Development of a 1.4-Mb BAC/PAC contig and physical map within the critical region for complete X-linked congenital stationary night blindness in Xp11.4

A physical map internal to the markers DXS1368 and DXS228 was developed for the p11.4 region of the human X chromosome. Twenty-four BACs and 10 PACs with an average insert size of 149 kb were aligned to form a contig across an estimated 1.4 Mb of DNA. This contig, which has on average fourfold clone coverage, was assembled by STS and EST content analysis using 46 markers, including 8 ESTs, two retinally expressed genes, and 22 new STSs developed from BAC- and PAC-derived DNA sequence. The average intermarker distance was 30 kb. This physical map provides resources for high-resolution mapping as well as suitable clones for large-scale sequencing efforts in Xp11.4, a region known to contain the gene for complete X-linked congenital stationary night blindness.     

A 2-Megabase Physical Contig Incorporating 43 DNA Markers on the Human X Chromosome at p11.23–p11.22 from ZNF21 to DXS255

A comprehensive physical contig of yeast artificial chromosomes (YACs) and cosmid clones between ZNF21 and DXS255 has been constructed, spanning 2 Mb within the region Xp11.23–p11.22. As a portion of the region was found to be particularly unstable in yeast, the integrity of the contig is dependent on additional information provided by the sequence-tagged site (STS) content of cosmid clones and DNA marker retention in conventional and radiation hybrids. The contig was formatted with 43 DNA markers, including 19 new STSs from YAC insert ends and an internalAlu-PCR product. The density of STSs across the contig ranges from one marker every 20 kb to one every 60 kb, with an average density of one marker every 50 kb. The relative order of previously known genes and expressed sequence tags in this region is predicted to be Xpter–ZNF21–DXS7465E (MG66)–DXS7927E (MG81)–WASP, DXS1011E, DXS7467E (MG21)–DXS- 7466E (MG44)–GATA1–DXS7469E (Xp664)–TFE3–SYP (DXS1007E)–Xcen. This contig extends the coverage in Xp11 and provides a framework for the future identification and mapping of new genes, as well as the resources for developing DNA sequencing templates.     

BAC contig development by fingerprint analysis in soybean.

We constructed a soybean bacterial artificial chromosome (BAC) library suitable for map-based cloning and physical mapping in soybean. This library consists of approximately 40 000 clones (4-5 genome equivalents) stored individually in 384-well microtiter dishes. A random sampling of 224 clones yielded an average insert size of 150 kb, giving a 98% probability of recovering any specific sequence. We screened the library for seven single or very low copy genie or genomic sequences using the polymerase chain reaction (PCR) and found between one and seven BACs for each of the seven sequences. When testing the library with a portion of the soybean psbA chloroplast gene, we found less than 1% chloroplast DNA representation. We also screened the library for eight different classes of disease resistance gene analogs (RGAs) and identified BACs containing all RGAs except class 8. We arranged nine of the class 1 RGA BACs and six of the class 3 RGA BACs into individual contigs based on fingerprint patterns observed after Southern probing of restriction digests of the member BACs with a class-specific sequence. This resulted in the partial localization of the different multigene family sequences without precise definition of their exact positions. Using PCR-based end rescue techniques and RFLP mapping of BAC ends, we mapped individual BACs of each contig onto linkage group J of the soybean public map. The class 1 contig mapped to the region on linkage group J that contains several disease resistance genes. The class 1 contig extended approximately 400 kb. The arrangement of the BACs within this contig has been confirmed using PCR. One end of the class 1 contig core BAC mapped to two positions on linkage group J and cosegregated with two class 1 RGA loci, suggesting that this segment is within an area of regional duplication.     

Physical and cDNA mapping in the DBH region of human chromosome 9q34

Chromosome 9q34 has been extensively studied and mapped due to the presence of known disease genes, principally tuberous sclerosis 1 (TSC1), in this region. During the course of our mapping of this region we constructed a 555-kb contig beginning approximately 50 kb proximal to the dopamine-beta-hydroxylase (DBH) gene and extending, with one small deletion, distal to the D9S114 marker. The contig consists of 11 P1 clones, four PAC clones, one BAC clone and six cosmid clones and contains 27 new nonpolymorphic STSs. We have found the region to be unstable in P1, PAC and BAC cloning vehicles and have identified several deleted genomic clones. In addition, we have isolated and mapped the 3' portions of three putative genes located within or immediately distal to the DBH gene, including one large gene that runs on the opposite strand to DBH and utilizes portions of two DBH exons. The genomic clones of the contig, cDNAs and new STSs will be useful reagents for the further study and mapping of this region.     

