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PubMed1999

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS): high-resolution physical and transcript map of the candidate region in chromosome region 13q11.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS or SACS) is a neurodegenerative disease frequent in northeastern Québec. In a previous study, we localized the disease gene to chromosome region 13q11 by identifying excess sharing of a marker allele in patients followed by linkage analysis and haplotyping. To create a detailed physical map of this region, we screened CEPH mega-YACs with 41 chromosome 13 sequence-tagged-sites (STSs) known to map to 13q11-q12. The YAC contig, composed of 27 clones, extends on the genetic map from D13S175 to D13S221, an estimated distance of at least 19.3 cM. A high-resolution BAC and PAC map that includes the ARSACS critical region flanked by D13S1275 and D13S292 was constructed. These YAC and BAC/PAC maps allowed the accurate placement of 29 genes and ESTs previously mapped to the proximal region of chromosome 13q. We confirmed the position of two candidate genes within the critical region and mapped the other 27 genes and ESTs to nearby intervals. Six BAC/PAC clones form a contig between D13S232 and D13S787 for sequencing within the ARSACS critical region.

Bacteriophage P1↗
PubMed2000

Construction of a physical and transcript map flanking the imprinted MEST/PEG1 region at 7q32.

MEST/PEG1, a gene expressed paternally in mesodermal derivatives in early embryonic stages, is the first imprinted gene mapped to chromosome 7q32. Since imprinted genes are clustered in general at a chromosomal region, we speculated that a similar imprinted-gene cluster may exist at chromosome region 7q32 and that the functions of some such genes may contribute to the phenotype of Silver-Russell syndrome including maternal uniparental disomy for chromosome 7 (maternal UPD7). As an initial step toward the isolation of imprinted genes at 7q32, we adopted an integrated approach involving the construction of a PAC contig and ESTs mapping in the vicinity of MEST. Here, we have constructed a complete contig of PAC and BAC clones and a transcript map spanning the entire approximately 1-Mb region between D7S530 and D7S649. We developed 60 novel STSs and precisely mapped 47 genes/ESTs. This map contains a putative autistic disorder locus that has been suggested to be localized near markers D7S530 and D7S684. This integrated physical and transcript map provides a valuable resource for identification of an imprinted gene(s) in this region as well as a candidate gene(s) for autistic disorder.

Base Sequence↗
PubMed1999

Isolation of human transcripts expressed in hamster cells from YACs by cDNA representational difference analysis.

Gene isolation methods used during positional cloning rely on physical contigs consisting of bacterial artificial chromosomes, P1, or cosmid clones. However, in most instances, the initial framework for physical mapping consists of contigs of yeast artificial chromosome (YACs), large vectors that are suboptimal substrates for gene isolation. Here we report a strategy to identify gene sequences contained within a YAC by using cDNA representational difference analysis (RDA) to directly isolate transcripts expressed from the YAC in mammalian cells. The RDA tester cDNAs were generated from a previously reported hamster cell line derived by stable transfer of a 590-kb YAC (911D5) that expressed NPC1, the human gene responsible for Niemann-Pick type C (NP-C). The driver cDNAs were generated from a control hamster cell line that did not contain the YAC that expressed NPC1. Among the gene fragments obtained by RDA, NPC1 was the most abundant product. In addition, two non-NPC1 fragments were isolated that were mapped to and expressed from 911D5. One of these RDA gene fragments (7-R) spans more than one exon and has 98% sequence identity with a human cDNA clone reported previously as an expressed sequence tag (EST), but not mapped to a chromosomal region. The other fragment (2-R) that had no significant sequence similarities with known mammalian genes or ESTs, was further localized to the region of overlap between YACs 911D5 and 844E3. The latter YAC is part of a contig across the NP-C candidate region, but does not contain NPC1. This two-part approach in which stable YAC transfer is followed by cDNA RDA should be a useful adjunct strategy to expedite the cloning of human genes when a YAC contig is available across a candidate interval.

Animals↗
PubMed1998

BAC/YAC contigs from the H2-M region of mouse Chr 17 define gene order as Znf173-Tctex5-mog-D17Tu42-M3-M2.

