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PubMed2002

Construction of a 1.2-Mb BAC/PAC contig of the porcine gene RYR1 region on SSC 6q1.2 and comparative analysis with HSA 19q13.13.

We screened a porcine bacterial artificial chromosome (BAC) and a P1 derived artificial chromosome (PAC) library to construct a sequence-ready approximately 1.2-Mb BAC/PAC contig of the ryanodine receptor-1 gene (RYR1) region on porcine chromosome (SSC) 6q1.2. This genomic segment is of special interest because it harbors the locus for stress susceptibility in pigs and a putative quantitative trait locus for muscle growth. Detailed physical mapping of this gene-rich region allowed us to assign to this contig 17 porcine genes orthologous to known human chromosome 19 genes. Apart from the relatively well-characterized porcine gene RYR1, the other 16 genes represent novel chromosomal assignments and 14 genes have been cloned for the first time in pig. Comparative analysis of the porcine BAC/PAC contig with the human chromosome (HSA) 19q13.13 map revealed a completely conserved gene order of this segment between pig and human. A detailed porcine-human-mouse comparative map of this region was constructed.

Animals↗
PubMed2003

Estimates of conserved microsynteny among the genomes of Glycine max, Medicago truncatula and Arabidopsis thaliana.

A growing body of research indicates that microsynteny is common among dicot genomes. However, most studies focus on just one or a few genomic regions, so the extent of microsynteny across entire genomes remains poorly characterized. To estimate the level of microsynteny between Medicago truncatula (Mt) and Glycine max (soybean), and also among homoeologous segments of soybean, we used a hybridization strategy involving bacterial artificial chromosome (BAC) contigs. A Mt BAC library consisting of 30,720 clones was screened with a total of 187 soybean BAC subclones and restriction fragment length polymorphism (RFLP) probes. These probes came from 50 soybean contig groups, defined as one or more related BAC contigs anchored by the same low-copy probe. In addition, 92 whole soybean BAC clones were hybridized to filters of HindIII-digested Mt BAC DNA to identify additional cases of cross-hybridization after removal of those soybean BACs found to be repetitive in Mt. Microsynteny was inferred when at least two low-copy probes from a single soybean contig hybridized to the same Mt BAC or when a soybean BAC clone hybridized to three or more low-copy fragments from a single Mt BAC. Of the 50 soybean contig groups examined, 54% showed microsynteny to Mt. The degree of conservation among 37 groups of soybean contigs was also investigated. The results indicated substantial conservation among soybean contigs in the same group, with 86.5% of the groups showing at least some level of microsynteny. One contig group was examined in detail by a combination of physical mapping and comparative sequencing of homoeologous segments. A TBLASTX similarity search was performed between 1,085 soybean sequences on the 50 BAC contig groups and the entire Arabidopsis genome. Based on a criterion of sequence homologues <100 kb apart, each with an expected value of < or =1e-07, seven of the 50 soybean contig groups (14%) exhibited microsynteny with Arabidopsis.

Arabidopsis↗
PubMed1999

Human KRML (MAFB): cDNA cloning, genomic structure, and evaluation as a candidate tumor suppressor gene in myeloid leukemias.

Members of the MAF family of basic region/leucine zipper transcription factors can affect transcription in either a positive or a negative fashion, depending on their partner protein(s) and the context of the target promoter. The KRML (MAFB) transcriptional regulator plays a pivotal role in regulating lineage-specific hematopoiesis by repressing ETS1-mediated transcription of erythroid-specific genes in myeloid cells. In previous studies, we mapped the human KRML gene within a genomic contig on human chromosome 20, bands q11.2-q13.1. We have isolated the human cDNA containing the full-length predicted open reading frame (ORF). Multiple KRML transcripts of approximately 1.8 and approximately 3 kb, which differ in the length of the 3' untranslated region, are ubiquitously expressed in hematopoietic tissues and encode a protein with 323 amino acids (MW 35,832). The protein has 84% identity and 92% similarity to the murine protein. The ORF of the human KRML gene contains no introns, and the gene spans approximately 3 kb. KRML maps within the smallest commonly deleted segment in malignant myeloid disorders characterized by a deletion of 20q; however, we detected no mutations of KRML in leukemia cells with loss of 20q. Thus, KRML is unlikely to be involved in the pathogenesis of malignant myeloid disorders characterized by abnormalities of chromosome 20.

Amino Acid Sequence↗
PubMed1994

Identification of YAC and cosmid clones encompassing the ZFX-POLA region using irradiation hybrid cell lines.

