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PubMed1995

A YAC contig map of Plasmodium falciparum chromosome 4: characterization of a DNA amplification between two recently separated isolates.

We have generated a physical map of Plasmodium falciparum chromosome 4 using yeast artificial chromosomes (YACs). The map is defined by a YAC contig spanning approximately 1.05 Mb, which has been restriction mapped to a resolution of 30 kb and is punctuated by 22 sequence-tagged sites. The physical information obtained has enabled us to compare and contrast the structure of chromosome 4 in detail between FCR3 and B8, two recently separated isolates of P. falciparum, leading to characterization of a novel chromosome polymorphism occurring in a subtelomeric region. Comparison of chromosomes 4 from 10 different isolates has shown that chromosome size polymorphisms are restricted to both subtelomeric regions. These analyses provide a high-resolution physical map that will be important to complement genetic analysis of this human pathogen.

Animals↗
PubMed1998

Contig maps and genomic sequencing identify candidate genes in the usher 1C locus.

Usher syndrome 1C (USH1C) is a congenital condition manifesting profound hearing loss, the absence of vestibular function, and eventual retinal degeneration. The USH1C locus has been mapped genetically to a 2- to 3-cM interval in 11p14-15.1 between D11S899 and D11S861. In an effort to identify the USH1C disease gene we have isolated the region between these markers in yeast artificial chromosomes (YACs) using a combination of STS content mapping and Alu-PCR hybridization. The YAC contig is approximately 3.5 Mb and has located several other loci within this interval, resulting in the order CEN-LDHA-SAA1-TPH-D11S1310-(D11S1888/KCNC1 )-MYOD1-D11S902D11S921-D11S 1890-TEL. Subsequent haplotyping and homozygosity analysis refined the location of the disease gene to a 400-kb interval between D11S902 and D11S1890 with all affected individuals being homozygous for the internal marker D11S921. To facilitate gene identification, the critical region has been converted into P1 artificial chromosome (PAC) clones using sequence-tagged sites (STSs) mapped to the YAC contig, Alu-PCR products generated from the YACs, and PAC end probes. A contig of >50 PAC clones has been assembled between D11S1310 and D11S1890, confirming the order of markers used in haplotyping. Three PAC clones representing nearly two-thirds of the USH1C critical region have been sequenced. PowerBLAST analysis identified six clusters of expressed sequence tags (ESTs), two known genes (BIR, SUR1) mapped previously to this region, and a previously characterized but unmapped gene NEFA (DNA binding/EF hand/acidic amino-acid-rich). GRAIL analysis identified 11 CpG islands and 73 exons of excellent quality. These data allowed the construction of a transcription map for the USH1C critical region, consisting of three known genes and six or more novel transcripts. Based on their map location, these loci represent candidate disease loci for USH1C. The NEFA gene was assessed as the USH1C locus by the sequencing of an amplified NEFA cDNA from an USH1C patient; however, no mutations were detected.

Bacteriophage P1↗
PubMed2000

Construction of a BAC contig map of chromosome 16q by two-dimensional overgo hybridization.

We have used sequence-based markers from an integrated YAC STS-content/somatic cell hybrid breakpoint physical map and radiation hybrid maps of human chromosome 16 to construct a new sequence-ready BAC map of the long arm of this chromosome. The integrated physical map was generated previously in our laboratory and contains 1150 STSs, providing a marker on average every 78 kb on the euchromatic arms of chromosome 16. The other two maps used for this effort were the radiation hybrid maps of chromosome 16 from Whitehead Institute and Stanford University. To create large sequenceable targets of this chromosome, we used a systematic approach to screen high-density BAC filters with probes generated from overlapping oligonucleotides (overgos). We first identified all available sequences in the three maps. These include sequences from genes, ESTs, STSs, and cosmid end sequences. We then used BLASTto identify 36-bp unique fragments of DNA for overgo probes. A total of 906 overgos were selected from the long arm of chromosome 16. Hybridizations occurred in three stages: (1) superpool hybridizations against the 12x coverage human BAC library (RPCI-11); (2) two-dimensional hybridizations against rearrayed positive BACs identified in the superpool hybridizations; and (3) pooled tertiary hybridizations for those overgos that had ambiguous positives remaining after the two-dimensional hybridization. For the superpool hybridizations, up to 236 overgos have been pooled in a single hybridization against the 12x BAC library. A total of 5187 positive BACs from chromosome 16q were identified as a result of five superpool hybridizations. These positive clones were rearrayed on membranes and hybridized with 161 two-dimensional subpools of overgos to determine which BAC clones were positive for individual overgos. An additional 46 tertiary hybridizations were required to resolve ambiguous overgo-BAC relationships. Thus, after a total of 212 hybridizations, we have constructed an initial probe-content BAC map of chromosome 16q consisting of 828 overgo markers and 3363 BACs providing >85% coverage of the long arm of this chromosome. The map has been confirmed by the fingerprinting data and BAC end PCR screening.

