Physical mapping and sequencing of class I genes in a 150-kb contig in the EC region.
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Amino Acid Sequence↗
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Technology development at the interface of proteome research and genomics: mapping nonpolymorphic proteins on the physical map of mouse chromosomes.
Data obtained from protein spots by peptide mass fingerprinting are used to identify the corresponding genes in sequence databases. The relevant cDNAs are obtained as clones from the Integrated Molecular Analysis of Genome Expression (I.M.A.G.E.) consortium. Mapping of I.M.A.G.E. clones is performed in two steps: first, cDNA clones are hybridized against a 10-hit genomic mouse bacterial artificial chromosome (BAC) library. Second, interspersed repetitive sequence polymerase chain reaction (IRS-PCR) using a single primer directed against the mouse B1 repeat element is performed on BACs. As each cDNA detects several BACs, and each individual BAC has a 50% chance to recover an IRS-PCR fragment, the majority of cDNAs produce at least a single IRS-PCR fragment. Individual IRS fragments are hybridized against high-density spotted filter grids containing the three-dimensional permutated pools of yeast artificial chromosome (YAC) library resources that are currently being used to construct a physical map of the mouse genome. IRS fragments that hybridize to YAC clones already placed into contigs immediately provide highly precise map positions. This technology therefore is able to draw links between proteins detected by 2-D gel electrophoresis and the corresponding gene loci in the mouse genome.
Animals↗
Anhidrotic ectodermal dysplasia gene region cloned in yeast artificial chromosomes.
Anhidrotic ectodermal dysplasia (EDA), an X-chromosomal recessive disorder, is expressed in a few females with chromosomal translocations involving bands Xq12-q13. Using available DNA markers from the region and somatic cell hybrids we mapped the X-chromosomal breakpoints in two such translocations. The breakpoints were further mapped within a yeast artificial chromosome contig constructed by chromosome walking techniques. Genomic DNA markers that map between the two translocation breakpoints were recovered representing putative portions of the EDA gene.
Base Sequence↗
Isolation and characterization of GT335, a novel human gene conserved in Escherichia coli and mapping to 21q22.3.
As part of efforts to identify candidate genes for disorders mapped to 21q22.3, we have constructed a 405-kb cosmid contig encompassing five tightly linked markers mapping to this region. A subset of these cosmids was used to identify cDNA fragments by the method of hybrid selection. We present here the cDNA sequence of one such gene (GT335) mapping to this region. The gene is expressed as a 1.7-kb transcript predominantly in heart and skeletal muscle, potentially displays alternate splicing, and is predicted to encode a protein 268 amino acids in length. GT335 spans an estimated 13 kb of genomic DNA and is split into seven exons. Five of the six introns conform to the GT . . . AG consensus for intronic splice junctions; the sixth contains nonconventional (AT . . . AC) intronic junctions. We screened this gene for single-basepair mutations using single-strand conformation polymorphism and sequence analysis of both cDNA and genomic DNA from a number of unrelated individuals and have identified several sequence variations, two of which cause conservative amino acid substitutions. This gene is well conserved evolutionarily, with homologs identified in zebrafish and Escherichia coli, suggesting that it plays an important role in basic cellular metabolism.
Amino Acid Sequence↗
Homozygosity and physical mapping of the autosomal recessive retinitis pigmentosa locus (RP14) on chromosome 6p21.3.
Retinitis pigmentosa (RP) is a heterogeneous genetic disorder with autosomal dominant, autosomal recessive, and X-linked forms. We previously mapped an additional arRP locus to chromosome 6p21 (RP14) in a single extended kinship from the Dominican Republic. Aided by a second linked RP pedigree from the same region of the Dominican Republic, we have refined the disease locus to a 2-cM region that is homozygous-by-descent in both pedigrees. A complete YAC, and a partial BAC, contig of the RP14 locus was constructed between the markers D6S1560 and D6S291, encompassing approximately 2.1 Mb. The contig contains 12 YACs and 31 BACs and is characterized by 45 markers including 8 microsatellite markers, 6 gene-derived sequences/ESTs obtained from the databases, and 28 new STSs and 4 new ESTs obtained by BLAST search using DNA sequence from the ends of the BAC and YAC inserts. With a STS density of approximately 1 every 20 kilobases, this contig significantly enhances available maps of the region.
