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。     

Characterization of polymorphic loci on a telomeric fragment of DNA from the long arm of human chromosome 7

The 240-kb yeast artificial chromosome (YAC) HTY146 (D7S427) containing the telomere from the q arm of human chromosome 7 was subcloned into the cosmid vector sCOS-1. Cosmid subclones were screened for DNA polymorphisms by Southern blot analysis of restriction digests of DNA from random individuals. Four distinct polymorphisms were characterized. These markers provide a resource for defining the end of the genetic map for the long arm of human chromosome 7.     

Construction of a 2.6-Mb contig in yeast artificial chromosomes spanning the human dystrophin gene using an STS-based approach.

A sequence tagged site (STS)-based approach has been used to construct a 2.6-Mb contig in yeast artificial chromosomes (YACs) spanning the human dystrophin gene. Twenty-seven STSs were used to identify and overlap 34 YAC clones. A DNA fingerprint of each clone produced by direct Alu-PCR amplification of YAC colonies and the isolation of YAC insert ends by vectorette PCR were used to detect overlaps in intron 1 (280 kb) where no DNA sequence data were available, thereby achieving closure of the map. This study has evaluated methods for mapping large regions of the X chromosome and provides a valuable resource of the dystrophin gene in cloned form for detailed analysis of gene structure and function in the future.     

Distribution of moderately repetitive sequences pTR5 and LF1 in Xq24-q28 human DNA and their use in assembling YAC contigs.

Xq24-q28 DNA, from a hamster/human hybrid cell containing only that portion of the human X chromosome, was found to contain 56 TaqI restriction fragments that hybridized to the moderately repetitive sequence pTR5. Using the pTR5 sequence as a probe in colony hybridization, 136 cognate yeast artificial chromosome (YAC) clones were detected among a collection of 820 containing about three genomic equivalents of the Xq24-q28 DNA. The YACs were then grouped into 48 contigs and single clones containing one or more of the TaqI fragments. Overlaps were confirmed both by fingerprinting YACs with AluI and L1 probes and by additional information. A less complete analysis was also carried out with a second moderately repetitive sequence, LF1, and some smaller contigs were merged into larger ones. Moderately repetitive sequences can thus be used as probes for multiple loci in single hybridization experiments and can help to organize and confirm YAC overlaps during the development of maps with long-range contiguity.     

Construction of a yeast artificial chromosome contig encompassing the human alpha 5(IV) collagen gene (COL4A5).

A PCR-based screening approach was used to isolate six yeast artificial chromosome (YAC) clones containing segments of the human alpha 5(IV) collagen gene (COL4A5). This gene is located at Xq22 and is known to be involved in the kidney disorder known as Alport syndrome (AS). By analyzing sequence-tagged sites, cDNA content, and rare-cutting restriction site patterns in these YAC clones, a contig that spans the entirety of the alpha 5(IV) gene was constructed. This contig may contain as much as 690 kb of DNA from the alpha 5(IV) locus. On the basis of the information obtained from these YAC clones, the genomic map and gene structure of the alpha 5(IV) gene have been refined. This study has also provided a valuable resource for subsequent studies of the alpha 5(IV) gene and its flanking DNA sequences.     

Unequal homologous recombination of human DNA on a yeast artificial chromosome.

We examined unequal homologous DNA recombination between human repetitive DNA elements located on a yeast artificial chromosome (YAC) and transforming plasmid molecules. A plasmid vector containing an Alu element, as well as a sequence identical to a unique site on a YAC, was introduced into yeast and double recombinant clones analyzed. Recombination occurs between vector and YAC Alu elements sharing as little as 74% identity. The physical proximity of an Alu element to the unique DNA segment appears to play a significant role in determining the frequency with which that element serves as a recombination substrate. In addition, cross-over points of the recombination reaction are largely confined to the ends of the repetitive element. Since a similar distribution of crossover sites occurs during unequal homologous recombination in human germ and somatic tissue, we propose that similar enzymatic processes may be responsible for the events observed in our system and in human cells. This suggests that further examination of the enzymology of unequal homologous recombination of human DNA within yeast may yield a greater understanding of the molecular events which control this process in higher eukaryotes.     