The human Xp21.3-p22.1 region is poorly mapped relative to other X chromosome regions. To target cosmid and YAC clones specifically from Xp21.3-p22.1 for rapid contig construction, a hybridization-based screening approach using irradiation hybrids has been used. Alu-PCR products generated from hybrid lines containing small overlapping fragments from Xp21-p22 were hybridized to an X chromosome cosmid library, and cosmids predicted by their hybridization pattern to map to the region of interest were analyzed by fluorescence in situ hybridization (FISH). Hybridization of the cosmids in pools to gridded YAC libraries identified 15 YACs, which were verified and tested for chimerism by FISH. Cosmid content analysis of the YACs defined two contigs, one with 12 YACs covering about 1.5 Mb and one with 3 YACs. Five YACs from the 12-YAC cluster had been previously recognized by DNA polymerase alpha (POLA). ZFX identified a single YAC; hence, the physical linkage of ZFX and POLA was demonstrated within the contig. Four YACs had been isolated previously with ZFX and these extend the contig to 2 Mb. Restriction mapping of several YACs demonstrates that ZFX and POLA are about 700 kb apart, a distance similar to that reported in the mouse between Zfx and Pola. The order of these two loci and two additional loci identified by homologous mouse linking clones was found to be conserved between human and mouse: tel-ZFX-DXCrc57-DXCrc140-POLA-cen. We have shown that YAC contigs can be rapidly constructed from targeted regions without the need for time-consuming YAC end rescue and chromosomal walking.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2001

Genetic mapping and DNA sequence-based analysis of deleted regions on chromosome 16 involved in progression of bladder cancer from occult preneoplastic conditions to invasive disease.

Histologic and genetic mapping with 30 hypervariable markers mapped to chromosome 16 were performed on 234 DNA samples of five cystectomy specimens from patients with invasive bladder cancer. Allelic losses of individual markers were related to microscopically identified precursor conditions in the entire bladder mucosa and invasive cancer. Their significance for the development and progression of neoplasia from in situ preneoplastic conditions to invasive disease was analysed by the nearest neighbor algorithm and binomial maximum likelihood analysis. Using this approach we identified five distinct regions of allelic losses defined by their flanking markers and predicted size as follows. p13.3(D16S418-D16S406, 1.2 cM), p13.1(D16S748-D16S287, 12.9 cM), q12 1(D16S409-D16S514, 24.0 cM), q22.1 (D16S496-D16S515, 5.4 cM), and q24 (D16S507-D16S511, 5.9 cM and D16S402-D16S413, 17.4 cM). The regions mapping to p13.1 and q24 were involved in early intraurothelial phases of bladder neoplasia such as mild to moderate dysplasia. On the other hand the deleted region mapping to p13.3 was involved in progression of severe dysplasia/carcinoma in situ to invasive bladder cancer. Testing of markers that exhibited statistically significant LOH in relation to progression of neoplasia from precursor conditions to invasive cancer on 28 tumors and voided urine samples from 25 patients with bladder cancer revealed that q12.1 showed LOH in 46.4% of tumor and 32.0% of voided urine samples. The LOH of a single marker D16S541 could be detected in approximately 28% of tumors and 20% of voided urine samples of patients with bladder cancer. These data imply that the deleted region centered around marker D16S541 spanning approximately 10 cM and flanked by D16S409 and D16S415 contains a novel putative tumor suppressor gene or genes playing an important role in the development of human bladder cancer. To facilitate more precise positional mapping and identification of pathogenetically relevant genes, we analysed of human genome contig and sequence databases spanning the deleted regions. Multiple known candidate genes and several smaller gene-rich areas mapping to the target regions of chromosome 16 were identified.

Aged↗
PubMed2005

Statistical Viewer: a tool to upload and integrate linkage and association data as plots displayed within the Ensembl genome browser.