A yeast artificial chromosome (YAC) contig from the C57BL/6 (H2(b)) mouse was created from the major histocompatibility complex (Mhc, H2 in mouse) class Ib subregion, H2-M. It spans approximately 1.2 megabase (Mb) pairs and unites the previous >1.5-Mb YAC contigs (Jones et al. 1995) into a single contig, which includes 21 Mhc class I genes distal to H2-T1. A bacterial artificial chromosome (BAC) contig from the 129 (H2(bc)) mouse, spanning approximately 600 kilobases, was also built from Znf173 (Afp, a gene for acid finger protein), through Tctex5 (t-complex testis expressed-5) and Mog (myelin oligodendrocyte glycoprotein), to H2-M2. Twenty-four sequence-tagged site (STS) markers were newly developed, and 35 markers were mapped in the YAC/BAC contigs, which define the marker order as Cen - Znf173 - Tctex5 - Mog - D17Tu42 - D17Mit232 - H2-M3 - D17Leh525 - H2-M2 - Tel. The gene order of Znf173 - Tctex5 - Mog - D17Tu42 is conserved between mouse and human, showing that the middle H2-M region corresponds to the subregion of the human Mhc surrounding HLA-A.

Animals↗
PubMed1996

Toward the construction of integrated physical and genetic maps of the mouse genome using interspersed repetitive sequence PCR (IRS-PCR) genomics.

Using two recently developed techniques, IRS-PCR YAC walking and IRS-PCR genotyping, a framework-integrated physical and genetic map of the mouse genome was constructed. The map consists of 821 contigs, containing 7746 YAC clones originating from three different YAC libraries. Three hundred eighty of the contigs have been anchored to the genetic map. Approximately 16% of the physical length of the mouse genome is estimated to be represented.

Animals↗
PubMed1998

Fine localization of the CMT4A locus using a PAC contig and haplotype analysis.

Charcot-Marie-Tooth disease type 4A (CMT4A) is a severe, autosomal recessive peripheral neuropathy linked to chromosome 8q13-q21. We have previously constructed a YAC contig across the CMT4A region and narrowed the disease-flanking interval to approximately three megabases. Subsequently, we constructed a PAC/BAC contig made of 44 clones and mapped 44 new and 30 previous STSs, ESTs, and polymorphic makers to the region. Using 13 polymorphic markers, we have now identified an ancestral haplotype segregating in three families, indicating a common founder mutation. Two ancestral recombination events in this haplotype significantly reduce the minimal candidate region to a minimal trailing path of five PAC/BAC clones, which will now allow direct investigation of candidate genes for CMT4A.

Alleles↗
PubMed2002

A fine physical map of the rice chromosome 4.

As part of an international effort to completely sequence the rice genome, we have produced a fine bacterial artificial chromosome (BAC)-based physical map of the Oryza sativa japonica Nipponbare chromosome 4 through an integration of 114 sequenced BAC clones from a taxonomically related subspecies O. sativa indica Guangluai 4 and 182 RFLP and 407 expressed sequence tag (EST) markers with the fingerprinted data of the Nipponbare genome. The map consists of 11 contigs with a total length of 34.5 Mb covering 94% of the estimated chromosome size (36.8 Mb). BAC clones corresponding to telomeres, as well as to the centromere position, were determined by BAC-pachytene chromosome fluorescence in situ hybridization (FISH). This gave rise to an estimated length ratio of 5.13 for the long arm and 2.9 for the short arm (on the basis of the physical map), which indicates that the short arm is a highly condensed one. The FISH analysis and physical mapping also showed that the short arm and the pericentromeric region of the long arm are rich in heterochromatin, which occupied 45% of the chromosome, indicating that this chromosome is likely very difficult to sequence. To our knowledge, this map provides the first example of a rapid and reliable physical mapping on the basis of the integration of the data from two taxonomically related subspecies.

Chromosomes↗
PubMed1996

Genetic and physical mapping of the Chediak-Higashi syndrome on chromosome 1q42-43.

The Chediak-Higashi syndrome (CHS) is a severe autosomal recessive condition, features of which are partial oculocutaneous albinism, increased susceptibility to infections, deficient natural killer cell activity, and the presence of large intracytoplasmic granulations in various cell types. Similar genetic disorders have been described in other species, including the beige mouse. On the basis of the hypothesis that the murine chromosome 13 region containing the beige locus was homologous to human chromosome 1, we have mapped the CHS locus to a 5-cM interval in chromosome segment 1q42.1-q42.2. The highest LOD score was obtained with the marker D1S235 (Zmax = 5.38; theta = 0). Haplo-type analysis enabled us to establish D1S2680 and D1S163, respectively, as the telomeric and the centromeric flanking markers. Multipoint linkage analysis confirms the localization of the CHS locus in this interval. Three YAC clones were found to cover the entire region in a conting established by YAC end-sequence characterization and sequence-tagged site mapping. The YAC contig contains all genetic markers that are nonrecombinant for the disease in the nine CHS families studied. This mapping confirms the previous hypothesis that the same gene defect causes CHS in human and beige pheno-type in mice and provides a genetic framework for the identification of candidate genes.