Remi-RFLP Mapping in the Dictyostelium Genome

A set of 147 Dictyostelium discoideum strains was constructed by random integration of a vector containing rare restriction sites. The strains were generated by transformation using restriction enzymemediated integration (REMI) which results in the integration of linear DNA fragments into randomly distributed genomic restriction sites. Restriction fragment length polymorphism (RFLP) was generated in a single genomic site in each strain. These REMI-RFLP strains were used to confirm gene linkages previously supported by two other physical mapping techniques: yeast artificial chromosome (YAC) contig construction, and megabase-scale restriction mapping. New linkages were uncovered when two or more hybridization probes identified the same RFLP fragments. Probes for 100 genes have marked 53% of the RFLPs, representing greater than 22 Mb of the 40 Mb Dictyostelium genome. Alignment of these and other large fragments along each chromosome should lead to a complete physical map of the Dictyostelium genome.     

PCR amplification and analysis of yeast artificial chromosomes

A strategy for the analysis of yeast artificial chromosome (YAC) clones that relies on polymerase chain reaction (PCR) amplification of small restriction fragments from isolated YACs following adapter ligation was developed. Using this method, termed YACadapt, we have amplified several YACs from a human Xq24-qter library and have used the PCR products for physical mapping by somatic cell hybrid deletion analysis and fluorescent in situ hybridization. One YAC, RS46, was mapped to band Xq27.3, near the fragile X mutation. The PCR product is an excellent renewable source of YAC DNA for analyses involving hybridization of YAC inserts to a variety of DNA/RNA sources.     

Linear order of new and established DNA markers around the fragile site at Xq27.3

We have used recombinant clones derived from microdissection of the fragile X region to characterize breakpoints around the fragile site at Xq27.3. So far, no microdissection markers derived from Xq28 material have been found, thus allowing a rapid screening for clones surrounding the fragile site by their presence in a somatic cell hybrid containing Xq27.2-Xqter. A total of 43 new DNA markers from Xq27 have been sublocalized within this chromosome band. Of these new DNA markers, 5 lie in an interval defined as containing the fragile X region. The saturation of Xq27 with DNA markers by microdissection demonstrates the power of this technique and provides the resources for generating a complete physical map of the region.     

The human HPRT gene on a yeast artificial chromosome is functional when transferred to mouse cells by cell fusion

A 680-kb yeast artificial chromosome (YAC) that contains a functional copy of the human hypoxanthine phosphoribosyltransferase (HPRT) gene has been isolated. This YAC, yHPRT, and another YAC, yXY837, which contains the 3' end of the HPRT gene, have been mapped with restriction enzymes that cleave human DNA infrequently. The HPRT gene lies near the center of yHPRT. Fusion of yHPRT-containing yeast spheroplasts with mouse L A-9 cells, which are HPRT-negative, gives rise to HPRT-positive colonies. These colonies contain the human HPRT gene and express human HPRT mRNA. Fusion of yeast with mammalian cells is an efficient way of testing the integrity and functionality of human DNA contained in YACs.     

Organization of the human monoamine oxidase genes and long-range physical mapping around them.

A 265-kb yeast artificial chromosome containing sequences for human monoamine oxidase A and B (MAO-A and MAO-B) genes has been characterized. These two genes are localized within a region of about 240 kb and are arranged in a tail-to-tail configuration, with the 3' coding sequences separated by about 50 kb. A region about 2.5 Mb around the MAO loci was mapped by pulsed-field gel electrophoresis (PFGE). Comparisons between the restriction maps derived from the YAC and the long-range map derived from genomic digestions were in general agreement. The important features identified include a CpG island at the 5' end of the MAO-A and MAO-B genes, respectively. The combined information supports the order of markers within this region to be DXS77-DXS7-MAOA-MAOB.     

Construction and characterization of a partial library of yeast artificial chromosomes from human chromosome 21

We report a protocol for cloning large DNA fragments in yeast artificial chromosomes (YAC). A partial library has been constructed from a somatic hybrid containing chromosome 21 as the single source of human DNA. About 4.0 Mb of human DNA was recovered in 17 YAC clones. Three clones were analyzed by in situ hybridization and mapped on chromosome 21. One clone hybridized with the chromosome 21 centromeric region and may provide new insight both on the molecular structure of centromere and on the localization of Alzheimer disease gene.     

Cloning and in vivo expression of the human GART gene using yeast artificial chromosomes.