BACKGROUND: To facilitate efficient selection and the prioritization of candidate complex disease susceptibility genes for association analysis, increasingly comprehensive annotation tools are essential to integrate, visualize and analyze vast quantities of disparate data generated by genomic screens, public human genome sequence annotation and ancillary biological databases. We have developed a plug-in package for Ensembl called "Statistical Viewer" that facilitates the analysis of genomic features and annotation in the regions of interest defined by linkage analysis. RESULTS: Statistical Viewer is an add-on package to the open-source Ensembl Genome Browser and Annotation System that displays disease study-specific linkage and/or association data as 2 dimensional plots in new panels in the context of Ensembl's Contig View and Cyto View pages. An enhanced upload server facilitates the upload of statistical data, as well as additional feature annotation to be displayed in DAS tracts, in the form of Excel Files. The Statistical View panel, drawn directly under the ideogram, illustrates lod score values for markers from a study of interest that are plotted against their position in base pairs. A module called "Get Map" easily converts the genetic locations of markers to genomic coordinates. The graph is placed under the corresponding ideogram features a synchronized vertical sliding selection box that is seamlessly integrated into Ensembl's Contig- and Cyto- View pages to choose the region to be displayed in Ensembl's "Overview" and "Detailed View" panels. To resolve Association and Fine mapping data plots, a "Detailed Statistic View" plot corresponding to the "Detailed View" may be displayed underneath. CONCLUSION: Features mapping to regions of linkage are accentuated when Statistic View is used in conjunction with the Distributed Annotation System (DAS) to display supplemental laboratory information such as differentially expressed disease genes in private data tracks. Statistic View is a novel and powerful visual feature that enhances Ensembl's utility as valuable resource for integrative genomic-based approaches to the identification of candidate disease susceptibility genes. At present there are no other tools that provide for the visualization of 2-dimensional plots of quantitative data scores against genomic coordinates in the context of a primary public genome annotation browser.

Chromosome Mapping↗
PubMed1995

Sequence analysis of the human hTg737 gene and its polymorphic sites in patients with autosomal recessive polycystic kidney disease.

DNA sequence analysis of the human Tg737 gene was performed in 36 patients with the autosomal recessive form of polycystic kidney disease (ARPKD). Coding exons and their adjacent splice sites were screened for mutations. Pathogenic exon or splice region mutations were not identified although one exonic and two intronic polymorphic sites were discovered. These results are in agreement with another study that has recently reported linkage to Chromosome (Chr) 6p21-cen in a set of 16 ARPKD families. STS mapping has localized the gene to a YAC contig that includes D13S175 on chromosome 13q12.1. The polymorphisms found in the htG737 gene will permit its future evaluation as a candidate gene for other recessive cystic renal diseases and as a modifier gene in human PKD.

Base Sequence↗
PubMed1996

A high-resolution annotated physical map of the human chromosome 13q12-13 region containing the breast cancer susceptibility locus BRCA2.

Various types of physical mapping data were assembled by developing a set of computer programs (Integrated Mapping Package) to derive a detailed, annotated map of a 4-Mb region of human chromosome 13 that includes the BRCA2 locus. The final assembly consists of a yeast artificial chromosome (YAC) contig with 42 members spanning the 13q12-13 region and aligned contigs of 399 cosmids established by cross-hybridization between the cosmids, which were selected from a chromosome 13-specific cosmid library using inter-Alu PCR probes from the YACs. The end sequences of 60 cosmids spaced nearly evenly across the map were used to generate sequence-tagged sites (STSs), which were mapped to the YACs by PCR. A contig framework was generated by STS content mapping, and the map was assembled on this scaffold. Additional annotation was provided by 72 expressed sequences and 10 genetic markers that were positioned on the map by hybridization to cosmids.

BRCA2 Protein↗
PubMed1993

A yeast artificial chromosome contig linking the steroid sulfatase and Kallmann syndrome loci on the human X chromosome short arm.

In this report we describe the construction of a yeast artificial chromosome (YAC) contig linking the steroid sulfatase (STS) and Kallmann syndrome (KAL) loci on Xp22.3. Four human YAC libraries were screened initially with sequences from DXS237 (GMGX9), DXS278 (S232B), and KAL and later with primers from exon 10 of the STS gene and the end fragment of a YAC clone YGX3 to fill the gaps. Fifteen clones were isolated and the sizes of their human inserts were determined by pulsed-field gel electrophoresis followed by Southern hybridization with labeled total human DNA. Overlaps between the YAC clones were evaluated using more than 20 DNA markers, including the screening probes, the end fragments, and the Alu-PCR products of the YAC clones. The extent of overlapping between the clones was further determined by long-range restriction mapping. In combination with our previously reported YAC contigs around STS and KAL, a total of 2 Mb of Xp22.3 have been isolated in YAC clones. These clones will facilitate the isolation of new genes and the characterization of deletions and translocations which occur at very high frequency in this region of the human X chromosome.

Arylsulfatases↗
PubMed1994

Cloning the human major histocompatibility complex in YACs.