Animals↗
PubMed1995

The organization of the human immunoglobulin lambda gene locus.

To elucidate the complex structure of the human immunoglobulin lambda gene locus, a 1020-kb contig was constructed using 184 cosmid clones and one bacterial artificial chromosome (BAC) clone. A high-resolution physical map of this contig revealed that the entire lambda gene locus is 911 kb in length. It contains seven constant region (C lambda) gene segments and 69 unique EcoRI-HindIII segments that hybridize to variable region gene (V lambda) probes. The VpreB gene, BCRL4, and gamma-glutamyl transpeptidase gene (GGT)-like sequences are also located within the lambda gene locus. Hybridization analysis suggested that the lambda gene locus has undergone extensive amplification events in evolution.

Base Sequence↗
PubMed2001

An integrated map of Arabidopsis thaliana for functional analysis of its genome sequence.

The genome of the model plant species Arabidopsis thaliana has recently been sequenced. To accelerate its current genome research, we developed a whole-genome, BAC/BIBAC-based, integrated physical, genetic, and sequence map of the A. thaliana ecotype Columbia. This new map was constructed from the clones of a new plant-transformation-competent BIBAC library and is integrated with the existing sequence map. The clones were restriction fingerprinted by DNA sequencing gel-based electrophoresis, assembled into contigs, and anchored to an existing genetic map. The map consists of 194 BAC/BIBAC contigs, spanning 126 Mb of the 130-Mb Arabidopsis genome. A total of 120 contigs, spanning 114 Mb, were anchored to the chromosomes of Arabidopsis. Accuracy of the integrated map was verified using the existing physical and sequence maps and numerous DNA markers. Integration of the new map with the sequence map has enabled gap closure of the sequence map and will facilitate functional analysis of the genome sequence. The method used here has been demonstrated to be sufficient for whole-genome physical mapping from large-insert random bacterial clones and thus is applicable to rapid development of whole-genome physical maps for other species.

Arabidopsis↗
PubMed1995

High-resolution DNA Fiber-FISH for genomic DNA mapping and colour bar-coding of large genes.

We have applied two-colour fluorescence in situ hybridization (FISH) to DNA fibers and combined it with digital imaging microscopy for the mapping of large cosmid contigs. The technique was validated using a set of unique plasmids and a cosmid contig both originating from the thyroglobulin (Tg) gene and previously mapped by restriction analysis. The resolution proved to be close to the theoretical lower limit of approximately 1 kb, ranging > or = 400 kb. Subsequently a 400 kb cosmid contig derived from a DMD-YAC was directly mapped by Fiber-FISH. The resulting map is in full agreement with the restriction map. Two-colour Fiber-FISH mapping thus showed to be capable for accurately sizing gaps and overlaps, and to identify chimeric or repeat sequence containing cosmids across a 400 kb region at once. The generated 400 kb 'colour bar-code' was subsequently used to map two DMD deletion breakpoints in patient DNA with an accuracy of 1-2 kb. The results underscore the value of this method for the delineation of chromosomal rearrangements for positional cloning and single patient clinical studies.

Chimera↗
PubMed2004

Sequence analysis of the long arm of rice chromosome 11 for rice-wheat synteny.

