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A yeast artificial chromosome library containing 33,000 clones with an average insert size of one megabase of human genomic DNA was extensively analysed by several different procedures for detecting overlaps and positional information. We developed an analysis strategy that resulted, after confirmatory tests, in a YAC contig map reliably covering about 75% of the human genome in 225 contigs having an average size of about ten megabases.
The murine Ly49 gene family is functionally analogous to the human killer cell Ig-like receptor (KIR) family of class I major histocompatibility complex (MHC) receptors. The number of KIR genes varies dramatically between individuals; however, the organization of the Ly49 genes has only been determined for the C57BL/6 (B6) mouse. The organization of the 129 Ly49 loci was determined from a BAC contig map by PCR and Southern blot analysis. In addition to the 10 Ly49 genes known from previous studies of the 129/J strain, 8 new genes were localized to the 129 Ly49 cluster. A gene order of Ly49q(1), e, (v, q(2)), e/c(2), l/r, s, t, e/c(1), r, u, u/i, i(1), g, p/d, (i(2), p), and o was determined. The 129 Ly49 gene cluster is predicted to span approximately 600 kb. These results indicate that Ly49 gene numbers can be significantly different between inbred mouse strains, analogous to the haplotype differences observed in the human KIR genes.
Genome physical mapping with large-insert clones by fingerprint analysis is becoming an active area of genomics research. Here, we report two new capillary electrophoresis-based fingerprinting methods for genome physical mapping and the effects of different fingerprinting methods and source clone genome coverage on quality physical map construction revealed by computer simulations and laboratory experiments. It was shown that the manual sequencing gel-based two-enzyme fingerprinting method consistently generated larger and more accurate contigs, followed by the new capillary electrophoresis-based three-enzyme method, the new capillary electrophoresis-based five-enzyme (SNaPshot) method, the agarose gel-based one-enzyme method, and the automatic sequencing gel-based four-enzyme method, in descending order, when 1% or fewer questionable clones were allowed. Analysis of clones equivalent to 5x, 8x, 10x, and 15x genomes using the fingerprinting methods revealed that as the number of clones increased from 5x to 10x, the contig length rapidly increased for all methods. However, when the number of clones was increased from 10x to 15x coverage, the contig length at best increased at a lower rate or even decreased. The results will provide useful knowledge and strategies for effective construction of quality genome physical maps for advanced genomics research.
Clone-based genome maps can be constructed by determining the presence or absence of sequence-tagged sites (STSs) in a redundant collection of yeast artificial chromosome clones (YACs). While STS-content mapping has proven to be an effective means of ordering clone ends and STSs along chromosomes, the exact physical map positions of these landmarks are not determined. This fundamental weakness can be overcome by RecA-assisted restriction endonuclease (RARE) cleavage, a method that exploits the binding specificity on duplex DNA of a RecA-protein-oligodeoxynucleotide complex to enhance the cleavage specificity of a restriction endonuclease. This technique allows selective cleavage at individual members of a large set of restriction sites. RARE-cleavage mapping was applied to a contig comprising 5 overlapping YACs spanning 580 kb on human chromosome 14. An STS-content map comprising 10 YAC-end specific STSs and one internal STS was constructed. RARE cleavage was performed on 2 YACs that span the entire contig at the EcoRI sites defining the vector-insert junctions of all 5 YACs, as well as at a HhaI site within the STS that was initially used to screen the YAC library for the clones in the contig. The sizes of the RARE-cleavage fragments were measured by pulsed-field gel electrophoresis and used to convert the STS-content map into a true physical map that indicates precise positions of clone ends and STSs.
We have constructed a physical map of human chromosome 22q using bacterial artificial chromosome (BAC) clones. The map consists of 613 chromosome 22-specific BAC clones that have been localized and assembled into contigs using 452 landmarks, 346 of which were previously ordered and mapped to specific regions of the q arm of the chromosome by means of chromosome 22-specific yeast artificial chromosome clones. The BAC-based map provides immediate access to clones that are stable and convenient for direct genome analysis. The approach to rapidly developing marker-specific BAC contigs is relatively straightforward and can be extended to generate scaffold BAC contig maps of the rest of the chromosomes. These contigs will provide substrates for sequencing the entire human genome. We discuss how to efficiently close contig gaps using the end sequences of BAC clone inserts.
A fine physical map of Arabidopsis thaliana chromosome 5 was constructed by ordering the clones from YAC, P1, TAC and BAC libraries of the genome using the sequences of a variety of genetic and EST markers and terminal sequences of clones. The markers used were 88 genetic markers, 13 EST markers, 87 YAC end probes, 100 YAC subclone end probes, and 390 end probes of P1, TAC and BAC clones. The entire genome of chromosome 5, except for the centromeric and telomeric regions, was covered by two large contigs 11.6 Mb and 14.2 Mb long separated by the centromeric region. The minimum tiling path of the chromosome was constituted by a total of 430 P1, TAC and BAC clones. The map information is available at the Web site http://www.kazusa.or.jp/arabi/.
