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Human Molecular Genetics Pages 501-507  

Genetic mapping of the copper toxicosis locus in Bedlington terriers to dog chromosome 10, in a region syntenic to human chromosome region 2p13-p16
Introduction
Results
   Isolation of BAC clones for the loci C04107, ATP7B, CTR1 and CTR2
   Co-localization experiments with the C04107-BAC N21-27
   Probe Wc1.gb10 identifies two loci in the canine genome
   Chromosomal assignment of BAC clones
   Comparative mapping between dog and human
   Accession numbers
Discussion
Materials And Methods
   DNA probes
   Obtaining C04107 flanking sequences by inverse PCR
   Isolation and restriction mapping of BAC clones
   FISH
   Chromosomal assignment
   PCR of Bedlington terrier genomic DNA and sequence analysis of the regions encoding the channel/transmembrane 6 (corresponding to exons 11-13 in human ATP7B)
   Comparative mapping
Acknowledgements
References

Genetic mapping of the copper toxicosis locus in Bedlington terriers to dog chromosome 10, in a region syntenic to human chromosome region 2p13-p16

Genetic mapping of the copper toxicosis locus in Bedlington terriers to dog chromosome 10, in a region syntenic to human chromosome region 2p13-p16

Bart J. A. van de Sluis, Matthew Breen3, Manoj Nanji4, Monique van Wolferen1, Pieter de Jong5, Matthew M. Binns3, Peter L. Pearson, Jeroen Kuipers2, Jan Rothuizen1, Diane W. Cox4, Cisca Wijmenga* and Bernard A. van Oost1

Department of Human Genetics, 1Department of Clinical Sciences of Companion Animals, and 2Department of Immunology, Utrecht University, PO Box 80030, 3508 TA Utrecht, The Netherlands, 3Center for Preventive Medicine, Animal Health Trust, Lanwades Park, Newmarket, Suffolk, UK, 4Department of Medical Genetics, University of Alberta, Edmonton, Alberta, Canada and 5Human Genetics Department, Roswell Park Cancer Institute, Buffalo, NY, USA

Received November 4, 1998; Revised and Accepted December 21, 1998

DDBJ/EMBL/GenBank accession nos AF087914 and AF113322

Abnormal hepatic copper accumulation is recognized as an inherited disorder in man, mouse, rat and dog. The major cause of hepatic copper accumulation in man is a dysfunctional ATP7B gene, causing Wilson disease (WD). Mutations in the ATP7B genes have also been demonstrated in mouse and rat. The ATP7B gene has been excluded in the much rarer human copper overload disease non-Indian childhood cirrhosis, indicating genetic heterogeneity. By investigating the common autosomal recessive copper toxicosis (CT) in Bedlington terriers, we have identified a new locus involved in progressive liver disease. We examined whether the WD gene ATP7B was also causative for CT by investigating the chromosomal co-localization of ATP7B and C04107, using fluorescence in situ hybridization (FISH). C04107 is an anonymous microsatellite marker closely linked to CT. However, BAC clones containing ATP7B and C04107 mapped to the canine chromosome regions CFA22q11 and CFA10q26, respectively, demonstrating that WD cannot be homologous to CT. The copper transport genes CTR1 and CTR2 were also excluded as candidate genes for CT since they both mapped to canine chromosome region CFA11q22.2-22.5. A transcribed sequence identified from the C04107-containing BAC was found to be homologous to a gene expressed from human chromosome 2p13-p16, a region devoid of any positional candidate genes.

INTRODUCTION

The toxic milk mouse (1), the Long-Evans Cinnamon (LEC) rat (2) and copper toxicosis (CT) in Bedlington terriers (3) exhibit similar, if not identical, copper storage diseases to those observed in human Wilson disease (WD). WD (OMIM 277900) is an autosomal recessive disorder of copper transport, characterized by an inability of the liver to excrete copper into bile and to incorporate copper into ceruloplasmin. This results initially in copper deposition in the liver and subsequently in the brain and other organs. The underlying defect in WD is a mutation in a copper transporting P-type ATPase (ATP7B) (4,5). It has recently been shown that mutations in the ATP7B cDNA from mouse and rat are responsible for the toxic milk mutation in mice (6) and the acute hepatitis in LEC rats (7), respectively. There are no reports of ATP7B mutations in Bedlington terriers suffering from CT and it is still unknown whether CT in Bedlington terriers is homologous to WD in humans. CT in Bedlington terriers is an autosomal recessive disorder characterized by a defect in the biliary excretion of copper resulting in accumulation of copper in the liver. This leads to chronic hepatitis and, ultimately, cirrhosis.

Circumstantial evidence exists to suggest that CT and WD may not be equivalent. Linkage studies between CT and esterase D (ESD) and retinoblastoma (RB), two genes tightly linked to WD (ATP7B) in both the human (8) and mouse (9), failed to demonstrate linkage (10). However, no linkage relationship between acute hepatitis and RB in LEC rats exists (11), demonstrating that the RB and WD genes are not syntenic in all mammalian species. The syntenic relationship of ESD, RB and WD still remains to be determined in the dog.

Although CT in Bedlington terriers resembles WD in many ways, there are also marked differences. In contrast to WD patients, Bedlington terriers with CT do not show neurological symptoms and Kayser-Fleisher rings (12,13). Moreover, normal serum ceruloplasmin levels are observed in Bedlington terriers with CT (12,13), whereas patients with WD typically show greatly reduced serum ceruloplasmin levels (14). Therefore, it cannot be concluded whether the basic defect underlying CT is different from that involved in WD.