Chromosomes, Bacterial↗
PubMed2000

A contig map of the Mhc class I genomic region in the zebrafish reveals ancient synteny.

In contrast to the human and mouse Mhc, in which the clusters of class I and class II loci reside in close vicinity to one another, in the zebrafish, Danio rerio, they are found in different linkage groups. Chromosome walking using BAC (bacterial artificial chromosome) and PAC (P1 artificial chromosome) clones reveals the zebrafish class I region to occupy a segment of approximately 450 kb and to encompass at least 19 loci. These include three class I (Dare-UDA, -UEA, -UFA), five proteasome subunit beta (PSMB8, -9A, -9C, -11, -12), two TAPs (TAP2A, TAP2B), and one TAP binding protein (TAPBP). This arrangement contrasts with the arrangements found in human and mouse Mhc, in which the orthologues of the PSMB, TAP, and TAPBP loci reside within the class II region. In addition to this main zebrafish class I contig, a shorter contig of about 150 kb contains two additional class I (UBA, UCA) and at least five other loci. It probably represents a different haplotype of part of the class I region. The previously identified UAA gene shares an identical 5' part with UEA, but the two genes differ in their 3' parts. One of them is probably the result of an unequal crossing over. The described organization has implications for the persistence of syntenic relationships, coevolution of loci, and interpretation of the origin of the human/mouse Mhc organization.

Animals↗
PubMed1996

Genetic and contig map of a 2200-kb region encompassing 5.5 cM on chromosome 1 of Arabidopsis thaliana.

In the course of the isolation of the MONOPTEROS (MP) gene, required for primary root formation in Arabidopsis thaliana, a yeast artificial chromosome (YAC) contig encompassing approximately 2200 kilobases corresponding to 5.5 cM on the top arm of chromosome 1 was established. Forty-six YAC clones were characterized and 12 new restriction fragment length polymorphism (RFLP) markers are presented. Three new codominant amplified polymorphic sequence (CAPS) markers were generated that enabled high resolution genetic mapping and correlation of physical and genetic distances along the contig. The map contributes to the completion of a physical map of the Arabidopsis genome and should facilitate positional cloning of other genes in the region as well as studies on genome organization. We also present another set of 11 physically linked probes, as well as mapping data for additional RFLP markers within a broader interval of 10.4 cM.

Arabidopsis↗
PubMed1999

A sequence-ready contig map of the top arm of Arabidopsis thaliana chromosome 3.

A fine physical map of the top arm of Arabidopsis thaliana chromosome 3 has been constructed by ordering P1, TAC and BAC clones using the sequences of a variety of DNA markers and end-sequences of clones. The marker sequences used in this study were derived from 58 DNA markers, 93 YAC end-sequences, and 807 end-sequences of P1, TAC and BAC clones. The entire top arm of chromosome 3, except for the centromeric and telomeric regions, was covered by a single contig 13.3 Mb long. This fine physical map will facilitate gene isolation by map-based cloning experiments as well as genome sequencing of the top arm of chromosome 3. The map and end-sequence information are available on the web site KAOS (Kazusa Arabidopsis data Opening Site) at [http://www.kazusa.or.jp/arabi/].