Chromosome Mapping↗
Detailed mapping and loss of heterozygosity analysis suggests a suppressor locus involved in sporadic breast cancer within a distal region of chromosome band 17p13.3.
The chromosome region 17p13.3 is thought to encode a tumour suppressor gene involved in sporadic breast cancer and other malignancies. Physical ordering of markers has been carried out by a series of multicolour fluorescent in situ hybridisation (FISH) experiments, using isolated yeast artificial chromosomes (YACs) and cosmids. Eight polymorphic markers ordered within this new physical map and one external marker were used to investigate the pattern of loss of heterozygosity in a panel of 40 sporadic breast tumour patients. The data revealed a region of high loss (60%) within distal 17p13.3, defined by markers D17S926, D17S695 and D17S849 which mapped close together. A contig of YACs was constructed physically linking these three markers.
Base Sequence↗
Fine mapping of the circling (cir) gene on the distal portion of mouse chromosome 9.
Circling mice manifest profound deafness, head-tossing, and bi-directional circling behavior, which they inherit in autosomal recessive manner. Histologic examination of the inner ear reveals abnormalities of the region around the organ of Corti, spiral ganglion neurons, and outer hair cells. A genetic linkage map was constructed for an intraspecific backcross between cir and C57BL/6J mice. The cir gene was mapped to a region between D9Mit116/D9Mit15 and D9Mit38 on mouse chromosome (Chr) 9. Estimated distances between cir and D9Mit116, and between cir and D9Mit38 were 0.70 +/- 0.40 and 0.23 +/- 0.23 cM, respectively. Order of the markers was defined as follows: centromere - D9Mit182 - D9Mit51/D9Mit79/D9Mit310 - D9Mit212/D184 - D9Mit116/D9Mit15 - cir - D9Mit38 - D9Mit20 - D9Mit243 - D9Mit16 - D9Mit55/D9Mit125 - D9Mit281. On the basis of genetic mapping, we constructed a yeast artificial chromosome (YAC) contig across the cir region. The cir gene is located between the lactotransferrin (ltf) and microtubule-associated protein (map4) genes. The distal portion of mouse Chr 9 encompassing the cir region is homologous with human chromosome 3p21, which contains the Deafness, form B: Autosomal Recessive Deafness (DFNB6) locus. Therefore, the circling mouse is a potential animal model for DFNB6 deafness in humans.
Animals↗
Isolation of a human gene (HES1) with homology to an Escherichia coli and a zebrafish protein that maps to chromosome 21q22.3.
Exon trapping was performed with chromosome 21 cosmids to identify those that may be involved in the pathogenesis of Down syndrome, or several of the genetic diseases that map to chromosome 21. BLASTX analysis revealed two exons with significant homology to a zebrafish protein (ES1) and an Escherichia coli protein (sigma cross-reacting protein 27A), both of unknown function. The exons also showed identity with several expressed sequence tags (ESTs). Sequences from all ESTs derived from this gene and reverse transcription-polymerase chain reaction (RT-PCR) analysis were used to determine the full cDNA sequence, which corresponded to an mRNA of 1.7 kb with an open reading frame of 268 amino acids. The mRNA from this gene, termed HES1, is ubiquitously expressed, but strongly so in heart and skeletal muscle. Potential mitochondrial targeting signals were found in both the human and zebrafish proteins, consistent with the high expression levels in muscle tissues. The strong homology between the E. coli, zebrafish and HES1 proteins suggests an important biological role. Hybridization of RT-PCR products to a cosmid contig in chromosome 21q22.3, mapped HES1 just proximal to D21S25, a critical mapping region for several genetic diseases. Given the mapping position, this gene is a candidate for involvement in these disorders, including autoimmune polyglandular disease type I and the autosomal nonsyndromic deafness loci, DFNB8 and DFNB10. In addition, the initial method of EST identification for gene isolation presented here is valid for many genes and can be used to obtain initial sequence contigs without cloning or library screening.
