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PubMed1998

Construction of a gene map of the nephronophthisis type 1 (NPHP1) region on human chromosome 2q12-q13.

A gene for the autosomal recessive kidney disorder juvenile nephronophthisis (NPH) is located on chromosome 2q between markers D2S1893 and D2S1888. Recently, the presence of large homozygous deletions was described in the majority of NPH patients. We constructed an integrated YAC/PAC contig of 54 markers and 30 PAC clones that encompasses this deletion and the flanking inverted duplication. Thirty-six novel sequence-tagged site markers were generated for this region of 2-3 Mb, 22 of which represent PAC ends. Ten of 18 multiplex NPH families show a homozygous deletion for 8 consecutive markers. BlastN database search and expressed sequence tag (EST) mapping led to the localization of 18 EST clones to the integrated contig, representing 11 putative transcribed sequences. Seven EST clones were localized to the NPHP1 region between D2S1893 and D2S1888. Two EST clones, zc07a11 from a human parathyroid tumor library and yy63e10 from a multiple sclerosis lesion library, are located in the deletion region. PCR amplification experiments indicate that zc07a11 represents a chimeric cDNA. Through FISH analysis the NPHP1 deletion region was localized to 2q12-q13. In summary, our study provides a high-resolution physical map of the NPHP1 region with 7 precisely localized expressed sequences, 2 of which have recently been shown to be part of a gene for NPH. These data will alleviate the identification of further genes of a homozygous gene deletion syndrome in patients with NPH and oculomotor apraxia and will be instrumental in the characterization of the molecular mechanism leading to the large homozygous deletion in this region. The data furthermore provide an important step toward the construction of a sequence-ready PAC contig of this region.

Adaptor Proteins, Signal Transducing↗

PubMed2004

An integrated approach for comparative mapping in rice and barley with special reference to the Rph16 resistance locus.

The accumulated sequence information of the almost completed rice genome and the transcriptome of other cereals provide an excellent starting point for comparative genome analysis. We performed targeted synteny-based marker saturation for the Rph16 leaf rust resistance locus in barley by extensively exploiting these newly available resources. Out of a collection of over 320,000 public barley ESTs 309 non-redundant candidate syntenic clones have been identified for this region in a two-step in silico selection procedure. For mapping, 54 barley cDNA-clones were selected due to the even distribution of their homologs on a putatively collinear 3-Mb rice BAC contig. Out of these, 97% (30) of the polymorphic markers could be genetically assigned in collinearity to the target region in barley and a set of 11 markers was integrated into an rph16 high-resolution map. Although, the collinear target region of rice does not contain an obvious candidate gene for rph16 the results demonstrate the potential of the presented procedure to efficiently utilize EST resources for synteny-based marker saturation. The systematic genome-wide exploitation of the increasing sequence data resources will strongly improve our current view of genome conservation and likely facilitate a synteny-based isolation of genes conserved across cereal species.

Chromosome Mapping↗

PubMed1996

Genetic analysis of the epidermal differentiation complex (EDC) on human chromosome 1q21: chromosomal orientation, new markers, and a 6-Mb YAC contig.

The epidermal differentiation complex (EDC) unites a remarkable number of structurally, functionally, and evolutionarily related genes that play an important role in terminal differentiation of the human epidermis. It is localized within 2.05 Mb of region q21 on human chromosome 1. We have identified and characterized 24 yeast artificial chromosome (YAC) clones by mapping individual EDC genes, sequence-tagged site (STS) markers (D1S305, D1S442, D1S498, D1S1664), and 10 new region-specific probes (D1S3619-D1S3628). Here we present a contig that covers about 6 Mb of 1q21 including the entire EDC. Fluorescence in situ hybridization on metaphase chromosomes with two YACs flanking the EDC determined its chromosomal orientation and established, in conjunction with physical mapping results, the following order of genes and STSs: 1cen-D1S442-D1S498-S100A10-THH-FLG- D1S1664-IVL-SPRR3-SPRR1-SPRR2-LOR- S100A9-S100A8-S100A7-S100A6-S100A5-S100 A4- S100A3-S100A2-S100A1-D1S305-1qtel. These integrated physical, cytogenetic, and genetic mapping data will be useful for linkage analyses of diseases associated with region 1q21 and for the identification of novel genes and regulatory elements in the EDC.

Cell Differentiation↗

PubMed2001

[Construction of a bacterial artificial chromosome (BAC) contig encompassing the bacterial blight resistance gene Xa4 locus in rice].