Arabidopsis↗
PubMed1995

A second-generation YAC contig map of human chromosome 3.

A map of human chromosome 3 which integrates both physical and genetic data has been developed from the fusion of two large collections of markers and corresponding yeast artificial chromosome (YAC) clones. The map contains 972 megabase-sized YACs identified with 593 primary markers, of which 162 are highly polymorphic sequence-tagged sites (STSs) and form a closely spaced genetic linkage map; the remaining markers are hybridization-based. Chromosome 3 is now represented by 24 large YAC contigs whose order and orientation is largely known. The map generated by fusion of these hybridization- and STS-based datasets covers about 80% (over 160 megabases) of the chromosome and will provide the foundation necessary for rapid development of a detailed genetic understanding for this large autosome.

Chromosome Mapping↗
PubMed1993

Refined localization and yeast artificial chromosome (YAC) contig--mapping of genes and DNA segments in the 7q21-q32 region.

The chromosome localizations for 159 gene and DNA segments have been refined to one of five intervals in the 7q21-132 region through hybridization analysis with a panel of somatic cell hybrid lines. Seventy-two of these chromosome 7 markers are also mapped on common or overlapping yeast artificial chromosome (YAC) clones. In addition, the breakpoints of chromosome rearrangement contained in five of the somatic cell hybrid lines have been defined by flanking probes within YAC contigs. To provide a framework for further mapping of the 7q21-q32 region, we have established the physical order of a set of reference markers: cen-(COL1A2-D7S15-CYP3A4-PON)-D7S456-(brea kpoint contained in cell hybrid 1EF2/3/K017)-GUSB-D7S186-ASL-(PGY1-PGY3 -GNB2-EPO-ACHE)-D7S238-(proximal breakpoint in GM1059-Rag5)-D7S240-(CUTL1-PLANH1)-(breakp oints in 1CF2/5/K016 and 2068Rag22-2)-(PRKAR2B-D7S13)-LAMB1-(breakpoint in JSR-17S)-DLD-D7S16-MET-WNT2-CFTR-D7S8-tel.

Base Sequence↗
PubMed1996

An EST and STS-based YAC contig map of human chromosome 9q22.3.

We have isolated 48 yeast artificial chromosome (YAC) clones from a 4 cM/27 cR region of human chromosome 9q22.3 encompassed by the markers cen-D9S196-D9S173-tel. Within this region, we have assembled a 4.3-Mb YAC contig across the interval cen-FACC-D9S173-tel containing 42 clones. As a first step toward completing the detailed transcription map of the region, we have mapped 9 gene sequences and 10 expressed sequence tags. Fifteen polymorphic microsatellite repeat markers and 17 novel sequence-tagged sites from the region are also described. The mapping of polymorphic simple tandem repeat markers has permitted the integration of existing genetic and physical maps of the region. Together these maps provide a valuable resource for fine structure mapping and DNA sequencing across the region as well as for the identification of disease gene loci and the isolation of novel coding sequences.

Base Sequence↗
PubMed1995

YAC contigs mapping the human COL4A5 and COL4A6 genes and DXS118 within Xq21.3-q22.

Sequence-tagged sites (STSs) were developed for three loci of uncertain X chromosomal localization (DXS122, DXS137, and DXS174) and were used to seed YAC contigs. Two contigs now total about 3.3 Mb formatted with 34 STSs. One contains DXS122 and DXS174 within 250 kb on single YACs; it is placed in Xq21.3-q22.1 by FISH analysis, which is consistent with somatic cell hybrid panel analyses and with the inclusion of a probe that detects polymorphism at the DXS118 locus already assigned to that general region. The other contig, which contains DXS137, is in Xq22.2 by FISH, consistent with cell hybrid analyses and with the finding that it covers the human COL4A5 and COL4A6 genes known to be in that vicinity. In addition to extending the cloned coverage of this portion of the X chromosome, these materials should aid, for example, in the further analysis of Alport syndrome.

Animals↗
PubMed1998

Integrated YAC contig map of the Prader-Willi/Angelman region on chromosome 15q11-q13 with average STS spacing of 35 kb.