Amino Acid Sequence↗
High-resolution mapping by YAC fragmentation of a 2.5-Mb Xp22 region containing the human RS, KFSD and CLS disease genes.
The disease loci for X-linked Retinoschisis (RS), Keratosis follicularis spinulosa decalvans (KFSD), and Coffin-Lowry syndrome (CLS) have been localized to the same, small region in Xp22 on the human X Chromosome (Chr). To generate a high-resolution map of the available contig in this area, we have used the YAC fragmentation vectors pBP108/ADE2 and pBP109/ADE2 and generated fragmented YACs from a 2.5-Mb YAC (y939H7) spanning the mentioned disease gene candidate regions. Forty-seven fragmented YACs were generated and analyzed, ranging in size from 170 kb to over 2400 kb. The resulting YAC fragmentation panel was used to construct a detailed restriction map of the region and has been used to bin clones and markers. As a deletion panel, it will present a valuable resource for further mapping.
Abnormalities, Multiple↗
Physical mapping of the human ATX1 homologue (HAH1) to the critical region of the 5q- syndrome within 5q32, and immediately adjacent to the SPARC gene.
The 5q- syndrome is a myelodysplastic syndrome with the 5q deletion as the sole karyotypic abnormality. The human ATX1 homologue (HAH1), encodes a copper-binding protein with a role in antioxidant defence. We have mapped this gene to the 3 Mb critical region of gene loss of the 5q- syndrome within 5q32, flanked by the genes for ADRB2 and IL12B, using gene dosage analysis. Fine physical mapping of the HAH1 gene within this genomic interval was then performed by screening YAC and BAC contigs spanning the critical region of the 5q- syndrome using PCR amplification. The HAH1 gene maps immediately adjacent to the SPARC gene at 5q32, and is flanked by the genetic markers D5S1838 and D5S1419. The HAH1 gene is expressed in haematological tissues and plays a role in antioxidant defence. Antioxidant levels are low in most cancers and the importance of antioxidant enzymes in cancer genesis is well recognised. Genomic localisation, function and expression would suggest that the HAH1 gene represents a candidate gene for the 5q-syndrome.
Carrier Proteins↗
Closing in on the Rieger syndrome gene on 4q25: mapping translocation breakpoints within a 50-kb region.
Rieger syndrome (RGS) is an autosomal dominant disorder of morphogenesis affecting mainly the formation of the anterior eye chamber and of the teeth. RGS has been localized to human chromosome 4q25 by linkage to epidermal growth factor (EGF). We have constructed a detailed physical map and a YAC contig of the genomic region encompassing the EGF locus. Using FISH, several YACs could be shown to cross the breakpoint in two independent RGS patients with balanced 4q translocations. Alu- and LINE-fragmentation of a 2.4-Mb YAC generated a panel of shorter YACs ranging in size from 2.4 Mb to 75 kb. Several fragmentation YACs were subcloned in cosmids, which were mapped to specific subregions of the original YAC by hybridization to the fragmentation panel to further refine the localization of the translocation breakpoints, allowing mapping of the breakpoints to within the most-telomeric 200 kb of the original 2.4-Mb YAC. FiberFISH of cosmids located in this 200-kb region mapped the two translocation breakpoints within a 50-kb region approximately 100-150 kb centromeric to D4S193, significantly narrowing down the candidate region for RGS. The mapping data and resources reported here should facilitate the identification of a gene implicated in Rieger syndrome.
Blotting, Southern↗
A 500-kb region on chromosome 16p13.1 contains the pseudoxanthoma elasticum locus: high-resolution mapping and genomic structure.
