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PubMed1997

A 2-Mb YAC contig and physical map of the natural killer gene complex on mouse chromosome 6.

We have constructed a physical map of a > 2-Mb region on mouse chromosome 6 that contains the natural killer gene complex (NKC). The map comprises a contig of 14 overlapping yeast artificial chromosomes onto which we positioned 25 NKC markers. NKC genetically linked genes encode > 17 proteins that directly control innate NK cell-mediated tumor lysis and disease resistance. Herein we show that Nkrp1 genes are clustered in a region flanked by A2m and Cd69 genes and that most Ly49 genes are clustered in a distal region -1 Mb distant. Importantly, syntenic intervals of mouse chromosome 6 and human chromosome 12p that include the NKC are conserved. NKC species conservation suggests that the human NKC may contain orthologues for the mouse viral disease resistance genes, Cmv1 and Rmp1. The high-resolution NKC map will facilitate investigation of NKC gene regulation and identification of phenotypically defined gene products that confer NK cell defense against viral pathogens.

Animals↗
PubMed1994

A high-resolution cytogenetic map of human chromosome 9: localization of 203 new cosmid markers by direct R-banding fluorescence in situ hybridization.

A high-resolution cytogenetic map of human chromosome 9 was constructed with 203 newly isolated cosmid markers by direct R-banding fluorescence in situ hybridization. The clones were localized preferentially to R-positive bands throughout chromosome 9. Although the number of clones was roughly proportional to the R-band size, many more clones (ca. 80) were mapped at the q34 region compared to its physical size. Based on these cytogenetic mapping data, one can establish physical contig maps with cosmids and yeast artificial chromosomes and a genetic linkage map essential for positional cloning of responsible genes.

Animals↗
PubMed1991

Current trends in mapping human genes.

The human is estimated to have at least 50,000 expressed genes (gene loci). Some information is available concerning about 5000 of these gene loci and about 1900 have been mapped, i.e., assigned to specific chromosomes (and in most instances particular chromosome regions). Progress has been achieved by a combination of physical mapping (e.g., study of somatic cell hybrids and chromosomal in situ hybridization) and genetic mapping (e.g., genetic linkage studies). New methods for both physical and genetic mapping are expanding the armamentarium. The usefulness of the mapping information is already evident; the spin-off from the Human Genome Project (HGP) begins immediately. The complete nucleotide sequence is the ultimate map of the human genome. Sequencing, although already under way for limited segments of the genome, will await further progress in gene mapping, and in particular creation of contig maps for each chromosome. Meanwhile the technology of sequencing and sequence information handling will be developed. It is argued that the HGP is a new form of coordinated, interdisciplinary science; that its primary objective must be seen as the creation of a tool for biomedical research--a source book that will be the basis of study of variation and function for a long time; that the impact on scientist training will be salutary by relieving graduate students of useless drudgery and by training scientists competent in both molecular genetics and computational science; and that the funding of the HGP will have an insignificant negative effect on science funding generally, and indeed may have a beneficial effect through economy of scale and a focusing of attention on the excitement of biology and medical science.

Chromosome Mapping↗
PubMed1999

Comparative mapping of the region of human chromosome 7 deleted in williams syndrome.

Williams syndrome (WS) is a complex developmental disorder resulting from the deletion of a large (approximately 1.5-2 Mb) segment of human chromosome 7q11.23. Physical mapping studies have revealed that this deleted region, which contains a number of known genes, is flanked by several large, nearly identical blocks of DNA. The presence of such highly related DNA segments in close physical proximity to one another has hampered efforts to elucidate the precise long-range organization of this segment of chromosome 7. To gain insight about the structure and evolutionary origins of this important and complex genomic region, we have constructed a fully contiguous bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) contig map encompassing the corresponding region on mouse chromosome 5. In contrast to the difficulties encountered in constructing a clone-based physical map of the human WS region, the BAC/PAC-based map of the mouse WS region was straightforward to construct, with no evidence of large duplicated segments, such as those encountered in the human WS region. To confirm this difference, representative human and mouse BACs were used as probes for performing fluorescence in situ hybridization (FISH) to metaphase and interphase chromosomes. Human BACs derived from the nonunique portion of the WS region hybridized to multiple, closely spaced regions on human chromosome 7q11.23. In contrast, corresponding mouse BACs hybridized to a single site on mouse chromosome 5. Furthermore, FISH analysis revealed the presence of duplicated segments within the WS region of various nonhuman primates (chimpanzee, gorilla, orangutan, and gibbon). Hybridization was also noted at the genomic locations corresponding to human chromosome 7p22 and 7q22 in human, chimpanzee, and gorilla, but not in the other animal species examined. Together, these results indicate that the WS region is associated with large, duplicated blocks of DNA on human chromosome 7q11.23 as well as the corresponding genomic regions of other nonhuman primates. However, such duplications are not present in the mouse.

