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PubMed1997

A high-resolution PAC and BAC map of the SCA2 region.

The spinocerebellar ataxia type 2 (SCA2) gene has been localized to chromosome 12q24.1. To characterize this region and to aid in the identification of the SCA2 gene, we have constructed a 3.9-Mb physical map, which covers markers D12S1328 and D12S1329 known to flank the gene. The map comprises a contig of 84 overlapping yeast artificial chromosomes (YACs), P1 artificial chromosomes (PACs), and bacterial artificial chromosomes (BACs) onto which we placed 82 PCR markers. We localized eight genes and expressed sequence tags on this map, many of which had not been precisely mapped before. In contrast to YACs, which showed a high degree of chimerism and deletions in this region, PACs and BACs were stable. Only 1 in 65 PACs contained a small deletion, and 2 in 18 BACs were chimeric. The high-resolution physical map, which was used in the identification of the SCA2 gene, will be useful for the positional cloning of other disease genes mapped to this region.

Ataxins↗

PubMed1996

Construction of high-resolution physical maps from yeast artificial chromosomes using restriction landmark genomic scanning (RLGS).

We have established a new system for chromosome-specific yeast artificial chromosome (YAC) contig construction using restriction landmark genomic scanning (RLGS-based YAC contig mapper). RLGS is a powerful tool for detecting more than 1000 restriction landmarks distributed on an entire genome in one procedure. In this system, RLGS is applied to sorted chromosomes to cover the target chromosome. Using these landmarks as guideposts, chromosome-specific YAC clones are then ordered. In this paper, we report the construction of a map for a human chromosome 21 YAC contig spanning q22.1 using this new approach. Applying RLGS to sorted chromosomes 21 enables detection of approximately 1400 spots (equivalent of 1050 PacI landmarks), covering the entire region of this chromosome. We constructed the 2.5-Mb YAC contig encompassing 21q22.1 with 66 spots (equivalent of 50 PacI landmarks). With this contig map, we could detect two deleted regions and chimerism in the YAC insert DNA. Our results demonstrated the usefulness of this approach for finding DNA alterations of YACs, such as deletions and chimerism.

Chimera↗

PubMed1999

An exon map of the AZFc male infertility region of the human Y chromosome.

STS markers spanning the AZFc region of human Y Chromosome (Chr) were used to isolate a series of 14 P1 artificial chromosome (PAC) clones covering at least 560 kb of DNA. End clone analysis of these PAC clones was used to derive 10 new STS markers. Together with other markers mapped to the region, an STS content analysis was used to assemble these PAC clones into three distinct contigs. A minimum tiling path of ten PAC clones was subjected to exon trapping to identify potentially new genes mapping to the AZFs region. In all, 39 potential exons were isolated, including 2 exons from the DAZ gene, 3 exons from the BPY2 gene, 2 exons from the PRY gene, and 1 exon from a member of the RBM II gene family; all these genes have been shown previously to map to the AZFc region. One further exon was found that shows homology to the DFFRY gene, which maps to Yq11. 2, indicating that there may be a further copy of this gene or a pseudogene in the distal Yq euchromatin. The majority of the remaining potential exons appear to be novel, suggesting that additional genes lie in the AZFc region. Mapping of these exons by PCR analysis of somatic cell hybrids has shown that six of these exons are homologous to autosomal sequences, and five to sequences on the X Chr. RT-PCR analysis of primary cDNA from adult testis, brain, liver, and skeletal muscle mRNA has shown that 11 of the novel exons are expressed in one or a combination of these tissues, indicating that they form parts of genuine transcripts.

Brain↗

PubMed1997

Yeast artificial chromosome clones of rice chromosome 2 ordered using DNA markers.

Yeast artificial chromosome (YAC) clones were ordered for the physical mapping of rice chromosome 2, the last of the 12 rice chromosomes to be assigned YACs by the Rice Genome Research Program. A total of 128 restriction fragment length polymorphism markers and 4 sequence-tagged site (STS) markers located on our high-density genetic map were used for YAC clone landing. By colony/Southern hybridization and polymerase chain reaction screening, a total of 239 individual YACs were selected from our YAC library of 6934 clones covering six genome equivalents. The YACs located on the corresponding marker positions in the linkage map formed 43 contigs and islands and were estimated to encompass about 50% of the length of rice chromosome 2.