We have recently mapped the genetic defect underlying pseudoxanthoma elasticum (PXE), an inherited disorder characterized by progressive calcification of elastic fibers in skin, eye, and cardiovascular system, to chromosome 16p 13.1. Here we report further data on the fine-mapping and genomic structure of this locus. Haplotype analysis of informative PXE families narrowed the locus to an interval of less than 500 kb located between markers D16B9621 and D16S764. Three overlapping YAC clones were found to cover this region through YAC-STS content mapping. An overlapping BAC contig was then constructed to cover this interval and the surrounding region. About 80% of this chromosomal region has been fully sequenced using the BAC shotgun technique. Gene content and sequence analysis predicted four genes (MRP1, MRP6, PM5, and a novel transcript) and two pseudogenes (ARA and PKDI) within this interval. By screening a somatic cell hybrid panel we were able to precision-map the breakpoint of Cy185 and the starting point of a chromosomal duplication within 20 kb of BAC A962B4. The present data further refine the localization of PXE, provide additional physical cloning resources, and will aid in the eventual identification of the genetic defect causing PXE.
Adult↗
Comparative analysis of a BAC contig of porcine chromosome 13q31-q32 and human chromosome 3q21-q22.
BACKGROUND: The gene(s) encoding the ETEC F4ab/ac receptors, involved in neonatal diarrhoea in pigs (a disease not yet described in humans), is located close to the TF locus on Sscr13. In order to reveal and characterize possible candidate genes encoding these receptors, a porcine physical map of the TF region is indispensable. RESULTS: A contig of 33 BAC clones, covering approximately 1.35 Mb surrounding the TF locus on Sscr13q31-q32, was built by chromosome walking. A total of 22,552 bp from the BAC contig were sequenced and compared with database sequences to identify genes, ESTs and repeat sequences, and to anchor the contig to the syntenic region of the human genome sequence (Hsap3q21-q22). The contig was further annotated based on this human/porcine comparative map, and was also anchored to the Sanger porcine framework map and the integrated map of Sscr13 by RH mapping. CONCLUSION: The annotated contig, containing 10 genes and 2 ESTs, showed a complete conservation of linkage (gene order and orientation) with the human genome sequence, based on 46 anchor points. This underlines the importance of the human/porcine comparative map for the identification of porcine genes associated with genetic defects and economically important traits, and for assembly of the porcine genome sequence.
Animals↗
Clustered and interspersed gene families in the mouse immunoglobulin kappa locus.
Although numerous solitary germ-line V kappa genes and two small V kappa contiguously cloned gene regions (contigs) are known, no attempts to systematically elucidate the structure of the kappa locus of the mouse have been reported so far. As a first step to this aim we screened a cosmid library of C57BL/6J mouse DNA with 18 probes that are more or less specific for the different V kappa gene families. Ninety-one V kappa gene-containing cosmid clones were characterized by detailed restriction mapping and hybridizations. Several contigs were constructed from overlapping clones. The contigs and the still unlinked cosmid clones cover 1.6 Mb. Many of the cosmid clones were localized on chromosome 6 where the kappa locus is known to reside; no evidence for the existence of dispersed V kappa genes (orphons) was obtained. Eighty-five strong hybridization signals were assigned to distinct V kappa gene families, while for 11 weak signals the assignment was less definite. As to the distribution of gene families within the locus the following situation emerged: there are both, groups of genes which belong to one V kappa gene family ("clusters") and groups in which genes of different families are interspersed. The interspersion of gene families seems to be more pronounced than has been assumed so far. Additional V kappa genes which are known to exist will have to be isolated from other gene libraries of the same mouse Ig kappa haplotype.
Animals↗
Large-scale identification and mapping of nuclear matrix-attachment regions in the distal imprinted domain of mouse chromosome 7.
Mammalian imprinted genes, which are expressed from only one of the parental alleles, have a tendency to form clusters and are regulated by long-range mechanisms. Nuclear matrix-attachment regions (MARs), the anchor points of loop domains, are involved in coordination of gene expression and could play a role in regulation of imprinted domains. We have identified and mapped a total of 52 MARs in a 1-Mb imprinted domain on mouse distal chromosome 7 using our cosmid contigs and an in vitro MAR assay. We find two MAR clusters (comprising 20 and 19 MARs), one of which is mapped in the Th-Ins2 intergenic region, coincident with the boundary between the two imprinted subdomains. However, the imprinted/non-imprinted boundaries are not associated with a MAR. Based on the sequence information, we find that many of the MARs are rich in long interspersed nuclear elements. In addition, comparisons of the results obtained with several MAR-prediction software programs reveal good performance of ChrClass in terms of both sensitivity and specificity. This study presents the first large-scale mapping of MARs in an imprinted domain and provides a platform for understanding the roles of MARs in imprinting.