The DNA sequence of 106 BAC/PAC clones in the minimum tiling path (MTP) of the long arm of rice chromosome 11, between map positions 57.3 and 116.2 cM, has been assembled to phase 2 or PLN level. This region has been sequenced to 10x redundancy by the Indian Initiative for Rice Genome Sequencing (IIRGS) and is now publicly available in GenBank. The region, excluding overlaps, has been predicted to contain 2,932 genes using different software. A gene-by-gene BLASTN search of the NCBI wheat EST database of over 420,000 cDNA sequences revealed that 1,143 of the predicted rice genes (38.9%) have significant homology to wheat ESTs (bit score >/= 100). Further BLASTN search of these 1,143 rice genes with the GrainGenes database of sequence contigs containing bin-mapped wheat ESTs allowed 113 of the genes to be placed in bins located on wheat chromosomes of different homoeologous groups. The largest number of genes, about one-third, mapped to the homoeologous group 4 chromosomes of wheat, suggesting a common evolutionary origin. The remaining genes were located on wheat chromosomes of different groups with significantly higher numbers for groups 3 and 5. Location of bin-mapped wheat contigs to chromosomes of all the seven homoeologous groups can be ascribed to movement of genes (transpositions) or chromosome segments (translocations) within rice or the hexaploid wheat genomes. Alternatively, it could be due to ancient duplications in the common ancestral genome of wheat and rice followed by selective elimination of genes in the wheat and rice genomes. While there exists definite conservation of gene sequences and the ancestral chromosomal identity between rice and wheat, there is no obvious conservation of the gene order at this level of resolution. Lack of extensive colinearity between rice and wheat genomes suggests that there have been many insertions, deletions, duplications and translocations that make the synteny comparisons much more complicated than earlier thought. However, enhanced resolution of comparative sequence analysis may reveal smaller conserved regions of colinearity, which will facilitate selection of markers for saturation mapping and sequencing of the gene-rich regions of the wheat genome.

Base Sequence↗
PubMed1998

Cloning and comparative mapping of the DiGeorge syndrome critical region in the mouse.

Chromosome deletions leading to the hemizygous loss of groups of contiguous genes are a major cause of human congenital defects. In some syndromes haploinsufficiency of a single gene causes the majority of the syndromal features, whereas other diseases are thought to be the consequences of a combined haploinsufficiency. In the case of the DiGeorge and velocardiofacial syndromes, caused by deletions within 22q11, the genetic analyses have so far failed to implicate a single gene. By virtue of FISH analysis and the creation of a BAC/P1 genomic clone contig we have mapped 19 murine homologues of genes and nine EST groups from the region deleted in DiGeorge syndrome and found them to be linked on mouse chromosome 16. Rearrangements during the divergence of mouse and human have led to differing gene orders in the two species, with implications for the most appropriate means of mimicking particular human deletions. The map confirms and extends previous analyses and the contig resources toward the generation of targeted deletions in the mouse.

Animals↗
PubMed2004

Fosmid-based physical mapping of the Histoplasma capsulatum genome.

A fosmid library representing 10-fold coverage of the Histoplasma capsulatum G217B genome was used to construct a restriction-based physical map. The data obtained from three restriction endonuclease fingerprints, generated from each clone using BamHI, HindIII, and PstI endonucleases, were combined and used in FPC for automatic and manual contig assembly builds. Concomitantly, a whole-genome shotgun (WGS) sequencing of paired-end reads from plasmids and fosmids were assembled with PCAP, providing a predicted genome size of up to 43.5 Mbp and 17% repetitive DNA. Fosmid paired-end sequences in the WGS assembly provide anchoring information to the physical map and result in joining of existing physical map contigs into 84 clusters containing 9551 fosmid clones. Here, we detail mapping the Histoplasma capsulatum genome comprehensively in fosmids, resulting in an efficient paradigm for de novo sequencing that uses a map-assisted whole genome shotgun approach.

Contig Mapping↗
PubMed1998

Construction of a 2.5-Mb integrated physical and gene map of distal 21q22.3.

The gene-rich telomeric region of 21q harbors several loci relevant to human diseases including autoimmune polyglandular disease type I, nonsyndromic deafness, Knobloch syndrome, holoprosencephaly, and bipolar affective disorder. A contig of genomic clones in this region would facilitate the isolation of these genes. However, distal 21q22.3 has yet been poorly mapped, presumably due to the presence of sequences that are underrepresented in yeast artificial chromosome (YAC) libraries. We generated a framework of YACs and used these clones as starting points for the isolation of a combination of bacterial artificial chromosome clones, P1-derived artificial chromosome clones, and cosmid clones by chromosome walking procedures. These studies resulted in the construction of a high-resolution contig map spanning the 2.5-Mb region from PFKL to the telomere, approximately 2 Mb of which are covered by ready-to-sequence contigs. Within this map we determined the location and relative distance of 21 markers. These include 9 established genetic markers, the order of which is cen-PFKL-D21S154-D21S170-D21S171-D21S1903- D21S1897- D21S112-D21S1446-D21S1575-tel. Moreover, we established the precise map position of 13 genes and 4 ESTs including the recently isolated genes C21ORF2, SMT3H1, RNA editing deaminase 1 (ADARB1), folate transporter (SLC19A1), COL18A1, lanosterol synthase (LSS-PEN), pericentrin (PCNT), and arginine methyltransferase (HRMT1L1). This integrated map provides a useful resource for the mapping and isolation of disease genes and for the construction of a complete transcription map of distal 21q as well as for large-scale sequencing efforts.

