A long-range physical map of human Chromosome 21q22.1 band from the YAC continuum

The human Chromosome (Chr) 21q22.1 region contains several genes for cytokines and neurotransmitters and the gene for superoxide dismutase (mutant forms of which can cause familial amyotrophic lateral sclerosis). A region of approximately 5.8 Mb encompassing D21S82 and the glycinamide ribonucleotide transformylase (GART) loci was covered by overlapping YAC clones, which were contiguously ordered by clone walking with sequence-tagged site (STSs). A total of 76 markers, including 29 YAC end-specific STSs, were unambiguously ordered in this 5.8-Mb region, and the average interval between markers was 76 kb. Restriction maps of the YAC clones with rare-cutting enzymes were simultaneously prepared, and the restriction sites were aligned to obtain a consensus restriction map of the proximal region of the 21q22.1 band. The restriction map made from 44 overlapping YACs contains 54 physically assigned STSs. By integrating the consensus map of the adjacent 1.8-Mb region, we obtained a fine physical map spanning 6.5 Mb of human Chr 21q22.1. This map contains 24 precisely positioned end-specific STSs and 12 NotI-linking markers. More than 39 potential CpG islands were identified in this region and were found to be unevenly distributed. This physical map and the YACs should be useful as a reference map and as a resource for further structural analysis of the Giemsa-negative band (R-band) of Chr 21q22.1. Received: 1 September 1995 / Accepted: 21 November 1995     

Co-amplification of L1 line elements with localised low copy repeats in Giemsa dark bands: implications for genome organisation.

A repeat sequence island, located at the A3 Giemsa dark band on the mouse X chromosome and consisting of 50 copies of a localised long complex repeat unit (LCRU), features an unusually high concentration of L1 LINE repeat sequences juxtaposed and inserted within the LCRU. Sequence analysis of three independent genomic clones containing L1 LINE elements juxtaposed with the LCRU demonstrates a common junction sequence at the L1/LCRU boundary, suggesting that the high concentration of L1 LINE sequences in the repeat sequence island has arisen by association of an L1 element with an LCRU followed by amplification. The LCRU target site at this common junction sequence bears no resemblance to the target site of an L1 element inserted within one LCRU, indicating there is no specific preferential target site for L1 integration. We propose that co-amplification of L1 LINE elements with localised low copy repeat families throughout the genome could have a major effect on the chromosomal distribution of L1 LINE elements.     

Sequence-ready physical map of the mouse Chromosome 16 region with conserved synteny to the human Velocardiofacial syndrome region on 22q11.2

Proximal mouse Chromosome (Chr) 16 shows conserved synteny with human Chrs 16, 8, 22, and 3. The mouse Chr 16/human Chr 22 conserved synteny region includes the DiGeorge/Velocardiofacial syndrome region of human Chr 22q11.2. A physical map of the entire mouse Chr 16/human Chr 22 region of conserved synteny has been constructed to provide a substrate for gene discovery, genomic sequencing, and animal model development. A YAC contig was constructed that extends ca. 5.4 Mb from a region of conserved synteny with human Chr 8 at Prkdc through the region conserved with human Chr 3 at DVL3. Sixty-one markers including 37 genes are mapped with average marker spacing of 90 kb. Physical distance was determined across the 2.6-Mb region from D16Mit74 to Hira with YAC fragmentation. The central region from D16Jhu28 to Igl-C1 was converted into BAC and PAC clones, further refining the physical map and providing sequence-ready template. The gene content and borders of three blocks of conserved linkage between human Chr 22q11.2 mouse Chr 16 are refined. Received: 4 November 1998 / Accepted: 21 December 1998     

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.     

Application of restriction fragment fingerprinting with a rice microsatellite sequence to assembling rice YAC clones.