Animals↗
PubMed1993

Characterisation of molecular DNA rearrangements within the Xq12-q13.1 region, in three patients with X-linked hypohidrotic ectodermal dysplasia (EDA).

A panel of somatic cell hybrids and X-linked hypohidrotic ectodermal dysplasia (EDA) patient-derived cell lines, containing different rearranged X chromosomes, have been used to refine the physical map of the Xq12-q13.1 region. The patient-derived material included genomic DNA from an EDA male (EDA family 1015) with an interstitial deletion, and a cell line GM0705A, obtained from an isolated female patient with a de novo balanced (X;9) translocation, and the somatic hybrid, AnLy, derived from this cell line. This map subdivides the region into at least 6 mapping-intervals. DNA probes from DXS732 and DXS453, identified as the closest flanking marker loci to the EDA locus, were used to identify homologous Yeast Artificial Chromosome (YAC) clones. Two of the DXS732-specific YACs were shown by fluorescent in situ hybridisation (FISH) analysis to bridge the (X;9) translocation breakpoint. These two YACs were also screened against the ICRF human X chromosome cosmid library and identified 36 cosmid clones. Direct cosmid-cosmid hybridisation analysis placed subsets of these clones within four different cosmid contigs. Mapping of anchor clones from each contig, against the mapping panel, localised all these contigs within the Xq12-q13.1 region. One cosmid, ICRFc104C03.184, identified potential junctional-fragments in several restriction digests of AnLy hybrid DNA. This was confirmed by FISH analysis of the GM0705A cell line with total cosmid ICRFc104C03.184, in which both chromosomal elements of the (X;9) translocation were identified. A single-copy probe pC03.184E2, derived from this cosmid, also identified the der(9)-derived junctional fragment when hybridised against AnLy DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Line↗
PubMed2000

ComboScreen facilitates the multiplex hybridization-based screening of high-density clone arrays.

MOTIVATION: The construction of physical maps based on bacterial clones [e.g. bacterial artificial chromosomes (BACs)] is valuable for a number of molecular genetics applications, including the high-resolution mapping of genomic regions of interest and the identification of clones suitable for systematic sequencing. A common approach for large-scale screening of bacterial clone libraries involves the hybridization of high-density arrays of immobilized, lysed colonies with collections of DNA probes. The use of a multiplex hybridization screening strategy, whereby pooled probes are analysed en masse, simplifies the effort by reducing the total number of parallel experiments required. However, this approach generates large amounts of hybridization-based data that must be carefully analysed, assimilated, and disambiguated in a careful but efficient manner. RESULTS: To facilitate the screening of high-density clone arrays by a multiplex hybridization approach, we have written a program called ComboScreen. This program provides an organizational framework and analytical tools required for the high-throughput hybridization screening of clone arrays with pools of probes. We have used this program extensively for constructing mouse sequence-ready BAC contig maps.

Chromosomes, Artificial, Bacterial↗
PubMed1993

A high-resolution cytogenetic map of human chromosome 12: localization of 195 new cosmid markers by direct R-banding fluorescence in situ hybridization.

We have constructed a high-resolution cytogenetic map of human chromosome 12 with 195 newly isolated cosmids by direct R-banding fluorescence in situ hybridization. The fluorescent signals of 195 clones were evenly distributed throughout chromosome 12, but sublocalized preferentially to R-positive bands. This high-resolution cytogenetic map with an average map distance of 0.73 Mb on bands can, in conjunction with a genetic linkage map, facilitate the analysis of chromosomal and molecular aberrations in genetic diseases and cancers. Moreover, the cytogenetic mapping data provide starting points for establishing contig maps with cosmid clones and yeast artificial chromosomes.

Animals↗
PubMed2001

Leucodystrophy and oculocutaneous albinism in a child with an 11q14 deletion.

We report a patient with an undetermined leucodystrophy associated with type 1A oculocutaneous albinism (OCA). Type 1 OCA results from recessive mutations in the tyrosinase gene (TYR) located in 11q14.3. The patient was found by FISH to carry a deletion of at least the first exon of the TYR gene on one chromosome and a (TG) deletion at codon 244/245 on the second chromosome. The existence of the microdeletion suggested that a gene responsible for leucodystrophy was located in the vicinity of the TYR gene. A combination of a test of hemizygosity and contig mapping studies allowed us to map the gene within a 0.6 cM region flanked by microsatellite markers D11S1780 and D11S931.

Adolescent↗
PubMed1999

Contig assembly of bacterial artificial chromosome clones through multiplexed fluorescence-labeled fingerprinting.