Chromosome Mapping↗
PubMed2005

An integrated map of Oryza sativa L. chromosome 5.

The developments of molecular marker-based genetic linkage maps are now routine. Physical maps based on contigs of large insert genomic clones have been established in several plant species. However, integration of genetic, physical, and cytological maps is still a challenge for most plant species. Here we present an integrated map of rice (Oryza sativa L.) chromosome 5, developed by fluorescence in situ hybridization mapping of 18 bacterial artificial chromosome (BAC) clones or PI-derived artificial chromosome (PAC) clones on meiotic pachytene chromosomes. Each BAC/PAC clone was anchored by a restriction fragment length polymorphism marker mapped to the rice genetic linkage map. This molecular cytogenetic map shows the genetic recombination and sequence information of a physical map, correlated to the cytological features of rice chromosome 5. Detailed comparisons of the distances between markers on genetic, cytological, and physical maps, revealed the distributions of recombination events and molecular organization of the chromosomal features of rice chromosome 5 at the pachytene stage. Discordance of distances between the markers was found among the different maps. Our results revealed that neither the recombination events nor the degree of chromatin condensation were evenly distributed along the entire length of chromosome 5. Detailed comparisons of the correlative positions of markers on the genetic, cytological, and physical maps of rice chromosome 5 provide insight into the molecular architecture of rice chromosome 5, in relation to its cytological features and recombination events on the genetic map. The prospective applications of such an integrated cytogenetic map are discussed.

Chromosome Mapping↗
PubMed1995

Efficient high-resolution genetic mapping of mouse interspersed repetitive sequence PCR products, toward integrated genetic and physical mapping of the mouse genome.

The ability to carry out high-resolution genetic mapping at high throughput in the mouse is a critical rate-limiting step in the generation of genetically anchored contigs in physical mapping projects and the mapping of genetic loci for complex traits. To address this need, we have developed an efficient, high-resolution, large-scale genome mapping system. This system is based on the identification of polymorphic DNA sites between mouse strains by using interspersed repetitive sequence (IRS) PCR. Individual cloned IRS PCR products are hybridized to a DNA array of IRS PCR products derived from the DNA of individual mice segregating DNA sequences from the two parent strains. Since gel electrophoresis is not required, large numbers of samples can be genotyped in parallel. By using this approach, we have mapped > 450 polymorphic probes with filters containing the DNA of up to 517 backcross mice, potentially allowing resolution of 0.14 centimorgan. This approach also carries the potential for a high degree of efficiency in the integration of physical and genetic maps, since pooled DNAs representing libraries of yeast artificial chromosomes or other physical representations of the mouse genome can be addressed by hybridization of filter representations of the IRS PCR products of such libraries.

Animals↗
PubMed1997

Framework YAC contig anchored into a 3.2-Mb high-resolution physical map in proximal 11q13.

Despite the presence on band q13 of chromosome 11 of a number of genes predisposing individuals to various human diseases, most of this genomic region remains loosely mapped. Moreover, there is a relative dearth of yeast artificial chromosome (YAC) contigs from genome-wide studies: YACs are irregularly distributed over this chromosomal region and have not been arranged into contigs. We have thus undertaken fine-scale mapping of a 3.2-Mb region flanked by ACTN3 and FGF3. Since this region has demonstrated a high degree of YAC instability, we have established a framework contig by anchoring YACs and cosmids into a high-resolution physical map based on fluorescence in situ hybridization and long-range restriction mapping. The 3.2-Mb area studied includes the boundaries of regions thought to contain genes predisposing individuals to osteoporosis-pseudoglioma syndrome and insulin-dependent diabetes mellitus, as well as genes driving amplification events in human carcinomas. Another feature of this genomic area is that it cross-hybridizes to nonsyntenic regions of the genome. In addition, it spans the region where syntenic conservation with mouse chromosome 19 ends, making clones that we have anchored there valuable tools in understanding genome evolution.

Animals↗