To clone the human major histocompatibility complex (MHC), 53 YACs, with an average size of 490 kb, were isolated and characterized from the CEPH YAC library. These YACs were organized in a single large contig covering more than 4000 kb. Furthermore, a complete physical map of the previously uncloned HLA class I region was established from partial and/or total digestions of 15 YACs spanning 2000 kb. This resulted in the establishment of the first YAC contig that spans the entire MHC region and constitutes an essential step in the isolation of all of the genes present in the region.

Animals↗
PubMed1994

Top-down construction of an ordered Schizosaccharomyces pombe cosmid library.

A very rapid and efficient method for sorting and ordering large numbers of clones is presented. This top-down mapping approach divides the entire ordering problem into many smaller tasks and analyzes in parallel a gridded membrane array of clones by hybridization with probe pools. The strategy was tested on a 15-fold-coverage Schizosaccharomyces pombe cosmid library. About 1600 clones were assigned to chromosomes and to regions defined by the Not I and Sfi I restriction maps. Then, the clones were ordered into 20 contigs, which is consistent with statistical expectations for the degree of genome coverage used. The parallel ordering of clones and the computer-based analysis of digitized images make this approach very efficient; it is about 8-fold faster than existing methods. Only 61 hybridizations were needed to order 1600 clones.

Base Sequence↗
PubMed1994

One-megabase yeast artificial chromosome and 400-kilobase cosmid-phage contigs containing the von Hippel-Lindau tumor suppressor and Ca(2+)-transporting adenosine triphosphatase isoform 2 genes.

We have isolated and ordered yeast artificial chromosomes (YACs) and cosmids surrounding the von Hippel-Lindau (VHL) tumor suppressor and plasma membrane Ca(2+)-transporting ATPase isoform 2 (PMCA-2) genes on chromosome 3p25-26. The YAC contig consists of six YACs and covers a region of 1 megabase. A cosmid-phage contig around VHL and PMCA-2 genes (400 kilobases) was established and integrated into the YAC map. Using these clones, we generated an EcoRI map of the 400-kilobase region. PMCA-2 and VHL complementary DNA were positioned entirely within the cosmid-phage contig as well as two polymorphic markers (D3S601 and D3S1317). This physical map of the cloned region will allow a detailed analysis of both the PMCA-2 and VHL genes. Some of the genomic clones may be useful for isolation of the full-length VHL complementary DNA.

Base Sequence↗
PubMed2001

Three chromosomal rearrangements in neuroblastoma cluster within a 300-kb region on 1p36.1.

Deletions in the short arm of chromosome 1 (1p36) and MYCN amplification are common in neuroblastoma. Previously we showed evidence of at least two different neuroblastoma tumor-suppressor loci on 1p. One is associated with MYCN single-copy tumors and maps distal on 1p36.3. A second, more proximal locus maps to 1p36.1 and is deleted in about 90% of neuroblastomas with MYCN amplification. The cell line UHG-NP has the smallest 1p36 deletion of all neuroblastoma cell lines with MYCN amplifications. We assume that the more proximal locus maps within this deletion, close to its proximal border. Here we present the exact localization of the 1p deletion breakpoint of UHG-NP. A 600-kb PAC contig spanning the breakpoint was analyzed for genes and aberrations. Two more neuroblastoma-associated aberrations were mapped within 150 kb of the UHG-NP breakpoint. Within the contig, we identified nine genes expressed in neuroblastoma cells. One of these genes, AML2, maps 200 kb distal to the UHG-NP breakpoint but is expressed only rarely in neuroblastoma and showed no mutations.

Base Composition↗
PubMed1999

Radiation hybrid and cytogenetic mapping of SOCS1 and SOCS2 to chromosomes 16p13 and 12q, respectively.

Suppressor of cytokine signaling (SOCS) proteins are involved in the negative regulation of cytokine-induced STAT (signal transducers and activators of transcription) factor signaling. We cloned genomic regions of SOCS1 and SOCS2 genes and mapped these genes to chromosome 16p12-p13.1 and chromosome 12q21.3-q23 regions, respectively, by cytogenetic and radiation hybrid mapping. In addition, we mapped SOCS2 by yeast artificial chromosome (YAC) pool screening to YAC contig WC 12.5 on chromosome 12 with an unambiguous hit to CHLC.ATA19H12 and WI-5940, which is 461.5 cR from the top of the map.

Carrier Proteins↗
PubMed1999

TTC4, a novel human gene containing the tetratricopeptide repeat and mapping to the region of chromosome 1p31 that is frequently deleted in sporadic breast cancer.