人Xp21．1－p21．3上3．5MbYAC重叠群构建及物理图谱分析

用Ａｌｕ－ＰＣＲ指纹图谱法分析了人Ｘｐ２１．１－ｐ２１．３上一系列的酵母人工染色体（ｙｅａｓｔａｒｔｉｆｉｃｉａｌｃｈｒｏｍｏｓｏｍｅ,ＹＡＣ）克隆,发现其中的两个ＹＡＣ克隆构成包含ＤＸＳ１６６位点的重叠群,而且这一重叠群与以前构建的包含ＤＭＤ基因全序列的ＹＡＣ重叠群相连接,ＹＡＣ克隆末端探针交叉杂交证实了这一重叠,使这一ＹＡＣ重叠群至少延伸至ＤＸＳ１６６位点,形成一个跨度为３．５Ｍｂ的ＹＡＣ重叠群。基于这些重叠的ＹＡＣ克隆绘制了这一区域的大尺度限制酶切图谱,并在这一图谱上定位了ＤＸＳ１６６位点,从而确定了ＤＸＳ１６６位点与ＤＭＤ基因的物理关系。这一工作为ＤＭＤ基因的５＇远端调控作用研究及该区域未知基因的克隆奠定了基础。     

A physical map of the 20q12-q13.1 region associated with type 2 diabetes

Several recent genetic studies have suggested linkage of Type 2 diabetes (non-insulin-dependent diabetes mellitus) susceptibility to a region of chromosome 20q12-q13.1. To facilitate the identification and cloning of a diabetes susceptibility gene(s) in this region, we have constructed correlated radiation hybrid and YAC/BAC contig physical maps of the region. A high-resolution radiation hybrid map encompassing 9.5 Mb between the PLC and the CEBPB genes was constructed using 68 markers: 25 polymorphic markers, 15 known genes, 21 ESTs, and 7 random genomic sequences. The physical order of the polymorphic markers within this radiation hybrid map is consistent with published genetic maps. A YAC/BAC contig that gives continuous coverage between PLC and CEBPB was also constructed. This contig was constructed from 24 YACs, 34 BACs, and 1 P1 phage clone onto which 71 markers were mapped: 23 polymorphic markers, 12 genes, 24 ESTs, and 12 random genomic sequences. The radiation hybrid map and YAC/BAC physical map enable precise mapping of newly identified transcribed sequences and polymorphic markers that will aid in linkage and linkage disequilibrium studies and facilitate identification and cloning of candidate Type 2 diabetes susceptibility genes residing in 20q12-q13.1.     

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 bacterial artificial chromosome library for the Chinese alligator (Alligator sinensis)

Chinese alligator (Alligator sinensis) is a rare and endangered species endemic to China. To better understand genetic details of the Chinese alligator genomic structure, a highly redundant bacterial artificial chromosome (BAC) library was constructed. This library consists of 216,238 clones with an average insert size of about 90kb, indicating that the library contains 6.8-fold genome equivalents. Subsequently, we constructed a 516kb contig map for the Chinese alligator olfactory receptor (OR) genes, which spans nine BAC clones, and subjected the BACs to full sequencing. The sequence analysis revealed that this contig contained 16 OR functional genes and meanwhile demonstrated that the nine BACs, which constituted the contig, overlapped correctly, proving the usability of this genome library. As a result, this BAC library could provide a useful platform for physical mapping, genome sequencing or complex analysis of targeted genomic regions for this rare species.     

A High-Resolution PAC and BAC Map of the SCA2 Region

The spinocerebellar ataxia type 2 (SCA2) gene has been localized to chromosome 12q24.1. To characterize this region and to aid in the identification of the SCA2 gene, we have constructed a 3.9-Mb physical map, which covers markers D12S1328 and D12S1329 known to flank the gene. The map comprises a contig of 84 overlapping yeast artificial chromosomes (YACs), P1 artificial chromosomes (PACs), and bacterial artificial chromosomes (BACs) onto which we placed 82 PCR markers. We localized eight genes and expressed sequence tags on this map, many of which had not been precisely mapped before. In contrast to YACs, which showed a high degree of chimerism and deletions in this region, PACs and BACs were stable. Only 1 in 65 PACs contained a small deletion, and 2 in 18 BACs were chimeric. The high-resolution physical map, which was used in the identification of the SCA2 gene, will be useful for the positional cloning of other disease genes mapped to this region.     

Identification of a U7snRNA homologue mapping to the human Xq27.1 region,between the DXS1232 and DXS119 loci

To contribute to the identification and analysis of novel genes, we undertook the study of a cosmid clone in the Xq27 region of human DNA. The cloned fragment was previously observed to have a high number of evolutionarily conserved sequences. In this genomic stretch of DNA we have identified sequence homologous to the U7 RNA gene including its potential regulatory elements. This paper describes the genomic organisation of this gene and its mapping to the Xq27.1 genomic sub-interval between the DXS1232 and DXS119 loci.     