Blotting, Southern↗

PubMed2003

Whole-genome shotgun optical mapping of Rhodobacter sphaeroides strain 2.4.1 and its use for whole-genome shotgun sequence assembly.

Rhodobacter sphaeroides 2.4.1 is a facultative photoheterotrophic bacterium with tremendous metabolic diversity, which has significantly contributed to our understanding of the molecular genetics of photosynthesis, photoheterotrophy, nitrogen fixation, hydrogen metabolism, carbon dioxide fixation, taxis, and tetrapyrrole biosynthesis. To further understand this remarkable bacterium, and to accelerate an ongoing sequencing project, two whole-genome restriction maps (EcoRI and HindIII) of R. sphaeroides strain 2.4.1 were constructed using shotgun optical mapping. The approach directly mapped genomic DNA by the random mapping of single molecules. The two maps were used to facilitate sequence assembly by providing an optical scaffold for high-resolution alignment and verification of sequence contigs. Our results show that such maps facilitated the closure of sequence gaps by the early detection of nascent sequence contigs during the course of the whole-genome shotgun sequencing process.

Chromosomes, Bacterial↗

PubMed2001

Construction of a high-resolution RH map of the human 2q35 region on TNG panel and comparison with a physical map of the porcine homologous region 15q25.

This article describes the construction of a high-resolution radiation hybrid map of Hsap 2q35 by using the TNG RH panel generated by irradiation with 50,000 rads. We were able to build a framework map of 1300 cR(50,000) including 34 markers ordered with odds higher than 1000:1. The comprehensive map includes 77 loci and describes a region of 3 Mb around the SLC11A1 gene. Because of the very small size of the fragments retained and a reduced retention frequency, it was difficult to build a high-resolution multi-point map of this region by using the TNG RH panel. Nevertheless, this study confirmed the very high potential of this RH panel for constructing a human, high-resolution physical map (2.3 kb/cR(50,000)). Moreover, human ESTs from Hsap 2q35 were hybridized with porcine BAC contigs to establish a porcine transcript map of the homologous region Sscr 15q25 (greater than 2.5 Mb). We identified 17 new genes in this porcine chromosomal region. We were able to compare the location of 26 genes mapped in both species. The gene order was similar except for two possible minor discrepancies in the Desmin sub-region, suggesting the existence of a porcine micro-region between TNP1 and IL8RB with an unknown origin.

Animals↗

PubMed1995

Refined genetic mapping of a gene for familial juvenile nephronophthisis (NPH1) and physical mapping of linked markers. APN Study Group.

We have recently assigned a gene for familial juvenile nephronophthisis (NPH1) to chromosome 2q between microsatellite markers at loci D2S135 and D2S110. Here we have extended and refined our previous linkage analysis by studying five additional NPH families and by testing five additional markers. By haplotype analysis in a large family yielding proof of linkage, D2S135 and D2S283 were defined with certainty as flanking the NPH1 critical region within a 14-cM interval. These data now allow cytogenetic assignment of the NPH1 critical region to 2q11.1-q21.1. Furthermore, haplotype analysis in 12 small families helped to define as flanking markers D2S293 and D2S363, which span an 8-cM interval. Multipoint linkage analysis by the location score method resulted in a maximum multipoint lod score of 10.30. The Zmax-1 support interval spans 6.9 cM and is flanked by marker loci D2S293 and D2S363. Since IL1A maps to this region and has been cytogenetically mapped to 2q13 in the literature, NPH1 can be assigned more closely to 2q13 or adjacent bands. Contigs of CEPH mega-YAC clones in the region were established by screening the clones with microsatellite markers, adding marker IL1A to the physical map as a novel assignment. We conclude that the NPH1 gene most probably localizes to an interval of 6.9 cM between marker loci D2S293 and D2S363 in the vicinity of 2q13. This contig mapping provides the basis for cloning of this interval and for isolation of the NPH1 gene.

Chromosome Mapping↗

PubMed1998

A HAPPY map of Cryptosporidium parvum.

