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At least 1,117 records · Page 62Linked to original sources
PubMed1996

Cosmids map two incontinentia pigmenti type 1 (IP1) translocation breakpoints to a 180-kb region within a 1.2-Mb YAC contig.

Incontinentia pigmenti (IP) is an X-linked dominant disorder of neuroectodermal development. Based on the observation of six unrelated females with clinical features of nonfamilial IP with constitutional de novo reciprocal X;autosome translocations, a putative incontinentia pigmenti type 1 locus (IP1; MIM No. 308300) was localized to region Xp11.21. Using available regional DNA markers, we constructed a yeast artificial chromosome (YAC) contig that contained 1.2 Mb of distal Xp11.21 and spanned two IP1 X-chromosomal breakpoints. This contig was used to generate a detailed molecular map of the region and identify three regional CpG islands. YAC-derived cosmids were used to clone and map the IP1 breakpoints to a 180-kb interval that was flanked by DNA markers DXS705 and DXS741. The physical map and genomic clones should facilitate the isolation and characterization of transcripts associated with the IP1 translocation breakpoints.

Base Sequence↗
PubMed2000

Multiple peptide synthetase gene clusters in Actinomycetes.

Two oligonucleotide probes derived from conserved motifs in peptide synthetases were hybridized with a cosmid library of Planobispora rosea genomic DNA. Detailed characterization of the physical organization of the positive cosmids indicated the existence of at least eight unlinked contigs containing multiple fragments that hybridized to both probes. Partial sequences of PCR products from the positive cosmids confirmed the existence of peptide synthetase genes. The combined results of hybridizations and physical mapping indicate that, in all likelihood, the isolated P. rosea contigs encode over 40 putative peptide synthetase modules. Similar results were obtained on screening a cosmid library of Actinoplanes teichomyceticus DNA. Furthermore, Southern hybridizations with several actinomycete strains, belonging to different genera, indicate that most strains contain multiple hybridizing bands well in excess of the number expected from the structure of the oligopeptides produced by these strains. Even strains not reported to produce oligopeptides gave clear positive signals when examined with the probes. These results strongly suggest that actinomycetes devote a notable fraction of their genomes to the non-ribosomal synthesis of peptides, and that most strains have the genetic potential to produce more oligopeptides than are currently described.

Actinomycetales↗
PubMed2000

A comparative transcript map and candidates for mutant phenotypes in the Tyrp1 (brown) deletion complex homologous to human 9p21-23.

The mouse Tyrp1 deletion complex is a valuable resource for high-resolution mapping of genes and phenotypes to the central region of Chromosome (Chr) 4. The distal part of the complex is homologous to human Chr 9p21-23, and we have used the available radiation hybrid maps to identify human transcripts in the region. We localize seven genes to a human YAC contig that spans the full extent of the distal deletion complex and show that the mouse homologs of four of these, including Cer1, map within the complex. On the basis of location and/or expression, we exclude genes as candidates for several known phenotypes in the region and identify a candidate transcript for the neonatal lethal phenotype l(4)Rn2.

Animals↗
PubMed2000

Cloning and characterization of the breakpoint regions of a chromosome 11;18 translocation in a patient with hamartoma of the retinal pigment epithelium.

Mutations of various tumor suppressor genes, e.g., PTEN, TSC1, and TSC2, are known to be responsible for different inherited diseases presenting with multiple hamartomas, a benign tumor resembling neoplasia that results from faulty organ development. Combined hamartoma of the retinal pigment epithelium (RPE) and retina is a rare, congenital, focal malformation of the fundus. So far, no disease gene has been associated with this disorder. By molecular analysis of an apparently balanced and reciprocal translocation between the short arms of chromosomes 11 and 18, t(11;18)(p13;p11.31), in a patient with hamartoma of the RPE and retina, we selected PAC clones crossing the breakpoints on both derivative chromosomes 11 and 18. For the overlapping chromosome 11 clone, two EST clusters were identified, suggesting the existence of at least two genes in the breakpoint region. We constructed a PAC contig and showed that at least three exons of a novel gene map to the breakpoint region on chromosome 18. Based on the results of FISH analysis with the PAC clones of this contig, we suggest the occurrence of a complex rearrangement.

Adult↗
PubMed2000

Characterization of a highly complex region in Xq13 and mapping of three isodicentric breakpoints associated with preleukemia.

