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1.
Nature ; 424(6950): 788-93, 2003 Aug 14.
Article in English | MEDLINE | ID: mdl-12917688

ABSTRACT

The systematic comparison of genomic sequences from different organisms represents a central focus of contemporary genome analysis. Comparative analyses of vertebrate sequences can identify coding and conserved non-coding regions, including regulatory elements, and provide insight into the forces that have rendered modern-day genomes. As a complement to whole-genome sequencing efforts, we are sequencing and comparing targeted genomic regions in multiple, evolutionarily diverse vertebrates. Here we report the generation and analysis of over 12 megabases (Mb) of sequence from 12 species, all derived from the genomic region orthologous to a segment of about 1.8 Mb on human chromosome 7 containing ten genes, including the gene mutated in cystic fibrosis. These sequences show conservation reflecting both functional constraints and the neutral mutational events that shaped this genomic region. In particular, we identify substantial numbers of conserved non-coding segments beyond those previously identified experimentally, most of which are not detectable by pair-wise sequence comparisons alone. Analysis of transposable element insertions highlights the variation in genome dynamics among these species and confirms the placement of rodents as a sister group to the primates.


Subject(s)
Conserved Sequence/genetics , Evolution, Molecular , Genomics , Vertebrates/genetics , Animals , Chromosomes, Human, Pair 7/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , DNA Transposable Elements/genetics , Genome , Humans , Mammals/genetics , Mutagenesis/genetics , Phylogeny , Sequence Alignment , Sequence Homology, Nucleic Acid , Species Specificity
2.
Mamm Genome ; 12(7): 508-12, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11420612

ABSTRACT

The comparative mapping and sequencing of vertebrate genomes is now a key priority for the Human Genome Project. In addition to finishing the human genome sequence and generating a 'working draft' of the mouse genome sequence, significant attention is rapidly turning to the analysis of other model organisms, such as the laboratory rat (Rattus norvegicus). As a complement to genome-wide mapping and sequencing efforts, it is often important to generate detailed maps and sequence data for specific regions of interest. Using an adaptation of our previously described approach for constructing mouse comparative and physical maps, we have established a general strategy for targeted mapping of the rat genome. Specifically, we constructed a framework comparative map of human Chromosome (Chr) 7 and the orthologous regions of the rat genome, as well as two large (>1-Mb) P1-derived artificial chromosome (PAC)-based physical maps. Generation of these physical maps involved the use of mouse-derived probes that cross-hybridized with rat PAC clones. The first PAC map encompasses the cystic fibrosis transmembrane conductance regulator gene (Cftr), while the second map allows a three-species comparison of a genomic region containing intra- and inter-chromosomal evolutionary rearrangements. The studies reported here further demonstrate that cross-species hybridization between related animals, such as rat and mouse, can be readily used for the targeted construction of clone-based physical maps, thereby accelerating the analysis of biologically interesting regions of vertebrate genomes.


Subject(s)
Chromosomes, Human, Pair 7/genetics , Physical Chromosome Mapping/methods , Sequence Analysis, DNA/methods , Animals , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Human Genome Project , Humans , In Situ Hybridization, Fluorescence , Mice , Nucleic Acid Hybridization , Rats
3.
Nature ; 409(6822): 934-41, 2001 Feb 15.
Article in English | MEDLINE | ID: mdl-11237014

ABSTRACT

The human genome is by far the largest genome to be sequenced, and its size and complexity present many challenges for sequence assembly. The International Human Genome Sequencing Consortium constructed a map of the whole genome to enable the selection of clones for sequencing and for the accurate assembly of the genome sequence. Here we report the construction of the whole-genome bacterial artificial chromosome (BAC) map and its integration with previous landmark maps and information from mapping efforts focused on specific chromosomal regions. We also describe the integration of sequence data with the map.