We have constructed a yeast artificial chromosome contig (YAC) map of human chromosome 4q21-q23 across the Parkinson's disease region by combining molecular and fluorescence in situ hybridization techniques. This map contains 55 YACs and 51 molecular markers, including 23 polymorphic markers. We have also isolated one P1 and 33 bacterial artificial chromosomes located within this contig. Plasmid libraries were generated from 11 of these BAC and P1 clones, and 614 random plasmid clones were sequenced for a total of about 200 kb. This contig allowed us to precisely determine the location of 18 transcripts within the D4S2460-D4S2986 interval, including the alpha-synuclein gene found to be mutated in some families with Parkinson's disease.
Draft sequence derived from the 46-Mb gene-rich euchromatic portion of human chromosome 19 (HSA19) was utilized to generate a sequence-ready physical map spanning homologous regions of mouse chromosomes. Sequence similarity searches with the human sequence identified more than 1000 individual orthologous mouse genes from which 382 overgo probes were developed for hybridization. Using human gene order and spacing as a model, these probes were used to isolate and assemble bacterial artificial chromosome (BAC) clone contigs spanning homologous mouse regions. Each contig was verified, extended, and joined to neighboring contigs by restriction enzyme fingerprinting analysis. Approximately 3000 mouse BACs were analyzed and assembled into 44 contigs with a combined length of 41.4 Mb. These BAC contigs, covering 90% of HSA19-related mouse DNA, are distributed throughout 15 homology segments derived from different regions of mouse chromosomes 7, 8, 9, 10, and 17. The alignment of the HSA19 map with the ordered mouse BAC contigs revealed a number of structural differences in several overtly conserved homologous regions and more precisely defined the borders of the known regions of HSA19-syntenic homology. Our results demonstrate that given a human draft sequence, BAC contig maps can be constructed quickly for comparative sequencing without the need for preestablished mouse-specific genetic or physical markers and indicate that similar strategies can be applied with equal success to genomes of other vertebrate species.
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.
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.
To characterize the bovine chromosome 6q21 for bovine chondrodysplastic dwarfism (BCD), we developed 48 new microsatellite markers from yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC) clones using a modified magnetic bead capture method. These new markers were used to construct a high-resolution physical map of the region with a total of 85 loci. The physical map will be a powerful tool for successful positional cloning experiments.
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.
A physical map of ordered bacterial artificial chromosome (BAC) clones was constructed to determine the genetic organization of the horse major histocompatibility complex. Human, cattle, pig, mouse, and rat MHC gene sequences were compared to identify highly conserved regions which served as source templates for the design of overgo primers. Thirty-five overgo probes were designed from 24 genes and used for hybridization screening of the equine USDA CHORI 241 BAC library. Two hundred thirty-eight BAC clones were assembled into two contigs spanning the horse MHC region. The first contig contains the MHC class II region and was reduced to a minimum tiling path of nine BAC clones that span approximately 800 kb and contain at least 20 genes. A minimum tiling path of a second contig containing the class III/I region is comprised of 14 BAC clones that span approximately 1.6 Mb and contain at least 34 genes. Fluorescence in situ hybridization (FISH) using representative clones from each of the three regions of the MHC localized the contigs onto ECA20q21 and oriented the regions relative to one another and the centromere. Dual-colored FISH revealed that the class I region is proximal to the centromere, the class II region is distal, and the class III region is located between class I and II. These data indicate that the equine MHC is a single gene-dense region similar in structure and organization to the human MHC and is not disrupted as in ruminants and pigs.
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.
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.
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/].
With genome sequencing efforts increasing exponentially, valuable information accumulates on genomic content of the various organisms sequenced. Projector 2 uses (un)finished genomic sequences of an organism as a template to infer linkage information for a genome sequence assembly of a related organism being sequenced. The remaining gaps between contigs for which no linkage information is present can subsequently be closed with direct PCR strategies. Compared with other implementations, Projector 2 has several distinctive features: a user-friendly web interface, automatic removal of repetitive elements (repeat-masking) and automated primer design for gap-closure purposes. Moreover, when using multiple fragments of a template genome, primers for multiplex PCR strategies can also be designed. Primer design takes into account that, in many cases, contig ends contain unreliable DNA sequences and repetitive sequences. Closing the remaining gaps in prokaryotic genome sequence assemblies is thereby made very efficient and virtually effortless. We demonstrate that the use of single or multiple fragments of a template genome (i.e. unfinished genome sequences) in combination with repeat-masking results in mapping success rates close to 100%. The web interface is freely accessible at http://molgen.biol.rug.nl/websoftware/projector2.
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.