A rapid multiplexed fingerprinting method has been developed for bacterial artificial chromosome (BAC) contig assembly. Defined subsets of BAC DNA fragments that result from digestion by three paired restriction endonucleases are labeled with unique fluorescent F-ddATP for each subset. Lists of the labeled fragment size are generated by an ABI 377 DNA sequencer and the GeneScan analysis software and then processed by an assembly program, FPC (Fingerprinted Contigs), to produce contig maps. Data obtained from the multiplexed labeling permit detection of smaller overlaps than is observed when data from a single double-digest are analyzed. The method has been tested on 98 BACs from chromosome 22 regions where large-scale sequencing is under way and also through simulation, using randomly generated BAC clones derived from existing DNA sequence data. In each case, contig assembly results demonstrated the advantages of multiplexed fingerprinting.

Chromosomes, Bacterial↗
PubMed1999

The CMT2D locus: refined genetic position and construction of a bacterial clone-based physical map.

Charcot-Marie-Tooth (CMT) disease is a progressive neuropathy of the peripheral nervous system, typically characterized by muscle weakness of the distal limbs. CMT is noted for its genetic heterogeneity, with four distinct loci already identified for the axonal form of the disease (CMT2). In 1996, linkage analysis of a single large family revealed the presence of a CMT2 locus on chromosome 7p14 (designated CMT2D). Additional families have been linked subsequently to the same genomic region, including one with distal spinal muscular atrophy (dSMA) and one with mixed features of dSMA and CMT2; symptoms in both of these latter families closely resemble those seen in the original CMT2D family. There is thus a distinct possibility that CMT2 and dSMA encountered in these families reflect allelic heterogeneity at a single chromosome 7 locus. In the study reported here, we have performed more detailed linkage analysis of the original CMT2D family based on new knowledge of the physical locations of various genetic markers. The region containing the CMT2D gene, as defined by the original family, overlaps with those defined by at least two other families with CMT2 and/or dSMA symptoms. Both yeast artificial chromosome (YAC) and bacterial clone-based [bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC)] contig maps spanning approximately 3.4 Mb have been assembled across the combined CMT2D critical region, with the latter providing suitable clones for systematic sequencing of the interval. Preliminary analyses have already revealed at least 28 candidate genes and expressed-sequence tags (ESTs). The mapping information reported here in conjunction with the evolving sequence data should expedite the identification of the CMT2D/dSMA gene or genes.

Bacteriophage P1↗
PubMed1997

Cosmid contig and transcriptional map of three regions of human chromosome 21q22: identification of 37 novel transcripts by direct selection.

Human chromosome 21 is associated with many disorders, including Down syndrome (DS). In an effort to identify genes involved in brain development or function and therefore implicated in the mental retardation associated with DS, we chose YACs from three regions of chromosome 21: a region within the so-called "Down syndrome critical region," a region proximal to it, and one distal to it. We made cosmid libraries from these YACs and generated high-resolution physical maps by constructing cosmid contigs. These are the first cosmid contigs on chromosome 21 outside the critical region. The cosmids were used for direct selection of cDNAs to isolate chromosome 21 expressed sequences. We have isolated 45 nonredundant partial cDNAs and mapped these back to the cosmid contigs. We isolated 3 nonoverlapping portions of DSCR1 and a part of GIRK2 and identified 3 nonoverlapping partial cDNAs with similarity to the rat Dyrk gene, which turned out to be the human homologue (MNB) of the Drosophila minibrain gene. Twelve sequences had matches with either STS or EST entries in the databases, including a chromosome 21 EST, a chromosome 21 STS, and 6 unmapped expressed sequence entries. Only 1 sequence resulted in a match with a protein entry. The remaining 25 sequences revealed no similarity to any database entry. All of these partial cDNAs are expressed as determined by Northern blotting or by RT-PCR.

Animals↗
PubMed1998

PAC and cosmid contig spanning the HOXA cluster on human chromosome 7p15.

To construct the PAC and cosmid contig map spanning the HOXA cluster on human chromosome 7, we used 9 DNA markers (D7S2243, D7S3010, HOXA1, EVX1, 750, pBH8, p60, p8.0, and HOXA11), among which the final 4 were generated in this study by shotgun cloning strategy. From the libraries, 5 PAC and 35 cosmid clones were screened and as a result, an overlapping continuous array of cosmid and PAC clones covering the genomic region (about 200 kb) spanning the entire cluster were constructed. The isolated cosmids contained several consecutive HOX genes of regional group, probably sharing the regulatory processes such as alternative splicing or polyadenylation, and thus could be used as useful materials for elucidating the molecular mechanism of HOX gene expression in the future.

Bacteriophage P1↗
PubMed1997

Detailed physical analysis of a 1.5-megabase YAC contig containing the MXI1 and ADRA2A genes.