We have constructed a HAPPY map of the apicomplexan parasite Cryptosporidium parvum. We have placed 204 markers on the 10.4-Mb genome, giving an average marker spacing of approximately 50 kb, with an effective resolution of approximately 40 kb. HAPPY mapping (an in vitro linkage technique based on screening approximately haploid amounts of DNA by the polymerase chain reaction) is fast and accurate and is not subject to the distortions inherent in cloning, meiotic recombination, or hybrid cell formation. In addition, little genomic DNA is needed as a substrate, and the AT content of the genome is largely immaterial, making it an ideal method for mapping otherwise intractable parasite genomes. The map, covering all eight chromosomes, consists of 10 linkage groups, each of which has been chromosomally assigned. We have verified the accuracy of the map by several methods, including the construction of a >140-kb PAC contig on chromosome VI. Less than 1% of our markers detect non-rDNA duplicated sequences.

Animals↗

PubMed1996

A 1.6-Mb P1-based physical map of the Down syndrome region on chromosome 21.

The Down syndrome (DS) region on chromosome 21, which is responsible for the main features of DS such as characteristic facial features, a congenital heart defect, and mental retardation, has been defined by molecular analysis of DS patients with partial trisomy 21. The 2. 5-Mb region around the marker D21S55 between D21S17 and ERG in 21q22 is thought to be important, although contributions of other regions cannot be excluded. In this region, we focused on a 1.6-Mb region between a NotI site, LA68 (D21S396, which is mapped distal to D21S17) and ERG, because analysis of a Japanese DS family with partial trisomy 21 revealed that the proximal border of its triplicated region was distal to LA68. We constructed P1 contigs with 46 P1 clones covering more than 95% of the 1.6-Mb region. A high-resolution restriction map using BamHI was also constructed for more detailed analysis. Our P1 contig map supplements other physical maps previously reported and provides useful materials for further analysis including gene isolation and sequencing of the DS region.

Base Sequence↗

PubMed1994

Organization of the region encompassing the human serum amyloid A (SAA) gene family on chromosome 11p15.1.

The four members of the human serum amyloid A protein (SAA) gene family are clustered on human chromosome 11p15.1. Three genes are differentially expressed and encode small apolipoproteins of M(r) 12-19 kDa: SAA1 and SAA2 encode the acute phase SAAs (A-SAAs), and SAA4 encodes the constitutively expressed SAA (C-SAA). A fourth locus, SAA3, is a pseudogene. The human SAA gene family encompasses approximately 150 kb contained on a 900-kb yeast artificial chromosome contig. SAA1 and SAA2 are 15-20 kb apart and are arranged in divergent transcriptional orientations. SAA4 is 9 kb downstream of SAA2 and in the same orientation. SAA3 is 110 kb downstream of SAA4, and its relative orientation could not be determined. All genes known to be in the same human and mouse syntenic linkage group as SAA were mapped within the contig. Interphase FISH was used to orientate the region relative to the centromere: cen-LDHC-LDHA-SAA1-SAA2-SAA4-SAA3-TP H-D11S18- KCNC1-MYOD1-pter.

Animals↗

PubMed2004

A physical map of large segments of pig chromosome 7q11-q14: comparative analysis with human chromosome 6p21.

The aim of this study was to establish a porcine physical map along the chromosome SSC7q by construction of BAC contigs between microsatellites Sw1409 and S0102. The SLA class II contig, located on SSC7q, was lengthened. Four major BAC contigs and 10 short contigs span a region equivalent to 800 cR measured by IMpRH7000 mapping. The BAC contigs were initiated by PCR screening with primers derived from human orthologous segments, extended by chromosome walking, and controlled and oriented by RH mapping with the two available panels, IMpRH7000Rad and IMNpRH12000Rad. The location of 43 genes was revealed by sequenced segments, either from BAC ends or PCR products from BAC clones. The 220 BAC end sequences (BES) were also used to analyze the different marks of evolution. Comparative mapping analysis between pigs and humans demonstrated that the gene organization on HSA6p21 and on SSC7p11 and q11-q14 segments was conserved during evolution, with the exception of long fragments of HSA6p12 which shuffled and spliced the SLA extended class II region. Additional punctual variations (unique gene insertion/deletion) were observed, even within conserved segments, revealing the evolutionary complexity of this region. In addition, 18 new polymorphic microsatellites have been selected in order to cover the entire SSC7p11-q14 region.