Subject(s)
Contig Mapping , Genome, Human , Chromosomes, Artificial, Bacterial , Cloning, Molecular , DNA Fingerprinting , Gene Duplication , Humans , In Situ Hybridization, Fluorescence , Repetitive Sequences, Nucleic Acid
4.
Genome Res ; 10(5): 624-33, 2000 May.
Article in English | MEDLINE | ID: mdl-10810084

ABSTRACT

The success of the ongoing Human Genome Project has resulted in accelerated plans for completing the human genome sequence and the earlier-than-anticipated initiation of efforts to sequence the mouse genome. As a complement to these efforts, we are utilizing the available human sequence to refine human-mouse comparative maps and to assemble sequence-ready mouse physical maps. Here we describe how the first glimpses of genomic sequence from human chromosome 7 are directly facilitating these activities. Specifically, we are actively enhancing the available human-mouse comparative map by analyzing human chromosome 7 sequence for the presence of orthologs of mapped mouse genes. Such orthologs can then be precisely positioned relative to mapped human STSs and other genes. The chromosome 7 sequence generated to date has allowed us to more than double the number of genes that can be placed on the comparative map. The latter effort reveals that human chromosome 7 is represented by at least 20 orthologous segments of DNA in the mouse genome. A second component of our program involves systematically analyzing the evolving human chromosome 7 sequence for the presence of matching mouse genes and expressed-sequence tags (ESTs). Mouse-specific hybridization probes are designed from such sequences and used to screen a mouse bacterial artificial chromosome (BAC) library, with the resulting data used to assemble BAC contigs based on probe-content data. Nascent contigs are then expanded using probes derived from newly generated BAC-end sequences. This approach produces BAC-based sequence-ready maps that are known to contain a gene(s) and are homologous to segments of the human genome for which sequence is already available. Our ongoing efforts have thus far resulted in the isolation and mapping of >3,800 mouse BACs, which have been assembled into >100 contigs. These contigs include >250 genes and represent approximately 40% of the mouse genome that is homologous to human chromosome 7. Together, these approaches illustrate how the availability of genomic sequence directly facilitates studies in comparative genomics and genome evolution.


Subject(s)
Chromosomes, Human, Pair 7/genetics , Human Genome Project , Physical Chromosome Mapping/methods , Sequence Analysis, DNA/methods , Animals , Humans
5.
Nat Genet ; 17(4): 411-22, 1997 Dec.
Article in English | MEDLINE | ID: mdl-9398842

ABSTRACT

Pendred syndrome is a recessively inherited disorder with the hallmark features of congenital deafness and thyroid goitre. By some estimates, the disorder may account for upwards of 10% of hereditary deafness. Previous genetic linkage studies localized the gene to a broad interval on human chromosome 7q22-31.1. Using a positional cloning strategy, we have identified the gene (PDS) mutated in Pendred syndrome and found three apparently deleterious mutations, each segregating with the disease in the respective families in which they occur. PDS produces a transcript of approximately 5 kb that was found to be expressed at significant levels only in the thyroid. The predicted protein, pendrin, is closely related to a number of known sulphate transporters. These studies provide compelling evidence that defects in pendrin cause Pendred syndrome thereby launching a new area of investigation into thyroid physiology, the pathogenesis of congenital deafness and the role of altered sulphate transport in human disease.


Subject(s)
Carrier Proteins/genetics , Hearing Loss, Sensorineural/genetics , Membrane Transport Proteins , Mutation , Sulfates/metabolism , Amino Acid Sequence , Animals , Biological Transport/genetics , Carrier Proteins/chemistry , Carrier Proteins/isolation & purification , Chromosome Mapping , Cloning, Molecular , Humans , Mice , Molecular Sequence Data , Pedigree , Rats , Sequence Alignment , Sequence Homology, Amino Acid , Sulfate Transporters , Syndrome
6.
Genome Res ; 7(7): 673-92, 1997 Jul.
Article in English | MEDLINE | ID: mdl-9253597

ABSTRACT

The construction of highly integrated and annotated physical maps of human chromosomes represents a critical goal of the ongoing Human Genome Project. Our laboratory has focused on developing a physical map of human chromosome 7, a approximately 170-Mb segment of DNA that corresponds to an estimated 5% of the human genome. Using a yeast artificial chromosome (YAC)-based sequence-tagged site (STS)-content mapping strategy, 2150 chromosome 7-specific STSs have been established and mapped to a collection of YACs highly enriched for chromosome 7 DNA. The STSs correspond to sequences generated from a variety of DNA sources, with particular emphasis placed on YAC insert ends, genetic markers, and genes. The YACs include a set of relatively nonchimeric clones from a human-hamster hybrid cell line as well as clones isolated from total genomic libraries. For map integration, we have localized 260 STSs corresponding to Genethon genetic markers and 259 STSs corresponding to markers orders by radiation hybrid (RH) mapping on our YAC contigs. Analysis of the data with the program SEGMAP results in the assembly of 22 contigs that are "anchored" on the Genethon genetic map, the RH map, and/or the cytogenetic map. These 22 contigs are ordered relative to one another, are (in all but 3 cases) oriented relative to the centromere and telomeres, and contain > 98% of the mapped STSs. The largest anchored YAC contig, accounting for most of 7p, contains 634 STSs and 1260 YACs. An additional 14 contigs, accounting for approximately 1.5% of the mapped STSs, are assembled but remain unanchored on either the genetic or RH map. Therefore, these 14 "orphan" contigs are not ordered relative to other contigs. In our contig maps, adjacent STSs are connected by two or more YACs in > 95% of cases. With 2150 mapped STSs, our map provides an average STS spacing of approximately 79 kb. The physical map we report here exceeds the goal of 100-kb average STS spacing and should provide an excellent framework for systematic sequencing of the chromosome.