The chromosomal abnormality represented by an isodicentric X chromosome [idic(X)(q13)] is associated with a subset of acute myeloid leukemia (AML) and preleukemia observed in elderly females. A previous study localized the breakpoints of two acquired isodicentric X chromosomes associated with myelodysplasia to a 450-kb region proximal to the XIST gene. Here we report the construction and extensive characterization of a reliable 1-Mb P1 artificial chromosome and bacterial artificial chromosome contig covering a highly problematic region in Xq13 that includes the previously described isodicentric breakpoint region. In addition to mapping of the brain-specific gene (NAP1L2) and the phosphoglyceryl kinase alpha subunit 1 gene (PHKA1) and generation and mapping of a large number of STSs throughout the contig, we have mapped a putative transcriptional regulatory protein (HDACL1), and 35 ESTs. Sequencing data, Southern blot analysis, and fiber-FISH analysis have permitted characterization of extensive region-specific duplications and triplications in addition to an unusually high concentration of long interspersed repeat elements, both of which could be implicated in isodicentric chromosome formation and other Xq13 chromosome aberrations. FISH analysis of metaphase chromosomes from two previously unpublished AML patients and one preleukemic patient using cosmid clones and selected subclones allowed mapping of the idic(X)(q13) breakpoints to a 100-kb interval, consistent with the involvement of an X-linked gene in the genesis of this form of preleukemia, disruption of which may represent a preliminary step in progression to AML. Assembly and physical mapping of this complex 1-Mb contig establish a foundation for ongoing sequencing and gene identification projects in the region.

Acute Disease↗
PubMed2000

Molecular cloning of the critical region for glomerulopathy with fibronectin deposits (GFND) and evaluation of candidate genes.

Glomerulopathy with fibronectin deposits (GFND, MIM 601894) is an autosomal dominant kidney disease that leads to terminal renal failure at a median age of 47 years. It represents a distinct entity of membranoproliferative glomerulonephritis (MPGN) type III and is characterized by the unique feature of massive glomerular deposits of fibronectin. We have recently localized a gene locus for GFND to human chromosome 1q32 by total genome linkage analysis in a large kindred, within a 4.1-cM critical interval between markers D1S2872 and D1S2891. This interval contains a cluster of genes for "regulators of complement activation" (RCA), which represent strong candidates for GFND. To identify positional candidate genes for GFND within the critical genetic interval, we here report the cloning of the entire critical GFND region in a complete YAC and partial PAC contig. We constructed a high-resolution transcriptional map, thereby defining positional and functional candidate genes for the disease. To evaluate their role in GFND, we performed functional studies on RCA proteins in GFND patients from the large kindred, as well as mutational analysis of the genes for complement receptor-2 (CR2), membrane cofactor protein (MCP), and decay accelerating factor (DAF). Although no loss-of-function mutation has been identified as yet, these data provide a basis for the examination of candidate genes for GFND and other genes for MPGN, which localize to the vicinity of the GFND region.

Adult↗
PubMed2000

Role of the AZFa candidate genes in male infertility.

The AZFa region on the Y-chromosome long arm has been recently assembled in a complete sequence map contained in a contig and shown to span more than 1 Mb. It contains three genes, USP9Y, DBY and UTY, but only the former two can be at present considered candidate genes for the infertile phenotype associated with deletion of this interval. These genes have X-homologues and are expressed in many tissues, even if DBY has a shorter transcript expressed in the testis only, strengthening its role in spermatogenesis. Only few patients with gene-specific deletion have been reported and a clear genotype-phenotype relation is still lacking. While deletions or even smaller mutations in USP9Y seem to be associated with a testicular phenotype of severe hypospermatogenesis, patients with deletions of DBY may present both Sertoli cell-only syndrome and severe hypospermatogenesis. On the contrary, the phenotype of patients with deletion of both USP9Y and DBY seem to be invariably azoospermia with a testicular histology of Sertoli cell-only.

Chromosome Mapping↗
PubMed2000

The genomic region encompassing the nephropathic cystinosis gene (CTNS): complete sequencing of a 200-kb segment and discovery of a novel gene within the common cystinosis-causing deletion.