Animals↗

PubMed1995

A physical map of the region spanning the chromosome 12 translocation breakpoint in a mesothelioma with a t(X;12)(q22;p13).

We have constructed a physical map of a 4.6-cM region of human chromosome band 12p13.3 that contains a translocation breakpoint from a mesothelioma with a t(X;12)(q22;p13). The map contains a contig of 22 yeast artificial chromosomes (YACs), onto which we have placed 18 sequence tagged site (STS) markers, including seven genes: D12S370, FGF6, KCAN1, KCNA5, KCNA6, NTF3, and VWF. A second YAC contig, comprised of 22 YAC clones, was located distal to the mesothelioma breakpoint and contained 12 STS markers, including four genes (CACNL1A1, D12S380E, D12S381E, and D12S382E). Based on STS content and fluorescence in situ hybridization experiments, two stable, nonchimeric YAC clones were found that span the mesothelioma breakpoint. A long-range restriction map of an 800-kb region was constructed and used to refine the mesothelioma breakpoint to a region of approximately 100 kb, flanked by the potassium channel genes KCNA1 and KCNA5. The latter was confirmed by direct visual hybridization (DIRVISH) experiments, using cosmids isolated for markers flanking the breakpoint as probes.

Animals↗

PubMed1998

Homology between human chromosome 2p13.3 and the wobbler critical region on mouse chromosome 11: comparative high-resolution mapping of STS and EST loci on YAC/BAC contigs.

Human Chr 2p13-14 and homologous regions on mouse Chrs 6 and 11 have been subjects of previous studies because they comprise the loci for several neuromuscular diseases. Here we report on high-resolution mapping of 55 STS and EST loci on human Chr 2p13.3 and of 47 markers on the corresponding region on proximal mouse Chr. 11. The maps comprise several known genes, MEIS1/Meis1, RAB1a/Rab1a, MDH1/Mor2, OTX1/Otx1, and REL on human 2p13.3 and mouse Chr 11, respectively, as well as the wobbler (wr) critical region of the mouse. Whereas a perfect correspondence was found in most of the 4-Mb region, a small rearrangement was discovered around the OTX1/Otx1 locus. The detailed STS and EST transcript maps of these regions and a further narrowing down of the mouse wr critical region to the interval between D11Mit79 and D11Mit19 allow for the selection of positional candidate genes for wr, and the exclusion of others.

Animals↗

PubMed1998

High resolution physical mapping and identification of transcribed sequences in the Down syndrome region-2.

The identification and mapping of genes within the Down syndrome region is an important step toward a complete understanding of the pathogenesis of this disorder. The objective of the present work is to identify and map genes within the Down syndrome region-2. Chromosome 21 cosmid clones corresponding to "cosmid pockets" 121-124 have been first used as a starting material for generation of a single high resolution integrated cosmid/PAC contig with full EcoRI/SmaI restriction map. The integrated contig has been further anchored to genetic and physical maps through the positioning of 6 markers in the following order: ACTL5-D21S3-684G2T7-D21S71-D21S343-D21S 268. The entire contig covers 342 kb of the Down syndrome region-2 of chromosome 21. Subsequently, we have isolated, identified, and mapped four novel cDNAs which we have named N143, N144, CHD/333, and 90/3H1 and a potentially transcribed genomic sequence (E05133T7). Additionally, we have accurately located a previously described gene, the WRB gene, between the markers ACTL5-D21S268 within this Down Syndrome Region-2.

Chromosome Mapping↗

PubMed1995

Visual mapping by fiber-FISH.

FISH techniques have opened new possibilities for high-resolution genome mapping. Effective utilization of these techniques for the rapid orientation and ordering of adjacent and overlapping probes as well as for the characterization of long-range genomic contigs would facilitate physical mapping and positional cloning efforts. Here, we have evaluated our recently developed improved fiber-FISH technique for the physical mapping of a 500-kb region at 1p32 as well as for the detection of genomic rearrangement affecting this region. Our fiber-FISH technique is based on the hybridization of probes to unfixed linearized DNA fibers on a microscope slide. Preparation of the target DNA from cells embedded in pulsed-field gel electrophoresis (PFGE) blocks makes it possible to obtain long intact DNA fibers that give an excellent signal-to-noise ratio in FISH. The linear range of the method reached from 2 to 500 kb with a measuring accuracy approaching that of PFGE. Fiber-FISH was used to establish the order, orientation, and distances for several probes for this region, including six large insert phage, cosmid, and P1 clones and seven genomic subclones. This has significantly facilitated our efforts to develop a genomic contig for this region, recently discovered to contain the gene for inherited neuronal ceroid lipofuscinosis (INCL). Finally, we also demonstrated how rearrangements affecting the L-myc gene at this locus in small-cell lung cancer can be visualized with fiber-FISH. In conclusion, fiber-FISH is very useful for high-resolution physical mapping and contig evaluation as well as for detecting genetic rearrangements.