To refine the current physical map of rice, we have established a restriction fragment fingerprinting method for identifying overlap between pairs of rice yeast artificial chromosome (YAC) clones and defining the physical arrangement of YACs within contiguous fragments (contigs). In this method, Southern blots of rice YAC DNAs digested with a restriction endonuclease are probed with a rice microsatellite probe, (GGC)5. The probe produces a unique fingerprint profile characteristic of each YAC clone. The profile is then digitized, processed in a computer, and a statistic that represents the degree of overlap between two YACs is calculated. The statistics have been used to detect overlaps among YAC clones, thereby filling a gap between two neighbouring contigs and organizing overlapping rice YAC clones into contiguous fragments. We applied this method to rearranging YACs that had previously been assigned to rice chromosome 6 by anchoring with RFLP markers.     

Walking, Cloning, and Mapping with YACs in 3q27: Localization of Five ESTs Including Three Members of the Cystatin Gene Family and Identification of CpG Islands

Using yeast artificial chromosomes, we have generated a high-resolution physical map for 2.7 Mb of human chromosomal region 3q27. The YAC clones group into three contigs, one of which has also been linked to the CEPH YAC contig map of human chromosome 3. Fluorescencein situhybridization has been used to order the contigs on the chromosome and to estimate the distance between them. Expressed sequence tags for five genes, including three members of the cystatin gene family and a gene thought to be involved in B-cell non-Hodgkin lymphoma, have been placed within the YAC contigs, and 12 putative CpG islands have been identified. These YACs provide a useful resource to complete the physical mapping of 3q27 and to begin identification and characterization of further genes that are located there.     

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。     

Comparative mapping on the mouse X Chromosome defines a myotubular myopathy equivalent region

The gene for X-linked myotubular myopathy (MTM1) has been localized to a 300-kb critical region in human Xq28 between IDS and GABRA3. As part of an effort to clone this gene, we developed a YAC contig on the mouse X Chromosome (Chr) which includes loci homologous to those within the human MTM1 critical region. The murine contig consists of 18 YACs and spans 2.5–3.0 Mb. We have aligned the human and murine physical maps by isolating conserved mouse genomic fragments, including CpG islands and trapped exons. We believe that the simultaneous isolation of genes from both mouse and human and continued comparative mapping will prove helpful in the eventual identification of MTM1 and other genes in the region. Received: 9 February 1996 / Accepted: 30 March 1996     

Construction of YAC Contigs at Human Chromosome 11q22.3-q23.1 Region Covering the Ataxia Telangiectasia Locus

Ataxia telangiectasia (AT) is an autosomal recessive diseaseof unknown etiology associated with cerebellar ataxia, telangiectasia,immune dysfunction, higher cancer risk, genomic instabilityand hypersensitivity to ionizing radiation. The major AT loci,AT-A and AT-C, are shown to be closely linked at chromosome11q22–q23. The most recent genetic linkage mapping andlinkage disequilibrium analysis have localized the major ATloci to a sequence of approximately 850 kb between the markersD11S1819 and D11S1818. The isolation of yeast artificial chromosomesspanning the AT region is an essential step to identify thegene or genes responsible for the mutation(s). We isolated atotal of 20 YAC clones from three independent YAC libraries,using sequence tagged sites mapped in the AT region as primersfor PCR-based YAC screening. The PCR assay for the presenceor absence of 16 different DNA markers allowed us to constructand to order four YAC contigs at the AT region. One of the contigswhich consists of the 10 YAC clones, covers about 2 Mb of DNAat the boundary between Giemsa-positive band 11q22.3 and Giemsa-negativeband 11q23.1 and includes the entire region of the major ATlocus between D11S1819 and D11S1818. Thus, the YAC contigs willfacilitate the positional cloning approach for searching transcribedsequences from the defined genomic region.     

YAC/P1 contigs defining the location of 56 microsatellite markers and several genes across a 3.4-cM interval on mouse Chromosome 11

The characterization of three YAC/P1 contigs from adjacent segments of the central region of mouse Chromosome (Chr) 11 is described. These contigs are based upon 63 YACs and 40 P1 recombinants. From these clones, 185 end sequences were obtained, of which 147 sequences could be converted into sequencetagged sites and mapped within the three contigs. Deletions were detected in 16 out of 63 YACs; 19 of 63 YACs were found to be chimeric. No such aberrations were found in P1 recombinants. A total of 22 public and 34 newly developed microsatellite markers were unambiguously localized to and ordered in the contigs. In the cryb1/Nf1 interval of the central contig, several new genes have been identified by exon trapping and precisely localized with respect to known STS markers.     