The 1p31 region shows loss of heterozygosity in up to 50% of human breast cancers, indicating the presence of a tumor suppressor gene in this location. We have mapped six novel ESTs to a 15-Mb contig of yeast artificial chromosomes spanning the critical region of 1p31. One of these ESTs was localized within the contig to the region most commonly undergoing loss of heterozygosity in breast cancer. The corresponding gene sequence for this EST was established by cDNA cloning and RACE procedures. This gene is 2 kb long and contains a tetratricopeptide repeat motif and a coiled-coil domain. This family of genes has been implicated in a wide variety of functions, including tumorigenesis. This is the fourth member of the human gene family, and so we have named this gene TTC4. Northern blot analysis demonstrates a ubiquitous pattern of gene expression that includes breast tissue. A preliminary screen of human breast cancer cell lines shows that TTC4 is expressed in all cases, but SSCP analysis of the coding region of this gene following RT-PCR failed to reveal any mutations. Clearly, because of its map location, a more extensive analysis is warranted to establish whether subtle mutations are present in breast cancers.

Amino Acid Sequence↗
PubMed1997

High-resolution mapping and transcript identification at the progressive epilepsy with mental retardation locus on chromosome 8p.

Progressive epilepsy with mental retardation (EPMR) is an autosomal recessive central nervous system disorder characterized by childhood onset epilepsy and subsequent mental retardation. The locus for EPMR has been mapped to human chromosome 8p23. We recently reported the construction of a YAC contig across the 4 centimorgan minimum genetic region that harbors the disease locus. We now report further delineation of the critical region to <700 kb. Our mapping strategy relied on the identification of nine novel microsatellite markers and the construction of a complete BAC contig across the critical region. Several partial gene sequences have been identified from the region and are being analyzed as candidate genes for EPMR.

Base Sequence↗
PubMed1993

Fine mapping of the FSHD gene region orientates the rearranged fragment detected by the probe p13E-11.

We have produced a fine restriction map around the locus D4F104S1 (previously designated D4S810); a probe to this locus, p13E-11, identifies a polymorphic EcoRI fragment containing 3.2kb tandem repeats and detects DNA rearrangements associated with facioscapulohumeral muscular dystrophy (FSHD). We developed an STS (D4F106S1) which maps 2kb proximal to D4F104S1, and used this to isolate a 470kb YAC (y25C2E) from the ICI YAC library and a 930kb YAC (y956A11) from the CEPH megabase library. Both YACs contain the loci D4S139, D4F35S1 and D4F104S1. A cosmid library was produced from YAC y25C2E and two cosmid contigs constructed; a 115kb contig encompassing D4S139, and one of 135kb linking D4F35S1 and D4F104S1 and extending distal to the EcoRI fragment detected by p13E-11. A fine restriction map of both these contigs has been generated, allowing the orientation of the EcoRI fragment rearranged in FSHD to be determined. YAC y956A11 was used to confirm the integrity of y25C2E and the map of this region. 9B6A, a probe to the homeobox region of the tandem repeat D4Z4, identified a cross-hybridising sequence proximal to D4F104S1, however, p13E-11 does not detect this additional locus. CpG islands were identified between D4S139 and D4F35S1 and within each copy of the tandem repeat. The probe 9B6A detected each copy of the repeat motif, suggesting there is homeobox present in every copy of the 3.2kb repeat.

Base Sequence↗
PubMed1999

High-resolution human/goat comparative map of the goat polled/intersex syndrome (PIS): the human homologue is contained in a human YAC from HSA3q23.

The genetic and cytogenetic map around the chromosome 1 region shown to be linked with polledness and intersexuality (PIS) in the domestic goat (Capra hircus) was refined. For this purpose, a goat BAC library was systematically screened with primers from human coding sequences, scraped chromosome 1 DNA, bovine microsatellites from the region, and BAC ends. All the BACs (n = 30) were mapped by fluorescence in situ hybridization (FISH) on goat chromosome 1q41-q45. The genetic mapping of 30 new goat polymorphic markers, isolated from these BACs, made it possible to reduce the PIS interval to a region of less than 1 cM on goat chromosome 1q43. The PIS locus is now located between the two genes ATP1B and COP, which both map to 3q23 in humans. Genetic, cytogenetic, and comparative data suggest that the PIS region is now probably circumscribed to an approximately 1-Mb DNA segment for which construction of a BAC contig is in progress. In addition, a human YAC contig encompassing the blepharophimosis-ptosis-epicanthus-inversus region was mapped by FISH to goat chromosome 1q43. This human disease, mapped to HSA 3q23 and affecting the development and maintenance of ovarian function, could be a potential candidate for goat PIS.

Animals↗