Subject(s)
Chromosome Mapping , Chromosomes, Human, Pair 7 , Genome, Human , Chromosomes, Artificial, Yeast , Humans , Molecular Sequence Data
7.
Genome Res ; 7(3): 281-92, 1997 Mar.
Article in English | MEDLINE | ID: mdl-9074931

ABSTRACT

The establishment and mapping of gene-specific DNA sequences greatly complement the ongoing efforts to map and sequence all human chromosomes. To facilitate our studies of human chromosome 7, we have generated and analyzed 2006 expressed-sequence tags (ESTs) derived from a collection of direct selection cDNA libraries that are highly enriched for human chromosome 7 gene sequences. Similarity searches indicate that approximately two-thirds of the ESTs are not represented by sequences in the public databases, including those in dbEST. In addition, a large fraction (68%) of the ESTs do not have redundant or overlapping sequences within our collection. Human DNA-specific sequence-tagged sites (STSs) have been developed from 190 of the ESTs. Remarkably, 180 (96%) of these STSs map to chromosome 7, demonstrating the robustness of chromosome enrichment in constructing the direct selection cDNA libraries. Thus far, 140 of these EST-specific STSs have been assigned unequivocally to YAC contigs that are distributed across the chromosome. Together, these studies provide > 2000 ESTs highly enriched for chromosome 7 gene sequences, 180 new chromosome 7 STSs corresponding to ESTs, and a definitive demonstration of the ability to enrich for chromosome-specific cDNAs by direct selection. Furthermore, the libraries, sequence data, and mapping information will contribute to the construction of a chromosome 7 transcript map.


Subject(s)
Chromosomes, Human, Pair 7 , DNA, Complementary , Gene Library , Brain/embryology , Chromosome Mapping , Chromosomes, Artificial, Yeast , Gene Expression , HeLa Cells , Humans , Molecular Sequence Data , Placenta , Sequence Analysis, DNA , Sequence Tagged Sites , Thymus Gland
8.
Genome Res ; 7(1): 59-64, 1997 Jan.
Article in English | MEDLINE | ID: mdl-9037602

ABSTRACT

An established goal of the ongoing Human Genome Project is the development and mapping of sequence-tagged sites (STSs) every 100 kb, on average, across all human chromosomes. En route to constructing such a physical map of human chromosome 7, we have generated 1814 chromosome 7-specific STSs. The corresponding PCR assays were designed by the use of DNA sequence determined in our laboratory (79%) or generated elsewhere (21%) and were demonstrated to be suitable for screening yeast artificial chromosome (YAC) libraries. This collection provides the requisite landmarks for constructing a physical map of chromosome 7 at < 100-kb average spacing of STSs.


Subject(s)
Chromosome Mapping , Chromosomes, Human, Pair 7 , Sequence Tagged Sites , Base Sequence , Chromosomes, Artificial, Yeast , Humans , Molecular Sequence Data
9.
Genomics ; 25(1): 170-83, 1995 Jan 01.
Article in English | MEDLINE | ID: mdl-7774915