Molecular cloning and analysis of the fragile X region in man.

The fragile X syndrome (FraX), the most common inherited form of mental retardation, has been located to Xq27.3. As a step in the molecular analysis of this mutation, we have cloned a contiguous 1.8 Mb region containing the entire fragile X region in YAC and cosmid clones. The cloned area defines a region of 50 kb containing a CpG island, found to be selectively methylated in patients expressing the fragile X phenotype. In this 50kb area we have localised the breakpoints of four somatic cell hybrids selected to break at the position of the fragile site. Fluorescence in-situ hybridisation of cosmids flanking this area shows that the breakpoints, the CpG island and the fragile site coincide.     

Use of interspersed repetitive sequences-PCR products for cDNA selection

In order to increase the efficiency of cDNA selection approaches, we describe the use of interspersed repetitive sequences-PCR (IRS-PCR) products to isolate genes from large-insert genomic clones. IRS-PCR is conducted on total yeast DNA containing a YAC of interest so that there is no need to purify the starting genomic clone. This enables the production of large amounts of genomic substrate for cDNA selection and allows the use of unstable YAC clones. Moreover, the hybridization of the IRS-PCR product to the cDNA clones after selection introduces a positive selection step. We tested these PCR products from YACs for the presence of exons, using cDNAs originating from seven different genes. In each case, at least one exon was present in the IRS-PCR product. We have applied this strategy to four YAC clones originating from the human X Chromosome (Chr). All the selected cDNAs, strongly positive with the IRS-PCR product, did indeed originate from a gene in the region covered by the YAC. In all cases, the previously known genes contained in the genomic clones have been isolated. In addition, we have isolated human genes that have already been described but not assigned to any chromosomal region.     

Large-insert clone/STS contigs in Xq11-q12, spanning deletions in patients with androgen insensitivity and mental retardation

An integrated large-insert clone map of the region Xq11-q12 is presented. A physical map containing markers within a few hundred kilobases of the centromeric locus DXZ1 to DXS1125 spans nearly 5 Mb in two contigs separated by a gap estimated to be approximately 100-250 kb. The contigs combine 75 yeast artificial chromosome clones, 12 bacterial artificial chromosome clones, and 17 P1-derived artificial chromosome clones with 81 STS or EST markers. Overall marker density across this region is approximately 1 STS/60 kb. Mapped within the contigs are 12 ESTs as well as 5 known genes, moesin (MSN), hephaestin (HEPH), androgen receptor (AR), oligophrenin-1 (OPHN1), and Eph ligand-2 (EPLG2). Orientation of the contigs on the X chromosome, as well as marker order within the contigs, was unambiguously determined by reference to a number of X chromosome breakpoints. In addition, the distal contig spans deletions from chromosomes of three patients exhibiting either complete androgen insensitivity (CAI) or a contiguous gene syndrome that includes CAI, impaired vision, and mental retardation.     

Methylation status of CpG-rich islands on active and inactive mouse X chromosomes

Single copy probes derived from CpG-rich island clones fromEag I andNot I linking libraries and nine rare-cutter restriction endonucleases were used to investigate the methylation status of CpG-rich islands on the inactive and active X chromosomes (Chr) of the mouse. Thirteen of the 14 probes used detected CpG-rich islands in genomic DNA. The majority of island CpGs detected by rare-cutter restriction endonucleases were methylated on the inactive X Chr and unmethylated on the active X Chr, but some heterogeneity within the cell population used to make genomic DNA was detected. The CpG-rich islands detected by two putative pseudoautosomal probes remained unmethylated on both the active and inactive X Chrs. Otherwise, distance from the X Chr inactivation center did not affect the methylation profile of CpG-rich islands. We conclude that methylation of CpG-rich islands is a general feature of X Chr inactivation.     