Carcinoma, Small Cell↗

PubMed2000

A physical and genetic map of the Mycoplasma hyopneumoniae strain J genome.

A macrorestriction map of the genome of Mycoplasma hyopneumoniae strain J, the type strain of the causative agent of enzootic pneumonia in pigs, was constructed using pulsed-field gel electrophoresis (PFGE) and DNA hybridization. The size of the genome as determined by PFGE was approximately 1070 kb. Assembly of the M. hyopneumoniae genomic map was facilitated and complimented by the simultaneous construction of an ordered cosmid library. Five contigs of overlapping cosmids were assembled, which together represent coverage of approximately 728 kb. Forty-two genetic markers (including three types of repeated elements) were placed on the M. hyopneumoniae map. Closer examination of an ApaI restriction fragment contained entirely within a single cosmid insert suggests that the genome size may be overestimated by PFGE.

Animals↗

PubMed1998

Imprinting of mouse Kvlqt1 is developmentally regulated.

Mouse distal chromosome 7 contains a cluster of at least five imprinted genes. The syntenic region in humans, at 11p15.5, has been implicated in several genetic disorders. Consistent with the imprinted status of the genes in the region, Beckwith-Wiedemann syndrome (BWS) and Wilms tumor are each associated with loss of maternal information. Also mapping to 11p15.5 are long QT and Jervell and Lange-Nielsen (JLN) syndromes. In contrast to BWS and Wilms tumor, these syndromes do not show any parent of origin bias. Recently positional cloning has identified KVLQT1 as the 11p15.5 gene responsible for increased susceptibility to long QT and JLN syndromes. Other studies associate KVLQT1 with BWS. Human KVLQT1 is paternally imprinted in embryos. In this study we present a contig and transcript map of distal mouse 7 and we physically and genetically map mouse Kvlqt1 to the region. Mouse Kvlqt1 is strongly expressed in heart, lung, gut, kidney and uterus. While its early developmental expression is maternal in origin, the paternal allele becomes increasingly active during development. Late juvenile and adult animals show complete biallelism, suggesting an explanation for the lack of parent of origin bias in JLN and long QT.

Aging↗

PubMed1994

A 1.8-Mb YAC contig in Xp11.23: identification of CpG islands and physical mapping of CA repeats in a region of high gene density.

The genes ARAF1, SYN1, TIMP, and PFC are clustered within 70 kb of one another, and, as reported in the accompanying paper (J. Knight et al., 1994, Genomics 21: 180-187), at least four more genes map within 400 kb: a cluster of Krüppel-type zinc finger genes (including ZNF21, ZNF41, and ZNF81) and ELK-1, a member of the ets oncogene superfamily. This gene-rich region is of particular interest because of the large number of disease genes mapping to Xp11.23: at least three eye diseases (retinitis pigmentosa type 2, congenital stationary night blindness CSNB1, and Aland Island eye disease), Wiskott-Aldrich syndrome, X-linked nephrolithiasis, and a translocation breakpoint associated with synovial sarcoma. We have constructed a 1.8-Mb YAC contig in this region, confirming the link between TIMP and OATL1 reported by Knight et al. (1994) and extending the map in the distal direction. To investigate the likelihood that more genes are located within this region, we have carried out detailed mapping of rare-cutter restriction sites in these YACs and identified seven CpG islands. At least six of these islands are located over 50 kb from any known gene locations, suggesting that the region contains at least this many as yet unidentified genes. We have also mapped the physical locations of six highly polymorphic CA repeats within the contig, thus integrating the physical, genetic, and transcriptional maps of the region and facilitating the mapping and identification of disease genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