Fine mapping and cloning of the breakpoint associated with Menkes syndrome in a female patient.

The gene responsible for Menkes syndrome has been assigned to Xq13 by a combination of comparative mapping and linkage analysis. A previous report has mapped the translocation breakpoint associated with the disease in a female patient to an interval delimited by PGK1 and a group of six more proximal Xq13 markers, including DXS56. We have characterized a number of PGK1- or DXS56-positive YACs, from which we have generated six new markers. One of them identifies a small overlap region between a PGK1-positive YAC and three DXS56-positive YACs, distal to the Menkes breakpoint. A 560-kb region covered by a DXS56-positive YAC has been restriction-mapped and subcloned, disclosing a 187-kb MluI fragment astride the breakpoint. A probe mapping distal to the rearrangement in the same interval reveals altered PGFE fragments in a hybrid constructed from the translocation patient's DNA. We describe the development of a cosmid contig extending 150 kb from a nearby CpG island across the breakpoint. This contig includes four adjacent clones displaying cross-specific hybridization.     

Refined Linkage Disequilibrium and Physical Mapping of the Gene Locus for X-Linked Dystonia–Parkinsonism (DYT3)

X-linked dystonia-parkinsonism (XDP) is a recessive disorder characterized by generalized dystonia with some patients exhibiting parkinsonism. The disease gene, DYT3, is located between DXS453 (DXS993) and DXS559, and strongest linkage disequilibrium is found distal to DXS7117 and proximal to DXS559. We have isolated and analyzed four novel polymorphic markers between DXS7117 and DXS559 and, by haplotype analysis, have narrowed the candidate interval to <350 kb. A sequence-ready contig of 700 kb has been constructed spanning DXS7117 to DXS559 and is composed of 35 PACs, BACs, and cosmids. Nine genes and novel ESTs have been mapped into this contig, and mutations in the coding regions and intron-exon borders of two genes have been excluded as the cause of XDP. Several of the other genes and ESTs located within the contig code for proteins implicated in normal brain development and function and are candidates for DYT3.     

A multipoint linkage map of the distal short arm of the human X chromosome.

The distal portion of the short arm of the human X chromosome (Xp) exhibits many unique and interesting features. Distal Xp contains the pseudoautosomal region, a number of disease loci, and several cell-surface markers. Several genes in this area have also been observed to escape X-chromosomal inactivation. The characterization of new polymorphic loci in this region has permitted the construction of a refined multipoint linkage map extending 15 cM from the Xp telomere. This interval is known to contain the loci for the diseases X-linked ichthyosis, chondrodysplasia punctata, and Kallmann syndrome, as well as the cell-surface markers Xg and 12E7. This region also contains the junction between the pseudoautosomal region and strictly X-linked sequences. The locus MIC2 has been demonstrated by linkage analysis to be indistinguishable from the pseudoautosomal junction. The steroid sulfatase locus has been mapped to an interval adjacent to the DXS278 locus and 6 cM from the pseudoautosomal junction. The polymorphic locus (STS) DXS278 was shown to be informative in all families studied, and linkage analysis reveals that the locus represents a low-copy repeat with at least one copy distal to the STS gene. The generation of a multipoint linkage map of distal Xp will be useful in the genetic dissection of many of the unique features of this region.     

High-Resolution Physical Map of the X-linked Retinoschisis Interval in Xp22

X-linked retinoschisis (RS) is the leading cause of macular degeneration in young males and has been mapped to Xp22 between DXS418 and DXS999. To facilitate identification of the RS gene, we have constructed a yeast artificial chromosome (YAC) contig across this region comprising 28 YACs and 32 sequence-tagged sites including seven novel end clone markers. To establish the definitive marker order, a PAC contig containing 50 clones was also constructed, and all clones were fingerprinted. The marker order is: Xpter–DXS1317–(AFM205yd12–DXS7175–DXS7992)–60N8T7–DXS1195–DXS7993–DXS7174–60N8-SP6–DXS418–DXS7994–DXS7995–DXS7996–HYAT2–25HA10R–HYAT1–DXS7997–DXS7998–DXS257–434E8R–3542R–DXS6762–DXS7999–DXS6763–434E8L–DXS8000–DXS6760–DXS7176–DXS8001–DXS999–3176R–PHKA2–Xcen. A long-range restriction map was constructed, and the RS region is estimated to be 1300 kb, containing three putative CpG islands. An unstable region was identified between DXS6763 and 434E8L. These data will facilitate positional cloning of RS and other disease genes in Xp22.