Animals↗
Application of AFLP technology to radiation hybrid mapping.
We have investigated the use of AFLP technology as a tool for the high throughput enrichment of Radiation Hybrid (RH) maps. The 3000 rad TM112 bovine RH panel was assayed with 37 EcoRI/TaqI AFLP primer combinations. The number of selective nucleotides used during PCR was increased to seven, to reduce the complexity of the AFLP profile and minimise the overlap between hamster and bovine bands co-amplified from hybrid cell clones. Seven-hundred-forty-seven bovine AFLP bands were amplified that could be distinguished following electrophoresis. Repeatability was tested within and between laboratories on independent template preparations and an error rate of 1.3% found. Two-point linkage analysis clustered 428 AFLP fragments in 39 linkage groups of at least 4 markers. Multi-point maps were constructed for 5 sample linkage groups. The study demonstrated that the AFLP approach could be used to rapidly screen for the most informative clones during panel construction and to increase the number of markers on RH maps, which could be useful for joining linkage groups formed by other markers. The use of AFLP markers as anchor points between existing RH maps and other physical maps, such as BAC contigs, is also discussed.
Animals↗
A sequence-ready BAC clone contig of human chromosome 10p15 spanning the loss of heterozygosity region in glioma.
Deletion of chromosome 10 is one of the most common chromosomal alterations in glioma. At 10p15, the telomeric region of the short arm of chromosome 10, loss of heterozygosity (LOH) has been frequently observed by microsatellite analysis, suggesting the presence of a tumor suppressor gene. We examined LOH in 34 gliomas on chromosome 10, and frequent LOH on 10p was detected on 10p15, in agreement with deletion mapping studies on chromosome 10. We then constructed a bacterial artificial chromosome (BAC) clone contig covering the critical region, which spanned the interval between D10S249 and D10S533 on 10p15. The map contained 68 BAC clones connected by 74 sequenced tag sites (STSs) and covered approximately 2.7 Mb, with one gap. A total of 74 STSs, including 6 microsatellite markers, 29 expressed sequenced tags (ESTs), and 39 BAC end STSs, were physically arranged. Twenty-eight ESTs were mapped in the interval between D10S249 and D10S559 (approximately 1200 kb), and another EST was mapped in the interval between D10S559 and D10S533 (approximately 1300 kb). This sequence-ready BAC clone contig map will be a basic resource for high-quality sequencing and positional cloning of the putative tumor suppressor gene at 10p15 in glioma.
Base Sequence↗
Assembly of high-resolution bacterial artificial chromosome, P1-derived artificial chromosome, and cosmid contigs.
The generation of contiguous physical maps is often complicated by a variety of factors including the type of cloning system used. Here we describe procedures for the isolation, rapid characterization, and physical mapping of large-insert recombinant bacterial clones from total human genomic BAC (bacterial artificial chromosome) and PAC (P1-derived artificial chromosome) libraries containing clones with an average insert size of 150 kbp. After initial isolation, the clones were subjected to a variety of fingerprinting procedures including inter-Alu PCR, semiautomated fluorescent finger-printing, and EcoRI restriction fragment mapping. Individual BAC and PAC clones were also used as probes to interrogate arrayed chromosome 19-specific cosmid libraries. The combination of analyses facilitated the identification of chromosome-specific large-insert clones as well as the construction of a large (1.2 Mb) high-resolution BAC, PAC, and cosmid contig in 19q13.2, spanning the region from the carcinoembryonic antigen gene family to the X-ray repair cross complementing 1 DNA repair gene. This type of approach directly demonstrates the utility of large-insert recombinant bacterial clones for the construction of contiguous physical maps of entire chromosomes.
Base Sequence↗