ABSTRACT

The paradigm of sequence-tagged site (STS)-content mapping involves the systematic assignment of STSs to individual cloned DNA segments. To date, yeast artificial chromosomes (YACs) represent the most commonly employed cloning system for constructing STS maps of large genomic intervals, such as whole human chromosomes. For developing a complete YAC-based STS-content map of human chromosome 7, we wished to utilize a limited set of YAC clones that were highly enriched for chromosome 7 DNA. Toward that end, we have assembled a human chromosome 7 YAC resource that consists of three major components: (1) a newly constructed library derived from a human-hamster hybrid cell line containing chromosome 7 as its only human DNA; (2) a chromosome 7-enriched sublibrary derived from the CEPH mega-YAC collection by Alu-polymerase chain reaction (Alu-PCR)-based hybridization; and (3) a set of YACs isolated from several total genomic libraries by screening for > 125 chromosome 7 STSs. In particular, the hybrid cell line-derived YACs, which comprise the majority of the clones in the resource, have a relatively low chimera frequency (10-20%) based on mapping isolated insert ends to panels of human-hamster hybrid cell lines and analyzing individual clones by fluorescence in situ hybridization. An efficient strategy for polymerase chain reaction (PCR)-based screening of this YAC resource, which totals 4190 clones, has been developed and utilized to identify corresponding YACs for > 600 STSs. The results of this initial screening effort indicate that the human chromosome 7 YAC resource provides an average of 6.9 positive clones per STS, a level of redundancy that should support the assembly of large YAC contigs and the construction of a high-resolution STS-content map of the chromosome.


Subject(s)
Chromosomes, Artificial, Yeast , Chromosomes, Human, Pair 7 , Animals , Base Sequence , Chromosome Mapping , Cloning, Molecular , Cricetinae , DNA/genetics , DNA/isolation & purification , DNA Primers , Electrophoresis, Agar Gel , Humans , Hybrid Cells , In Situ Hybridization, Fluorescence , Molecular Sequence Data , Molecular Weight , Polymerase Chain Reaction , Restriction Mapping , Sequence Tagged Sites
10.
Hum Mol Genet ; 3(3): 489-501, 1994 Mar.
Article in English | MEDLINE | ID: mdl-8012362

ABSTRACT

An important goal for the human genome project is to assemble fully integrated physical, genetic and cytogenetic maps for each human chromosome. Towards that end, we have isolated yeast artificial chromosome (YAC) clones containing 117 of the 119 genetic markers that constitute a recently constructed, detailed genetic map of human chromosome 7. Analysis of these clones reveals numerous examples where adjacent genetic markers have been physically connected, either in individual YACs or in multi-YAC contigs. At present, the 117 genetic markers are contained in fewer than 80 YAC contigs, with most of these contigs uniquely ordered relative to one another based on the genetic map positions of the corresponding markers. These YACs and YAC contigs are estimated to contain approximately 60-85% of the DNA from human chromosome 7. YACs representing 36 genetic markers were mapped by fluorescence in situ hybridization (FISH) to metaphase chromosomes, allowing assignment of these genetic markers to cytogenetic bands along chromosome 7 and placement of the centromere within the genetic map. Together, these studies provide genetically and cytogenetically anchored YAC clones covering the majority of chromosome 7 that will be useful both for the positional cloning of genes and as a framework for assembling a complete YAC-based physical map of the chromosome.


Subject(s)
Chromosomes, Human, Pair 7 , Genetic Markers , Chromosome Mapping , Chromosomes, Artificial, Yeast , Cloning, Molecular , Humans , In Situ Hybridization, Fluorescence
11.
Genomics ; 11(3): 548-64, 1991 Nov.
Article in English | MEDLINE | ID: mdl-1837788

ABSTRACT

Basic to the development of long-range physical maps of DNA are the detection and localization of landmarks within recombinant clones. Sequence-tagged sites (STSs), which are short stretches of DNA that can be specifically detected by the polymerase chain reaction (PCR), can be used as such landmarks. Our interest is to construct physical maps of whole human chromosomes by localizing STSs within yeast artificial chromosome (YAC) clones. Here we describe a generalized strategy for the systematic generation of large numbers of STSs specific for human chromosome 7. These STSs can be detected by PCR assays developed following the sequencing of anonymous pieces of chromosome 7 DNA, which was derived from flow-sorted chromosomes or from lambda clones made from DNA of a human-hamster hybrid cell line. Our approach for STS generation is tailored for the development of PCR assays capable of screening a large YAC library. In this study, we report the generation of 100 new STSs specific to human chromosome 7.


Subject(s)
Chromosome Mapping/methods , Chromosomes, Human, Pair 7 , Oligodeoxyribonucleotides/genetics , Sequence Tagged Sites , Animals , Bacteriophage lambda/genetics , Base Sequence , Chromosomes, Fungal , Cloning, Molecular , Cricetinae , Humans , Hybrid Cells , Molecular Sequence Data , Polymerase Chain Reaction , Temperature
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