Prader-Willi syndrome and Angelman syndrome are associated with parent-of-origin-specific abnormalities of chromosome 15q11-q13, most frequently a deletion of an approximately 4-Mb region. Because of genomic imprinting, paternal deficiency of this region leads to PWS and maternal deficiency to AS. Additionally, this region is frequently involved in other chromosomal rearrangements including duplications, triplications, or supernumerary marker formation. A detailed physical map of this region is important for elucidating the genes and mechanisms involved in genomic imprinting, as well as for understanding the mechanism of recurrent chromosomal rearrangments. An initial YAC contig extended from D15S18 to D15S12 and was comprised of 23 YACs and 21 STSs providing an average resolution of about one STS per 200 kb. To close two gaps in this contig, YAC screening was performed using two STSs that flank the gap between D15S18 and 254B5R and three STSs located distal to the GABRA5-149A9L gap. Additionally, we developed 11 new STSs, including seven polymorphic markers. Although several groups have developed whole-genome genetic and radiation hybrid maps, the depth of coverage for 15q11-q13 has been somewhat limited and discrepancies in marker order exist between the maps. To resolve the inconsistencies and to provide a more detailed map order of STSs in this region, we have constructed an integrated YAC STS-based physical map of chromosome 15q11-q13 containing 118 YACs and 118 STSs, including 38 STRs and 49 genes/ESTs. Using an estimate of 4 Mb for the size of this region, the map provides an average STS spacing of 35 kb. This map provides a valuable resource for identification of disease genes localized to this region as well as a framework for complete DNA sequencing.

Angelman Syndrome↗
PubMed1998

Construction and validation of yeast artificial chromosome contig maps by RecA-assisted restriction endonuclease cleavage.

RecA-assisted restriction endonuclease (RARE) cleavage is an "Achilles' heel" approach to restriction mapping whereby a RecA-protein-oligodeoxynucleotide complex protects an individual restriction site from methylation, thus limiting subsequent digestion to a single, predetermined site. We have used RARE cleavage to cut yeast artificial chromosomes (YACs) at specific EcoRI sites located within or adjacent to sequence-tagged sites (STSs). Each cleavage reaction produces two YAC fragments whose sizes are a direct measure of the position of the STS in the YAC. In this fashion, we have positioned 45 STSs within a contig of 19 independent YACs and constructed a detailed RARE-cleavage map that represents 8.4 Mbp of human chromosome 6p21.3-22. By comparing maps of overlapping YACs, we were able to detect seven internal deletions that ranged from approximately 75 kbp to approximately 1 Mbp in size. Thirteen pairs of EcoRI sites were targeted for double RARE cleavage in uncloned total human DNA. The excised fragments, up to 2 Mbp in size, were resolved by pulsed-field gel electrophoresis and were detected by hybridization. In general, the genomic RARE-cleavage results support the YAC-based map. In one case, the distance in uncloned DNA between the two terminal EcoRI sites of a YAC insert was approximately 1 Mbp larger than the YAC itself, indicating a major deletion. The general concept of RARE-cleavage mapping as well as its applications and limitations are discussed.

Chromosome Mapping↗
PubMed1995

A second-generation YAC contig map of human chromosome 12.

Human chromosome 12 constitutes approximately 4.5% of the human genome and has an estimated size of 135 million base pairs (Mb). We have started to construct a high-resolution physical map of chromosome 12 as overlapping yeast artificial chromosomes (YACs), using as a foundation the first-generation physical map of this chromosome covers nearly 102 Mb of DNA and includes 426 highly polymorphic, monomorphic and gene-based markers. We also mapped 119 of the YACs, most of which are part of the physical map, by cytogenetic methods. Thus the map integrates genetic, physical and cytogenetic data and provides information about the organization of this chromosome and will help in the localization and cloning of disease-related genes. The strategy used here to generate the chromosome-12 map could be applied for the rapid construction of physical and expression maps for other human chromosomes.

Base Sequence↗
PubMed1995

A high-density YAC contig map of human chromosome 22.