The gene Xa4 confers dominantly resistance to rice bacterial blight, which has been finely mapped between RFLP markers G181 and L1044, and co-segregated with the resistance gene homologues sequence marker RS13. The three markers were used to screen a rice Bacterial Artificial Chromosome (BAC) library constructed from IRBB56, a Xa4-harborring indica variety, resulting in the detection of totally 128 positive clones. Of the 18 positive clones picked out by RS13, 4 and 6 clones were simultaneously detected by G181 and L1044, respectively. Based on their HindIII restriction patterns, 12 clones were selected out to construct a contig that spanned about 420 kb covering the Xa4 locus, which is a solid base for the isolation of Xa4 gene.

Chromosomes, Artificial, Bacterial↗

PubMed1998

High-resolution genetic, physical, and transcript map of the mnd2 region of mouse chromosome 6.

The autosomal recessive mutation mnd2 is responsible for a lethal neuromuscular wasting disorder in the mouse. A high-resolution genetic map of the mnd2 region of mouse chromosome 6 was generated by analysis of 1147 F2 offspring from an intersubspecific cross between strains C57BL/6J-mnd2/+ and CAST/Ei. The results localize mnd2 to the 0.2-cM interval between D6Mit164 and D6Mit128. A contig of overlapping YAC, BAC, and P1 clones spanning the nonrecombinant interval was constructed. One novel gene isolated from the contig, D6Mm3e, is a new member of the WD repeat gene family. The observed gene order for the five positional candidate genes previously mapped to the region and five newly isolated genes is centromere-Hexokinase II-D6Mm5e-p62 Dok-Aup1-Rhotekin, D6Mm3e-Dynactin 1-Smooth muscle gamma actin-D6Mm4e-beta-adducin-telomere. Seven of these genes are located within the 400-kb nonrecombinant interval for mnd2. Comparison between wildtype and mutant failed to detect any differences in mRNA size, abundance, or coding sequence for these seven genes. The genes described here are positional candidates for the Parkinson disease susceptibility locus PARK3 that was recently mapped to the corresponding region of human chromosome band 2p13.1.

Amino Acid Sequence↗

PubMedDate not supplied

A YAC contig spanning the blepharophimosis-ptosis-epicanthus inversus syndrome and propionic acidemia loci.

Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) is an autosomal dominant condition consisting of congenital dysplasia of the eyelids with a reduced horizontal diameter of the palpebral fissures, droopy eyelids and epicanthus inversus. Two clinical entities have been described: type I and type II. The former is distinguished by female infertility, whereas the latter presents without other symptoms. Both type I and type II were recently mapped on the long arm of chromosome 3 (3q22-q23), suggesting a common gene may be affected. The centromeric and the telomeric limits of this region are well defined between loci D3S1316 and D3S1615, which reside approximately 5 cM apart. Here, we present the construction of a YAC contig spanning the entire BPES locus using 17 polymorphic markers, 2 STS and 28 ESTs. This region of approximately 5 Mb was covered by 31 YACs, and was supported by detailed FISH analysis. In addition, we have precisely mapped the propionyl-CoA carboxylase beta polypeptide (PCCB), the gene mutated in propionic acidemia, within this contig. Apart from providing a framework for the identification of the BPES gene, this contig will also be useful for the future identification of defects and genes mapped to this region, and for developing template resources for genomic sequencing.

Amino Acid Metabolism, Inborn Errors↗

PubMed1992

A structural analysis of the Sxr region of the mouse Y chromosome.

Three genetic functions have been mapped to the minute Sxr (sex-reversed) region of the mouse Y chromosome. These are Tdy, the primary testis determinant; Hya, the locus (either structural or regulatory) controlling the expression of the male-specific minor histocompatibility antigen H-Y; and Spy, a spermatogenic gene. Hya and Spy map to DNA deleted from the Sxr region in the deletion variant Sxrb (the delta Sxrb DNA). With the object of cloning Hya and Spy, we initiated chromosome walking in the delta Sxrb DNA. From three independent loci--Sx1, Zf2, and T5--we have isolated approximately 270 kb of delta Sxrb DNA lying in three contigs of 145, 60, and 65 kb, respectively. Within 17 kb of the 3' end of the Zfy-2 gene, lowcopy repeat elements were found in a region that extends for approximately 35 kb. Probes isolated from this region detect multiple Sxr loci, some of which map to the delta Sxrb DNA present in the T5 contig DNA. Three of these multicopy probes detect delta Sxrb loci not represented in our three contigs, which means that six distinct delta Sxrb loci have now been identified. Here we present a preliminary model of the molecular structure of the DNA in this unique region.