Nephropathic cystinosis is an autosomal recessive disorder caused by the defective transport of cystine out of lysosomes. Recently, the causative gene (CTNS) was identified and presumed to encode an integral membrane protein called cystinosin. Many of the disease-associated mutations in CTNS are deletions, including one >55 kb in size that represents the most common cystinosis allele encountered to date. In an effort to determine the precise genomic organization of CTNS and to gain sequence-based insight about the DNA within and flanking cystinosis-associated deletions, we mapped and sequenced the region of human chromosome 17p13 encompassing CTNS. Specifically, a bacterial artificial chromosome (BAC)-based physical map spanning CTNS was constructed by sequence-tagged site (STS)-content mapping. The resulting BAC contig provided the relative order of 43 STSs. Two overlapping BACs, which together contain all of the CTNS exons as well as extensive amounts of flanking DNA, were selected and subjected to shotgun sequencing. A total of 200,237 bp of contiguous, high-accuracy sequence was generated. Analysis of the resulting data revealed a number of interesting features about this genomic region, including the long-range organization of CTNS, insight about the breakpoints and intervening DNA associated with the common cystinosis-causing deletion, and structural information about five genes neighboring CTNS (human ortholog of rat vanilloid receptor subtype 1 gene, CARKL, TIP-1, P2X5, and HUMINAE). In particular, sequence analysis detected the presence of a novel gene (CARKL) residing within the most common cystinosis-causing deletion. This gene encodes a previously unknown protein that is predicted to function as a carbohydrate kinase. Interestingly, both CTNS and CARKL are absent in nearly half of all cystinosis patients (i.e., those homozygous for the common deletion). [The sequence data described in this paper have been submitted to the GenBank data library under accession nos. AF168787 and AF163573.]

Amino Acid Transport Systems, Neutral↗
PubMed2001

Integration of cytogenetic landmarks into the draft sequence of the human genome.

We have placed 7,600 cytogenetically defined landmarks on the draft sequence of the human genome to help with the characterization of genes altered by gross chromosomal aberrations that cause human disease. The landmarks are large-insert clones mapped to chromosome bands by fluorescence in situ hybridization. Each clone contains a sequence tag that is positioned on the genomic sequence. This genome-wide set of sequence-anchored clones allows structural and functional analyses of the genome. This resource represents the first comprehensive integration of cytogenetic, radiation hybrid, linkage and sequence maps of the human genome; provides an independent validation of the sequence map and framework for contig order and orientation; surveys the genome for large-scale duplications, which are likely to require special attention during sequence assembly; and allows a stringent assessment of sequence differences between the dark and light bands of chromosomes. It also provides insight into large-scale chromatin structure and the evolution of chromosomes and gene families and will accelerate our understanding of the molecular bases of human disease and cancer.

Chromosome Aberrations↗
PubMed1997

A 3-Mb contig from D11S987 to MLK3, a gene-rich region in 11q13.

We have combined genetic, radiation-reduced somatic cell hybrid (RRH), fluorescent in situ hybridization (FISH), and physical mapping methods to generate a contig of overlapping YAC, PAC, and cosmid clones corresponding to > 3 continuous Mb in 11q13. A total of 15 STSs [7 genes (GSTP1, ACTN, PC, MLK3, FRA1, SEA, HNP36), 4 polymorphic loci (D11S807, D11S987, GSTP1, D11S913), 3 ESTs (D11S1956E, D11S951E, and W1-12191), and 1 anonymous STS (D11S703)], mapping to three independent RRH segregation groups, identified 26 YAC, 7 PAC, and 16 cosmid clones from the CGM, Roswell Park, CEPH Mark I, and CEPH MegaYAC YAC libraries, a 5 genome equivalent PAC library, and a chromosome II-specific cosmid library. Thirty-six Alu-PCR products derived from 10 anonymous bacteriophage lambda clones, a cosmid containing the polymorphic marker D11S460, or STS-positive YAC or cosmid clones were identified and used to screen selected libraries by hybridization, resulting in the identification of 19 additional clones. The integrity and relative position of a subset of clones was confirmed by FISH and were found to be consistent with the physical and RRH mapping results. The combination of STS and Alu-PCR-based approaches has proven to be successful in attaining contiguous cloned coverage in this very GC-rich region, thereby establishing for the first time the absolute order and distance between the markers: CEN-MLK3-(D11S1956E/D11S951E/W1-12191)-FRA1-D 11S460-SEA-HNP36/ D11S913-ACTN-PC-D11S703-GSTP1-D11S987-TEL.