The distal long arm of chromosome 10 harbors genes of biomedical interest such as MXI1, a putative tumor suppressor gene, and those encoding the adrenergic receptors alpha2A (ADRA2A) and beta1 (ADRB1). As part of a physical and genetic study of this genomic region, we constructed a 1.5-Mb YAC contig mapping to 10q25 that contains MXI1 and ADRA2A as well as a number of STSs. Rare cutting restriction site analysis of overlapping YACs allowed fine mapping of these genes and markers along the contig and revealed the presence of four CpG islands. MXI1 and ADRA2A appear to be about 600 kb apart, whereas ADRB1 is separated from ADRA2A by a distance larger than previously reported.

Basic Helix-Loop-Helix Proteins↗
PubMed1994

A fast random cost algorithm for physical mapping.

Ordering clones from a genomic library into physical maps of whole chromosomes presents a central computational/statistical problem in genetics. Here we present a physical mapping algorithm for creating ordered genomic libraries or contig maps by using a random cost approach [Berg, A. (1993) Nature (London) 361, 708-710]. This random cost algorithm is 5-10 times faster than existing physical mapping algorithms and has optimization performance comparable to existing procedures. The speedup in the algorithm makes practical the widespread use of bootstrap resampling to assess the statistical reliability of links in the physical map as well as the use of more elaborate physical mapping criteria to improve map quality. The random cost algorithm is illustrated by its application in assembling a physical map of chromosome IV from the filamentous fungus Aspergillus nidulans.

Aspergillus nidulans↗
PubMed1994

Isolation of human chromosome 21-specific cosmids and their uses in mapping of cosmid contigs on chromosomal subregions.

A cosmid library of 3 x 10(5) clones has been constructed from a human x hamster hybrid cell line, 153E9a3, which contains human chromosome 21 (HC21) as the only human chromosome. From 56,500 clones of this library, 229 HC21-specific cosmids have been isolated by their hybridization to total human DNA and by their failure to hybridize to total Chinese hamster DNA. The cosmids isolated were then characterized, of these, 28 cosmids (12.2% of those tested) contained Not1 site(s), and 41 cosmids were localized on the eight subregions of HC21 by differential hybridization with Alu-PCR products obtained from a hybrid mapping panel. The cosmids localized were further integrated into the existing contigs using the end-specific probes of the clone insert. Therefore, they provided useful anchor points for contig mapping and walking.

Animals↗
PubMed1995

A high-resolution cytogenetic map of human chromosome 2: localization of 434 cosmid markers by direct R-banding fluorescence in situ hybridization.

We have constructed a high-resolution cytogenetic map of human chromosome 2 with 434 newly isolated cosmid markers by means of direct R-banding fluorescence in situ hybridization. Two markers were mapped to the centromeric region, 173 to the short arm, and 259 to the long arm. The clones were evenly distributed along the entire chromosome, although a tendency toward clustering in R-positive bands was observed. The mapped cosmids provide useful landmarks for construction of a contig map and for positional cloning of cancer-associated genes, as well as genes responsible for hereditary diseases.

Animals↗
PubMed1996

A yeast artificial chromosome contig and NotI restriction map that spans the tumor suppressor gene(s) locus, 11q22.2-q23.3.

Human chromosome 11q22-q23 is a pathologically important region in which a high level of loss of heterozygosity has been reported for breast, ovary, cervical, colon, and lung carcinomas, malignant melanomas, and hematologic malignancies. This strongly indicates that one or more tumor suppressor genes reside within the deleted region. In this report, we report the development of a contig map that covers most of the deleted regions found in these malignancies. The map comprises a contig of 66 overlapping yeast artificial chromosomes (YACs) and spans a region of 17 Mb from the PGR gene at 11q22.2 to the MLL gene at q23.3. In the process of screening the YACs, 50 new sequence-tagged site markers were developed from the termini of the YAC inserts. These markers were used for chromosome walking, and the data were then integrated into the contig map. NotI restriction mapping of these YACs revealed the presence of at least 26 NotI sites in the region. Using 22 of them, a NotI restriction map of the region from PGR to D11S939 was developed. This YAC contig will provide efficient tools for identification of the putative tumor suppressor gene(s).

Base Sequence↗
PubMed1997

A physical map at 1p31 encompassing the acute insulin response locus and the leptin receptor.

Recently, we reported genetic linkage in Pima Indians between the acute insulin response to an intravenous glucose challenge and the short tandem repeat marker D1S198, indicative of a genetic element in this region that controls the phenotypic variation in the first phase of insulin secretion. As a first step to isolating the gene responsible for the acute insulin response, we have constructed a yeast artificial chromosome (YAC) contig map that spans the DNA microsatellites D1S438 through D1S464. The contig comprises 34 YACs on which we have mapped 44 ends of the genomic DNA inserts from the 34 YACs, 13 short tandem repeats, eight expressed sequence tags, and six genes. In addition, we have used this contig to construct a physical map encompassing approximately 9 Mb of DNA in this region.

Carrier Proteins↗