Animals↗

PubMed1995

Isolation and characterization of a candidate gene for progressive myoclonus epilepsy on 21q22.3.

The Unverricht-Lundborg type of progressive myoclonus epilepsy (EPM1) and autoimmune polyglandular disease type I (APECED) have been mapped to human chromosome 21q22.3 by genetic linkage analysis and/or linkage disequilibrium studies. In order to isolate the genes for these disorders, we have constructed BAC contigs in this region and a 14 week trisomy 21 fetal brain cDNA library. A direct cDNA selection technique, modified to permit the recovery 5' and 3' ends of cDNA, was applied to gene identification using the BAC contigs. We have isolated and characterized a novel gene defined by three overlapping but distinct cDNAs of 5, 3, and 3 kb in size all named EHOC-1 (Epilepsy, HOloprosencephaly Candidate-1). This gene maps less than 45 kb centromeric of D21S25, and spans at least 56 kb of genomic DNA. Northern analysis of the 5 kb cDNA revealed that 8, 7.5 and 5.3 kb transcripts are ubiquitously expressed in adult tissues. DNA sequence analysis of the 5 kb cDNA showed a complete coding sequence of 3570 bp that has multiple putative transmembrane domains and has partial homologies to transmembrane proteins including sodium channel proteins. This gene (EHOC-1) is a good candidate for APECED, and particularly for EPM1 because of the location, size, structure and homologies.

Amino Acid Sequence↗

PubMed2000

Concurrent activation of a novel putative transforming gene, myeov, and cyclin D1 in a subset of multiple myeloma cell lines with t(11;14)(q13;q32).

Through the application of the NIH/3T3 tumorigenicity assay to DNA from a gastric carcinoma, we have identified a novel transforming gene, designated myeov (myeloma overexpressed gene in a subset of t[11;14]-positive multiple myelomas). Sequence analyses did not reveal any homology with sequences present in the GenBank, except the deduced protein structure predicts a transmembrane localization. Myeov was mapped to chromosome 11q13 and localized by DNA fiber fluorescence in situ hybridization (FISH) 360-kilobase (kb) centromeric of cyclin D1. In 3 of 7 multiple myeloma (MM) cell lines with a t(11;14)(q13;q32) and cyclin-D1 overexpression, Northern blot analysis revealed overexpression of myeov as well. In all 7 cell lines, the translocation breakpoint was mapped within the 360-kb region between myeov and cyclin D1. DNA fiber FISH with a contig of probes covering the constant region of the immunoglobulin heavy chain (IgH) revealed that exclusively in the 3 myeov-overexpressing cell lines (KMS-12, KMS-21, and XG-5), either the 5' E(mu) enhancer or the most telomeric 3' Ealpha enhancer was juxtaposed to myeov. Although cyclin D1 overexpression represents a characteristic feature of all MM cell lines with t(11;14), our results demonstrate aberrant expression of a second putative oncogene in a subset of these cases, due to juxtaposition to IgH enhancers. The clinical relevance of this dual activation remains to be elucidated. (Blood. 2000;95:2691-2698)

Amino Acid Sequence↗

PubMed2001

Mapping of quantitative trait locus related to submergence tolerance in rice with aid of chromosome walking.

The major QTL for submergence tolerance was locate in the 5.9 cM interval between flanking RFLP markers. To narrow down this region, a physical map was constructed using YAC and BAC clones. A 400-kb YAC was identified in this region and later its end fragments were used to screen a rice BAC library. Through chromosome walking, 24 positive BAC clones formed two contigs around linked-RFLP markers, R1164 and RZ698. Using one YAC end, six BAC ends and three RFLP markers, a fine-scale map was constructed of the 6.8-cM interval of S10709-RZ698 on rice chromosome 9. The submergence tolerance and related trait were located in a small, well-defined region around BAC-end marker 180D1R and RFLP marker R1164. The physical-to-map distance ratio in this region is as small as 172.5 kb/cM, showing that this region is a hot spot for recombination in the rice genome.

Chromosome Walking↗

PubMed1999

Confirmation of linkage of type 1 hereditary sensory neuropathy to human chromosome 9q22.