Two Yeast Artificial Chromosomes (YACs) were isolated each with a full-length copy of the human gene that encodes the trifunctional protein containing phosphoribosylglycinamide synthetase (GARS), phosphoribosylglycinamide formyltransferase (GART) and phosphoribosylaminoimidazole synthetase (AIRS). The YACs were characterized by restriction mapping and by in situ hybridization of cosmid subclones containing the YAC ends to human metaphase chromosomes. One of the YACs contains co-cloned non-contiguous DNA whereas the other appears to have a single 600 kbp insert from 21q22.1, the location of the GART gene. A restriction map of the gene was obtained from two cosmid subclones which together span the 40 kb gene. The gene is functional when YAC DNA is transferred into GARS- or GARS-and-AIRS-deficient Chinese Hamster Ovary cells. The gene transfer was carried out both by lipofection using purified yeast DNA and by fusion between yeast spheroplasts and the hamster cells. Restriction analysis of DNA from cell lines whose purine auxotrophy was complemented by the YAC showed that with either method a complete and unrearranged copy of the gene can be transferred. The majority of the fusion cell lines appear to contain at least 80% of the YAC.     

Integrated YAC/STS Physical and Genetic Map of 22.5 Mb of Human Xq24–q26 at 56-kb Inter-STS Resolution

A yeast artificial chromosome sequence-tagged site-based (YAC/STS) physical map of 22.5 Mb of the Xq24–q26 cytogenetic band region of the human X chromosome has been assembled. DNA coverage includes 857 large-insert clones formatted with 405 STSs to provide ninefold depth of DNA. At five points, no bridging clones have been recovered from 20 X-chromosome equivalents of human DNA in YACs or bacterial clones, but the placement of 25 (“CA”)_npolymorphic markers permits the ordering of contigs by comparison with the genetic linkage map and radiation hybrid data. The map localizes the X3000 translocation breakpoint and six genes (ANT2, NDUFA1, LAMP2, OCRL, IGSF1, and HDGF) at better than 100-kb resolution. The relatively gene-poor nature of the region is consistent with relatively low uniform 34–42% GC content in STSs across nearly all of the region.     

Mapping human chromosomes by walking with sequence-tagged sites from end fragments of yeast artificial chromosome inserts.

Sequence-tagged sites (STSs) derived from end fragments of chromosome-specific yeast artificial chromosomes (YACs) can facilitate the assembly of an overlapping YAC/STS map. Contigs form rapidly by iteratively screening YAC collections with end-fragment STSs from YACs that have not yet been detected by any previous STS. The map is rendered rapidly useful during its assembly by incorporating supplementary STSs from genes and genetic linkage probes with known locations. Methods for the systematic development and testing of the end-fragments STSs are given here, and a group of 100 STSs is presented for the X chromosome. The mapping strategy is shown to be successful in simulations with portions of the X chromosome already largely mapped into overlapping YACs by other means.     

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.     

Construction of a human chromosome 4 YAC pool and analysis of artificial chromosome stability.

In order to construct a human chromosome 4-specific YAC library, we have utilized pYAC4 and a mouse/human hybrid cell line HA(4)A in which the only human chromosome present is chromosome 4. From this cell line, approximately 8Mb of chromosome 4 have been cloned. The library includes 65 human-specific clones that range in size from 30kb to 290kb, the average size being 108kb. In order to optimize the manipulation of YAC libraries, we have begun to investigate the stability of YACs containing human DNA in yeast cells; these studies will also determine if there are intrinsic differences in the properties of chromosomes containing higher eukaryotic DNAs. We are examining two kinds of stability: 1] mitotic stability, the ability of the YAC to replicate and segregate properly during mitosis, and 2] structural stability, the tendency of the YAC to rearrange. We have found that the majority of YACs examined are one to two orders of magnitude less stable than authentic yeast chromosomes. Interestingly, the largest YAC analyzed displayed a loss rate typical for natural yeast chromosomes. Our results also suggest that increasing the length of an artificial chromosome improves its mitotic stability. One YAC that showed a very high frequency of rearrangement by mitotic recombination proved to be a mouse/human chimera. In contrast to studies using total human DNA, the frequency of chimeras (i.e., mouse/human) in the YAC pool appeared to be low.