Base Sequence↗
PubMed1998

Rapid mapping of markers applying vectorette technology to YAC fragmentation allows easy assembly of a high-density STS bacterial clone contig spanning the markers D6S1260-D6S1918.

We have generated a detailed physical map of the 6p21.3/p22.1 boundary, using a combination of yeast artificial chromosome (YAC) fragmentation and high-resolution sequence tagged site (STS) content mapping. YACs from the CEPH, St. Louis, and ICRF libraries have been used to construct a 4.5-Mb contig spanning the markers D6S306 to D6S1571. YAC insert sizes were determined by pulsed field gel electrophoresis (PFGE). Chimerism of YACs was determined by fluorescent in situ hybridization (FISH), and their integrity was determined by fingerprinting with Alu-PCR. We have identified 10 new CA repeat loci in this region as well as over 50 novel STSs, several tRNA genes, a new histone H2B gene and the phospholipase D gene. Using these new markers, we have rapidly generated a bacterial clone contig of over 250 kb, spanning the markers D6S1260 to D6S1918 (WI-3111) with STSs spaced on average every 6 kb.

Animals↗
PubMed1999

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.

Chromosome Mapping↗
PubMed2003

A high resolution physical and RH map of pig chromosome 6q1.2 and comparative analysis with human chromosome 19q13.1.

BACKGROUND: The generation of BAC/PAC contigs in targeted genome regions is a powerful method to establish high-resolution physical maps. In domestic animal species the generation of such contigs is typically initiated with the screening of libraries with probes derived from human genes that are expected to be located in the region of interest by comparative mapping. However, in many instances the available gene-derived probes are too far apart to allow the cloning of BAC/PAC contigs larger than a few hundred kb. High resolution physical mapping allows to estimate the sizes of gaps and to control the orientation of the individual sub-contigs, which helps to avoid errors during the assembly of smaller contigs into final Mb-sized contigs. The recently constructed porcine IMNpRH2 panel allowed us to use this approach for the construction of high-resolution physical maps of SSC 6q1.2. RESULTS: Two sequence-ready BAC/PAC contigs of the gene-rich region on porcine chromosome 6q1.2 (SSC 6q1.2) containing the RYRl gene were constructed. The two contigs spanned about 1.2 Mb and 2.0 Mb respectively. The construction of these contigs was monitored by the results provided by the mapping of 15 markers on the IMpRH(7000rad) and 35 markers on the IMNpRH2(12000rad) radiation hybrid panels. Analyses on the IMpRH panel allowed us to globally link and orientate preliminary smaller contigs, whereas analyses on the high resolution IMNpRH2 panel allowed us to finally identify the order of genes and markers. CONCLUSIONS: A framework map of 523 cR12000 was established covering the whole studied region. The order of markers on the framework 1000:1 RH map was found totally consistent with the data deduced from the contig map. The kb/cR ratio was very constant in the whole region, with an average value of 6.6 kb/cR. We estimate that the size of the remaining gap between the two contigs is of about 300 kb. The integrated physical and RH map of the investigated region on SSC 6q1.2 was used for a comparative analysis with respect to the syntenic regions on HSA 19q13.1 and MMU 7 and revealed a perfectly conserved gene order across the entire studied interval.

Animals↗
PubMed1996

Bacterial artificial chromosome cloning and mapping of a 630-kb human extrachromosomal structure.

We have cloned and mapped a circular 630-kb human extrachromosomal structure (termed amplisome) using the bacterial artificial chromosome (BAC) cloning system. Twenty-one BACs were isolated from an amplisome-enriched library by colony hybridization. The insert sizes range from 25 to 143 kb, with an average size of 82 kb. The coverage of the amplisome in clones is approximately 2.7-fold. To construct a physical map of the amplisome, we used three different but complementary methods: hybridization, STS content mapping, and fingerprinting. In addition, we compared the advantages and the drawbacks of these techniques in mapping the amplisomal BACs. The 21 BACs were grouped into two contigs and the two small gaps (3.5 and 26.5 kb) were filled by screening of a human genomic BAC library. The organization of the amplisome revealed by the BAC-based physical map is consistent with the long-range restriction map reported previously. Our results demonstrate that a 630-kb region can be rapidly cloned and mapped into contigs by use of the BAC system. Because of the low frequency (<0.1%) of chimerism and rearrangement, these BAC clones are ready for DNA sequencing and functional analysis.