OBJECTIVES: 1) To confirm linkage of hereditary sensory neuropathy type 1 (HSN-I) to human chromosome 9q22 in a large American family of German origin. 2) To construct a yeast artificial chromosome (YAC) contig spanning the HSN-I candidate interval. 3) To investigate the HSN-I contig for potential candidate genes. BACKGROUND: HSN-I is a rare peripheral neuropathy characterized by loss of temperature sensation, ulceration and osteomyelitis of the digits, and subtle distal weakness. A gene for HSN-I has previously been mapped to human chromosome 9q22.1-q22.3 between markers D9S318 and D9S176 in an 8-cM interval in four Australian families. METHODS: In a large German-American family with HSN-I, genome-wide linkage analysis was performed on 68 family members extending over five generations and including 17 affected members. Genotyping was performed with PCR, and the resulting genotypes were analyzed with two-point linkage analysis with Fastlink. A YAC contig was constructed based on the Whitehead Institute YAC contig WC9.3. RESULTS: Two-point linkage analysis resulted in a maximum lod score of 8.2 at theta = 0 for marker D9S1815. Haplotype analysis locates the HSN-I gene between markers D9S1797 and D9S197. Using YAC clones from the Centre d'Etude du Polymorphism Humain YAC Library, we constructed a YAC contig spanning these markers. Based on the radiation hybrid map of the human genome, we estimate that the size of this interval is less than 2,500 kb. CONCLUSIONS: Our study confirms linkage of a putative HSN-I gene to chromosome 9q22, considerably narrows the HSN-I locus, and provides a basis for identification of the HSN-I gene.

Chromosome Mapping↗

PubMed1995

Physical map of the region containing the gene for Batten disease (CLN3).

CLN3 has been mapped genetically to 16p12, to the interval between D16S288 and D16S383, a sex-averaged genetic distance of 2.1 cM. Analysis of disease haplotypes for four microsatellite markers in this interval, D16S288, D16S299, D16S298, and SPN, has shown significant allelic association between one allele at each of these loci and CLN3. All four of the associated markers were used as nucleation sites in the isolation of genomic clones (YACs). A contig was assembled which contains 3 of the 4 associated markers and which confirmed the relative order of these markers. Marker D16S272 has been located on the physical map between D16S288 and D16S299. Restriction mapping has demonstrated the location of possible CpG islands. One gene, STP, has been localised on the YAC contig proximal to D16S298 and is therefore a candidate for CLN3. Other genes, including IL4R, SGLT2, and UQCRC2, have been excluded from this region.

Alleles↗

PubMed1995

Mapping of the gracile axonal dystrophy (gad) gene to a region between D5Mit197 and D5Mit113 on proximal mouse chromosome 5.

The gracile axonal dystrophy (gad) mouse, which shows hereditary sensory ataxia and motor paresis, has been morphologically characterized by the dying back type of axonal degeneration in the nerve terminals of dorsal root ganglion cells and motor neurons. In the present study, using an intraspecific backcross between gad and C57BL/6J mice, the gracile axonal dystrophy (gad) gene was mapped to a region between D5Mit197 and D5Mit113. Estimated distances between gad and D5Mit197 and between gad and D5Mit113 are 0.4 +/- 0.3 and 5.0 +/- 1.0 cM, respectively. The gene order was defined: centromere-D5Mit81-D5Mit233-D5Mit184/D5Mit254- D5Mit256-D5Mit197-gad-D5Mit113-D5Mit7. The mouse map location of the gad locus appears to be in a region homologous to human 4p15-p16. Our present data suggest that the nearest flanking marker D5Mit197 provides a useful anchor for the isolation of the gad gene in a yeast artificial chromosome contig.

Alleles↗

PubMed1998

An expression map from human chromosome 14q24.3.

We have constructed an expression map of chromosome 14q24.3 between markers D14S42 and D14S63. cDNA selection with YACs from 14q24.3 was used to generate expressed sequence tags (ESTs). The localization of ESTs was confirmed on a YAC contig. PCR products of ESTs were used as probes to screen cDNA libraries leading to the isolation of transcripts for known and unknown genes. In total, the expression map contains 7 known genes previously mapped to 14q24.3, 6 cDNA transcripts, and 15 anonymous ESTs. The addition of 21 unique transcribed loci from an approximately 5- to 7-Mb region of chromosome 14q24.3 will facilitate future efforts to identify human disease genes from this region.

Chromosome Mapping↗

PubMed2000

Positional cloning utilizing genomic DNA microarrays: the Niemann-Pick type C gene as a model system.

