

A closed YAC contig spanning the mh locus was assembled by STS content mapping with seven microsatellite markers, eight genes or EST, and nine STS corresponding to YAC ends. The contig comprises 27 YACs, has an average depth of 4.3 YACs, and spans an estimated 1.2 Mb. A linkage map was constructed based on five of the microsatellite markers anchored to the contig and shown to span 7 cM, yielding a ratio of 160 kb/1 cM for the corresponding chromosome region. Comparative mapping data indicate that the constructed contig spans an evolutionary breakpoint connecting two chromosome segments that are syntenic but not adjacent in the human. Consolidation of human gene order by means of whole genome radiation hybrids and its comparison with the bovine order as inferred from the contig confirm conservation of gene order within segments. Received: 6 August 1998 / Accepted: 28 October 1998     

The Mla (powdery mildew) resistance cluster is associated with three NBS-LRR gene families and suppressed recombination within a 240-kb DNA interval on chromosome 5S (1HS) of barley

Powdery mildew of barley, caused by Erysiphe graminis f. sp. hordei, is a model system for investigating the mechanism of gene-for-gene interaction between large-genome cereals and obligate-fungal pathogens. A large number of loci that confer resistance to this disease are located on the short arm of chromosome 5(1H). The Mla resistance-gene cluster is positioned near the telomeric end of this chromosome arm. AFLP-, RAPD-, and RFLP-derived markers were used to saturate the Mla region in a high-resolution recombinant population segregating for the (Mla6 + Mla14) and (Mla13 + Ml-Ru3) resistance specificities. These tightly linked genetic markers were used to identify and develop a physical contig of YAC and BAC clones spanning the Mla cluster. Three distinct NBS-LRR resistance-gene homologue (RGH) families were revealed via computational analysis of low-pass and BAC-end sequence data derived from Mla-spanning clones. Genetic and physical mapping delimited the Mla-associated, NBS-LRR gene families to a 240-kb interval. Recombination within the RGH families was at least 10-fold less frequent than between markers directly adjacent to the Mla cluster.     

A YAC contig encompassing the recessive Stargardt disease gene (STGD) on chromosome 1p.

Stargardt disease (STGD) and fundus flavimaculatus are infrequent autosomal recessive conditions characterized by a juvenile macular dystrophy and variable degrees of peripheral retinal changes. Linkage analysis performed in 47 STGD/fundus flavimaculatus families demonstrated significant linkage to 13 polymorphic DNA markers on chromosome 1p. The maximum combined two-point lod score was 32.7 (maximum recombination fraction [phi max] = .006) with the polymorphic marker D1S188. Our data demonstrate that STGD and fundus flavimaculatus are the same disorder clinically and genetically and provide further evidence for genetic homogeneity of this phenotype. Analysis of recombination events on disease chromosomes placed the STGD gene within a 4-cM interval between markers D1S435 and D1S236. A physical map was constructed of a YAC contig flanking STGD, from markers D1S500 to D1S495, and includes the critical interval delineated by historical recombinants. This contig spans approximately 31 cM, with one gap (3-5 cM) that is outside the 4-cM critical region. Localization of STGD to a single YAC contig will facilitate its positional cloning.     

人Xp11.2区4.3MbYAC重叠群：大尺度限制图与CpG岛分析

人Ｘｐ１１．２区域具有重要的医学遗传学和基础遗传学价值,它包含很多遗传疾病基因,且至少包含一个逃避Ｘ染色体失活的位点,非常规的基化多态也有发现。我们利用这一区域已知的一系列ＤＮＡ位标,从我们构建的ＹＡＣ库中筛选出一系列ＹＡＣ克隆。     