We have constructed a high-resolution clone map of human chromosome 22 which integrates the available physical and genetic information, establishing a single consensus. The map consists of all classes of DNA landmarks ordered on 705 yeast artificial chromosomes (YACs) at an average landmark density of more than one per 70 kilobases. This map represents the practical limits of currently available YAC resources and provides the basis for determination of the entire gene content and genomic DNA sequence of human chromosome 22.

Chromosome Mapping↗
PubMed1996

Comparative genomic analysis of the Haloferax volcanii DS2 and Halobacterium salinarium GRB contig maps reveals extensive rearrangement.

Anonymous probes from the genome of Halobacterium salinarium GRB and 12 gene probes were hybridized to the cosmid clones representing the chromosome and plasmids of Halobacterium salinarium GRB and Haloferax volcanii DS2. The order of and pairwise distances between 35 loci uniquely cross-hybridizing to both chromosomes were analyzed in a search for conservation. No conservation between the genomes could be detected at the 15-kbp resolution used in this study. We found distinct sets of low-copy-number repeated sequences in the chromosome and plasmids of Halobacterium salinarium GRB, indicating some degree of partitioning between these replicons. We propose alternative courses for the evolution of the haloarchaeal genome: (i) that the majority of genomic differences that exist between genera came about at the inception of this group or (ii) that the differences have accumulated over the lifetime of the lineage. The strengths and limitations of investigating these models through comparative genomic studies are discussed.

Blotting, Southern↗
PubMed1998

Application of fiber-FISH in physical mapping of Arabidopsis thaliana.

Arabidopsis thaliana has become a model plant species for genetic studies because of its small genome and short juvenility period. However, the small chromosomes of this species are not suitable for classical cytogenetic studies. Here we demonstrate that the fluorescence in situ hybridization (FISH) technique using extended DNA fibers can be a powerful tool in the physical mapping of the A. thaliana genome. Using a refined fiber-FISH technique we were able to measure DNA clusters as long as 1.71 Mb, more than 1% of the A. thaliana genome. Several small DNA loci, including the telomeres and a dispersed repetititve DNA sequence, mi167, were also analyzed with this technique. The results show that without known adjacent DNA markers such small DNA loci cannot be mapped precisely using fiber-FISH. One of the most difficult obstacles in physical mapping by contig assembly is closing the gaps that are present between adjacent contigs. Currently available molecular techniques are not sufficient to accurately estimate the physical sizes of these gaps. We isolated bacterial artificial chromosome (BAC) clones bordering gaps 2 and 3 on the physical contig map of A. thaliana chromosome II. The BAC clones were used in fiber-FISH analysis and the physical sizes of the two gaps were estimated as 31 kb and more than 500 kb, respectively. Thus, we have demonstrated that fiber-FISH is an efficient technique for determining the physical size of gaps on molecular contig maps.

Arabidopsis↗
PubMed1992

Evaluation of a cosmid contig physical map of human chromosome 16.

A cosmid contig physical map of human chromosome 16 has been developed by repetitive sequence finger-printing of approximately 4000 cosmid clones obtained from a chromosome 16-specific cosmid library. The arrangement of clones in contigs is determined by (1) estimating cosmid length and determining the likelihoods for all possible pairwise clone overlaps, using the fingerprint data, and (2) using an optimization technique to fit contig maps to these estimates. Two important questions concerning this contig map are how much of chromosome 16 is covered and how accurate are the assembled contigs. Both questions can be addressed by hybridization of single-copy sequence probes to gridded arrays of the cosmids. All of the fingerprinted clones have been arrayed on nylon membranes so that any region of interest can be identified by hybridization. The hybridization experiments indicate that approximately 84% of the euchromatic arms of chromosome 16 are covered by contigs and singleton cosmids. Both grid hybridization (26 contigs) and pulsed-field gel electrophoresis experiments (11 contigs) confirmed the assembled contigs, indicating that false positive overlaps occur infrequently in the present map. Furthermore, regional localization of 93 contigs and singleton cosmids to a somatic cell hybrid mapping panel indicates that there is no bias in the coverage of the euchromatic arms.

Chromosome Banding↗