Construction of YAC Contigs on Rice Chromosome 5

A physical map of rice chromosome 5 was constructed with yeastartificial chromosome (YAC) clones along a high-resolution molecularlinkage map carrying 118 DNA markers distributed over 123.7cM of genomic DNA. YAC clones have been identified by colonyand Southern hybridization for 105 restriction fragment lengthpolymorphism (RFLP) markers and by polymerase chain reaction(PCR) screening for 8 sequence-tagged site (STS) markers and5 randomly amplified polymorphic DNA (RAPD) markers. Of 458YACs, 235 individual YACs with an average insert length of 350kb were selected and ordered on chromosome 5 from the YAC library.Forty-eight contigs covering nearly 21 Mb were formed on thechromosome 5; the longest one was 6 cM and covered 1.5 Mb. Thelength covered with YAC clones corresponded to 62% of the totallength of chromosome 5. There were many multicopy sequencesof expressed genes on chromosome 5. The distribution of manycopies of these expressed gene sequences was determined by YACSouthern hybridization and is discussed. A physical map withthese characteristics provides a powerful tool for elucidationof genome structure and extraction of useful genetic informationin rice.     

High-resolution comparative physical mapping of mouse Chromosome 10 in the region of homology with human Chromosome 21

Comparative mapping of human and mouse chromosomes can be used to predict locations of homologous loci between the species, provides the substrate to examine the process of chromosomal evolution, and facilitates the continuing development of mouse genetic models for human disorders. A YAC contig of the region of mouse Chromosome (Chr) 10 (MMU10) that demonstrates conserved linkage with the distal portion of human Chr 21 (HSA21) has been constructed. The contig contains all known genes mapped in both species, defines the proximal region of homology between MMU10 and HSA22, and contains the evolutionary junction between HSA21 and HSA22 on MMU10. It consists of 23 YACs and 2 PACs, and covers 3.2 Mb of MMU10. The average marker density for this region is 1 marker/69 kb. Nine of 22 expressed sequences are mapped here for the first time in mouse, and two are newly characterized expressed sequences. The contig also contains 12 simple sequence repeats (SSRs) and 16 YAC and PAC endclone markers. YAC fragmentation analysis was used to create a physical map for the proximal 2.2 Mb of the contig. Cloning of the corresponding region of HSA21 has proven difficult, and the mouse contig includes segments absent from previously described sequence ready maps of HSA21. Received: 22 July 1998 / Accepted: 13 November 1998     

Identification of novel simple sequence length polymorphisms (SSLPs) in mouse by interspersed repetitive element (IRE)-PCR.

Interspersed repetitive element (IRE)-PCR is a useful method for identification of novel human or mouse sequence tagged sites (STSs) from contigs of genomic clones. We describe the use of IRE-PCR with mouse B1 repetitive element primers to generate novel, PCR amplifiable, simple sequence length polymorphisms (SSLPs) from yeast artificial chromosome (YAC) clones containing regions of mouse chromosomes 13 and 14. Forty-two IRE-PCR products were cloned and sequenced from eight YACs. Of these, 29 clones contained multiple simple sequence repeat units. PCR analysis with primers derived from unique sequences flanking the simple sequence repeat units in seven clones showed all to be polymorphic between various mouse strains. This novel approach to SSLP identification represents an efficient method for saturating a genomic interval with polymorphic genetic markers that may expedite the positional cloning of genes for traits and diseases.     

A 2-Mb YAC Contig Encompassing Three Loci (DXF34, DXS14, and DXS390) That Lie between Xp11.2 Translocation Breakpoints Associated with Incontinentia Pigmenti Type 1

We demonstrate that all the repeat elements representing the conserved loci DXF34 and DXS390 lie between the X;9 and the X;17 translocation breakpoints associated with incontinentia pigmenti type 1 (IP1). Sequence-tagged sites (STSs) at DXF34S1, DXS14, and DXS390 have been used to isolate YAC clones containing these loci, and a contig of approximately 2 Mb has been constructed. Patterns of hybridization observed in the YAC clones indicate that DXS390 comprises two distinct regions (A and B). The STS at DXS390 detects the A region and includes a polymorphic CA repeat (PIC = 0.25). This expansion of the cloned region around DXF34 and DXS390 will enable the isolation of additional conserved sequences that will help in understanding both the lesions underlying the pathogenesis of IP1 and the size and extent of the man-mouse homologous block defined by DXF34.