Chromosome landing at the tomato Bs4 locus

The tomato (Lycopersicon esculentum) Bs4 gene confers resistance to strains of Xanthomonas campestris pathovar vesicatoria that express the avirulence protein AvrBs4. As part of a map-based cloning strategy for the isolation of Bs4, we converted Bs4-linked amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) markers into locus-specific sequence-tagged-site (STS) markers. The use of these markers for the analysis of 1972 meiotic events allowed high-resolution genetic mapping within a 1.2-cM interval containing the target gene. Two tomato yeast artificial chromosome (YAC) clones, each harboring inserts of approximately 250 kb, were identified using the marker most closely linked to Bs4. YAC end-specific markers were established and employed to construct a local YAC contig. The ratio of physical to genetic distance at Bs4 was calculated to be 280 kb/cM, revealing that recombination rates in this region are about three times higher than the genome-wide average. Mapping of YAC end-derived markers demonstrated that the Bs4 locus maps within a region of 250 kb, corresponding to a genetic interval of 0.9 cM.     

Construction of a 1.5-Mb Bacterial Artificial Chromosome Contig in Xp11.23, a Region of High Gene Content

To generate sequence-ready templates for the gene-rich Xp11.23 region, we have constructed a 1.5-Mb bacterial artificial chromosome (BAC) contig spanning the interval between the DNA markers OATL1 and DXS255. The contig includes 28 BACs, ranging in size from 58 to 285 kb with an average size of 135 kb, which provide 2.5-fold coverage of the region. The BAC contig was constructed based entirely on the content of 40 DNA markers from a previously established YAC contig and 11 new markers developed from BAC-end DNA sequences, 4 of which were required to close gaps in the map. There was no evidence of rearrangement, instability, or chimerism in any of the BAC clones. The BAC cloning system appears to provide robust and total physical coverage of this gene-rich region with clones that are suitable for DNA sequencing.     

A YAC, P1, and Cosmid Contig and 17 New Polymorphic Markers for the Werner Syndrome Region at 8p12–p21

A yeast artificial chromosome (YAC), P1, and cosmid clone contig was constructed for the Werner syndrome (WRN) region of chromosome 8p12–p21 and used to clone a candidate gene forWRN.This region also possibly contains a familial breast cancer locus. The contig was initiated by isolating YACs for the glutathione reductase (GSR) gene and extended in either direction by walking techniques. Sequence-tagged site (STS) markers were generated from subclones of 2GSRYACs and used to identify P1 and cosmid clones. Additional STSs were generated from P1 and cosmid clones and from potential expressed sequences identified by cDNA selection and exon amplification methods. The final contig was assembled by typing 17 YACs, 20 P1 clones, and 109 cosmids for 54 STS markers. TheWRNregion could be spanned by 2 nonchimeric YACs covering approximately 1.4 Mb. A P1/cosmid contig was established covering the core 700–800 kb of theWRNregion. Fifteen new short tandem repeat polymorphisms and 2 biallelic polymorphic markers were identified and included as STSs in the contig. Analysis of these markers in Werner syndrome subjects demonstrates that the candidate WRN gene is in a region of linkage disequilibrium.     

Physical Mapping of Rice Chromosomes 8 and 9 with YAC Clones

First efforts for physical mapping of rice chromosomes 8 and9 were carried out by ordering YAC clones of a rice genomicDNA library covering six genome equivalents with mapped DNAmarkers. A total of 79 and 74 markers from chromosomes 8 and9, respectively, were analyzed by YAC colony and Southern hybridizationusing RFLP markers of cDNA and genomic clones, and by polymerasechain reaction (PCR) screening using PCR-derived and sequence-taggedsite (STS) markers. As a result, 252 YAC clones were confirmedto contain the mapped DNA fragments on both chromosomes. A contigmap was constructed by ordering these YAC clones and about 53%and 43% genome coverage was obtained for chromosomes 8 and 9,respectively, assuming a YAC clone size of 350 kb and overlapbetween neighboring YACs of 50%. A continuous array of YAC cloneswith minimum overlap gave a total size of 18.9 Mb for chromosome8 and 15.6 Mb for chromosome 9, which are close to previousestimates. These contig maps may provide valuable informationthat can be useful in understanding chromosome structure andisolating specific genes by map-based cloning.