Chromosome Mapping↗
PubMed1994

Yeast kar1 mutants provide an effective method for YAC transfer to new hosts.

Yeast artificial chromosome (YAC) clones propagate large segments of exogenous DNA in a host organism with well-developed classical and molecular genetics. Most extant YAC clones are from libraries created in a single yeast host (AB1380). The application of techniques allowing the manipulation and/or restructuring of these cloned DNA segments often requires a change in the yeast genetic background to introduce desirable genetic markers. Transfer methods in current use require extremely high yeast transformation efficiencies or require access to equipment for yeast tetrad analysis. We have developed an alternative method for moving YAC clones from one yeast strain to another, taking advantage of the properties of kar1 mutants altered in a gene required for normal karyogamy (nuclear fusion) during mating. Transfer by this method requires generally accessible methods, including yeast cell culture, replica plating, and pulsed-field gel electrophoresis. We present data demonstrating efficient transfer of nine different YACs from their original host (AB1380) to a kar1 recipient strain (YPH925) with genetic markers that facilitate the use of existing homologous recombination-based modification methods. The enhanced ability to transfer clones to this new host will accelerate the pace of refinement and fine-structure mapping of the YAC contigs currently under construction and facilitate gene manipulation on YACs for subsequent functional analysis.

Chromosomes, Artificial, Yeast↗
PubMed1995

A YAC contig encompassing the XRCC5 (Ku80) DNA repair gene and complementation of defective cells by YAC protoplast fusion.

The Chinese hamster ovary xrs mutants are sensitive to ionizing radiation, defective in DNA double-strand break rejoining, and unable to carry out V(D)J recombination effectively. Recently, the gene defective in these mutants, XRCC5, has been shown to encode Ku80, a component of the Ku protein and DNA-dependent protein kinase. We present here a YAC contig involving 25 YACs mapping to the region 2q33-q34, which encompasses the XRCC5 gene. Eight new markers for this region of chromosome 2 are identified. YACs encoding the Ku80 gene were transferred to xrs cells by protoplast fusion, and complementation of all the defective phenotypes has been obtained with two YACs. We discuss the advantages and disadvantages of this approach as a strategy for cloning human genes complementing defective rodent cell lines.

Animals↗
PubMed1997

An integrated physical and genetic map spanning chromosome band 10q24.

Chromosome band 10q24 is rich in genes involved in development, tumorigenesis, neurological disorders, hormone metabolism, and environmentally induced disease susceptibility. We have constructed an STS-based integrated physical and genetic map of 10q24 derived from the CEPH-Généthon mega-YAC contig data for this region. This map consists of 42 fluorescence in situ hybridization-mapped overlapping CEPH mega-YACs spanning approximately 15 Mb to which 49 STS markers have been assigned, including 24 Généthon CA repeat genetic markers, 10 known gene loci from the 10q24 region (IFI56, IDE, PDE6C, RBP4, CYP2C, CD39, DNTT, GOT1, WNT8B, and PAX2) and 11 additional expressed sequences of unknown function.

Chromosome Banding↗
PubMed1999

Optical PCR: genomic analysis by long-range PCR and optical mapping.

Optical mapping is an approach for the rapid, automated, non-electrophoretic construction of ordered restriction maps of DNA from ensembles of single molecules. Previously, we used optical mapping to make high-resolution maps of large insert clones such as bacterial artificial chromosomes (BAC) and large genomic DNA molecules. Here, we describe a combination of optical mapping and long-range polymerase chain reaction (PCR), in a process we term optical PCR, which enables automated construction of ordered restriction maps of long-range PCR products spanning human genomic loci. Specifically, we amplified three long PCR products, each averaging 14.6 kb in length, which span the 37-kb human tissue plasminogen activator (TPA) gene. PCR products were surface mounted in gridded arrays, and samples were mapped in parallel with either ScaI, XmnI, HpaI, ClaI, or BglII. A contig of overlapping high-resolution maps was generated, which agreed closely with maps predicted from sequence data. The data demonstrate an approach to construct physical maps of genomic loci where very little prior sequence information exists, since the only sequence needed is that required to anchor PCR primers. Large segments of genomic DNA (within the practical limits imposed by long-range PCR) can be mapped quickly and to high resolution without the use of cloning vectors.

DNA Restriction Enzymes↗