A major obstacle in positional cloning is identifying the specific mutated gene from within a large physical contig. Here we describe the application of DNA microarray technology to a defined genomic region (physical map) to identify: (i) exons without a priori sequence data and (ii) the disease gene based on differential gene expression in a recessive disorder. The feasibility was tested using resources from the positional cloning of the Neimann-Pick Type C (NP-C) disease gene, NPC1. To identify NPC1 exons and optimize the technology, an array was generated from genomic fragments of the 110-kb bacterial artificial chromosome, 108N2, which encodes NPC1. First, as a test case for blindly identifying exons, fluorescently labeled NPC1 cDNA identified 108N2 fragments that contained NPC1 exons, many of which also contained intronic sequences and could be used to determine part of the NPC1 genomic structure. Second, to demonstrate that the NPC1 disease gene could be identified based upon differential gene expression, subarrays of 108N2 fragments were hybridized with fluorescently labeled cDNA probes generated from total RNA from hamster cell lines differentially expressing NPC1. A probe derived from the NP-C cell line CT60 did not detect NPC1 exons or other genomic fragments from 108N2. In contrast, several NPC1 exons were detected by a probe generated from the non-NP-C cell line 911D5A13, which was derived from CT60, and expressed NPC1 as a consequence of stable transduction with a YAC that contains NPC1 and encompasses 108N2. Thus, the array technology identified NPC1 as a candidate gene based on a physical contig and differential NPC1 expression between NP-C and non-NP-C cells. This technique should facilitate gene identification when a physical contig exists for a region of interest and mutations result in changes in the mRNA level of the disease gene or portions thereof.

Animals↗

PubMed1999

Genetic and physical mapping of the dreher locus on mouse chromosome 1.

Mutations in the mouse dreher (dr) gene cause skeletal defects, hyperactivity, abnormal gait, deafness, white belly spotting, and hypoplasia of Müllerian duct derivatives. To map dr to high resolution, we utilized two crosses. Initially, we analyzed an intersubspecific intercross to construct a detailed genetic map of simple sequence length polymorphism markers within a 6.3-cM region surrounding the dr locus. Subsequently, we analyzed a second intersubspecific intercross segregating for the dr(6J) allele, which positioned dr within a 0.13-cM region between Rxrg and D1Mit370. A physical contig of BAC clones spanning the dr critical region was constructed, and eight potential dr candidate genes were excluded by genetic or physical mapping. Together these results lay the foundation for positional cloning of the dr gene.

Abnormalities, Multiple↗

PubMed1996

A 2-megabase physical contig incorporating 43 DNA markers on the human X chromosome at p11.23-p11.22 from ZNF21 to DXS255.

A comprehensive physical contig of yeast artificial chromosomes (YACs) and cosmid clones between ZNF21 and DXS255 has been constructed, spanning 2 Mb within the region Xp11.23-p11.22. As a portion of the region was found to be particularly unstable in yeast, the integrity of the contig is dependent on additional information provided by the sequence-tagged site (STS) content of cosmid clones and DNA marker retention in conventional and radiation hybrids. The contig was formatted with 43 DNA markers, including 19 new STSs from YAC insert ends and an internal Alu-PCR product. The density of STSs across the contig ranges from one marker every 20 kb to one every 60 kb, with an average density of one marker every 50 kb. The relative order of previously known genes and expressed sequence tags in this region is predicted to be Xpter-ZNF21-DXS7465E (MG66)-DXS7927E (MG81)-WASP, DXS1011E, DXS7467E (MG21)-DXS- 7466E (MG44)-GATA1-DXS7469E (Xp664)-TFE3-SYP (DXS1007E)-Xcen. This contig extends the coverage in Xp11 and provides a framework for the future identification and mapping of new genes, as well as the resources for developing DNA sequencing templates.

Animals↗

PubMed1998

Transcript map of a 900-kb genomic region in Xp22.1-p22.2: identification of 12 novel genes.

The Xp22.1-p22.2 interval is a focus of interest as a number of hereditary disease loci have been mapped to this region, including X-linked nonsyndromic sensorineural deafness (DFN6), X-linked juvenile retinoschisis (RS), and several X-linked mental retardation syndromes. In the course of cloning the RS gene we have assembled YAC and PAC contigs of the 900-kb candidate region delimited by DXS418 and DXS999. In this study, we now report the construction of a first transcript map of this chromosomal interval by combining exon trapping, EST mapping, and computational gene identification methods. Overall, this strategy has led to the assembly of at least 12 novel transcripts positioned within the DXS418-DXS999 region, one of these encoding a putative protein kinase motif with significant homology to the rat p58/GTA protein kinase domain and another a putative neuronal protein with strong homology to a Drosophila transcriptional repressor.

Amino Acid Sequence↗