The detailed mechanisms by which copper is exchanged and transported in mammalian cells are largely unknown. Fortuitously, the recent cloning of the genes human ATOX1 (previously known as Hah1) (15) and human CTR1 and CTR2 (16) has expanded our knowledge of copper metabolism in mammalian cells. In analogy to yeast, human CTR1 is assumed to be responsible for the high affinity cellular uptake of copper and CTR2 for the low affinity copper uptake (16). ATOX1 is a copper chaperone that directs copper to the WD protein in the trans-Golgi compartment for final incorporation into ceruloplasmin (15).

The purpose of this study was to determine if CT in Bedlington terriers is caused by one of the copper-associated genes ATP7B, CTR1 or CTR2. Recently, a polymorphic microsatellite marker (C04107) was found to be closely linked (0 cM) to the CT locus in Bedlington terriers (17). The C04107 marker as well as the copper-associated genes ATP7B, CTR1 and CTR2 were isolated in bacterial artificial chromosome (BAC) clones. Co-localization between the C04107-positive BAC and each of the BAC clones containing a copper-associated gene was performed by dual colour fluorescence in situ hybridization (FISH). The precise chromosomal localization of C04107, Atp7b, CTR1 and CTR2 was made by hybridization of the corresponding BAC clones onto high resolution metaphase spreads and the chromosome identification confirmed by subsequent co-hybridization with a chromosome-specific cosmid clone. Moreover, comparative mapping was performed between the region of the dog genome containing the CT locus and the human genome.

RESULTS

Isolation of BAC clones for the loci C04107, ATP7B, CTR1 and CTR2

The CT-linked marker C04107 contains a (CA)n repeat polymorphism. Hybridization of the C04107 PCR product onto digested dog DNA showed a smear, indicating cross-hybridization to the many different loci in the canine genome containing CA repeats (data not shown). Therefore, to isolate specific C04107 BAC clones from the canine BAC library we first generated a unique sequence probe, C04107ipcr, which lies next to the CA polymorphic sequence, by inverse PCR. Screening the BAC library with C04107ipcr yielded one positive BAC clone (N21-27). Restriction enzyme digests of N21-27 were hybridized with C04107ipcr and the sizes of the hybridizing fragments were identical to those seen in total genomic dog DNA (Fig. 1A). Also, when the C04107 marker was amplified with BAC N21-27 DNA as template a 164 bp fragment was obtained (Fig. 1D), thereby confirming the identity of the clone. For CTR1 and CTR2 three different BAC clones were identified (D14-1, E2-56 and K8-22). After Southern blot analysis it was shown that BAC clones E2-56 and K8-22 were positive for both the ctr1pcr and ctr2pcr probes, whereas BAC D14-1 was only positive for the ctr1pcr probe (Fig. 1B). The fragment sizes observed for these three CTR-BAC clones were in agreement with the fragments observed in total genomic dog DNA (Fig. 1; data not shown).


Figure 1. Southern blot analysis of genomic dog DNA and BAC DNA digested with BamHI (B), EcoRI (E) or EcoRI/BamHI (E/B). Hybridization of the locus-specific BAC clones to the probes C04107ipcr, ctr1pcr and ctr2pcr and Wc1.gb10 are shown in (A-C), respectively. (D) Ethidium bromide staining of PCR amplification of the C04107 polymorphic marker from BAC N21-27 and genomic dog DNA.

Probe Wc1.gb10, corresponding to exons 13-15 of the ATP7B gene, was used to isolate BAC clones containing the Atp7b gene. Eighteen positive BAC clones were identified and six of the BAC clones further characterized. Surprisingly, two types of Atp7b-positive BAC clones could be distinguished by hybridization analysis. After EcoRI and/or BamHI digestion Wc1.gb10 was found to map to a 21 kb EcoRI and a 12 kb BamHI or EcoRI-BamHI fragment in BAC N9-57, whereas it mapped to a 7 kb EcoRI and a 4 kb BamHI or EcoRI-BamHI fragment in the remaining five BAC clones (Fig. 1C; data not shown). Both fragments were present in total genomic dog DNA (Fig. 1C; data not shown). For all further experiments the BAC clones N9-57 and N11-276 were used.

Co-localization experiments with the C04107-BAC N21-27

To determine whether a physical relationship exists between CT in Bedlington terriers and any of the copper-associated genes ATP7B, CTR1 or CTR2, co-localization experiments between the copper-associated genes and C04107 were carried out. By detecting the C04107-positive BAC N21-27 with one colour and each of the BACs containing Atp7b, CTR1 or CTR2 with another colour, we were able to simultaneously detect two different probes by FISH. In all observations, however, the two probes mapped to different chromosomes (Fig. 2).


Figure 2. Chromosomal assignment and co-localization experiments of the C04107-BAC N21-27 and BAC clones containing one of the copper-associated genes Atp7b, CTR1 or CTR2. (A) FISH of the BAC clone containing the C04107 polymorphic marker (red) and a chromosome 10-specific cosmid clone (green) maps C04107 to CFA10q26 (inset). (B) FISH mapping of C04107-BAC (red) and the CTR1-BAC D14-1 (green) onto different chromosomes. BAC D14-1 was assigned to CFA11q22.2-22.5 using a chromosome 11-specific cosmid clone (inset). (C) FISH mapping of C04107-BAC (green) and the Atp7b-BAC N11-276 (red) onto different chromosomes. BAC N11-276 was assigned to CFA4q23 using a chromosome 4-specific cosmid clone (inset). (D) FISH mapping of C04107-BAC (red) and the Atp7b-BAC N9-57 (green) onto different chromosomes. BAC N9-57 was assigned to CFA22q11 using a chromosome 22-specific cosmid clone (inset).


Probe Wc1.gb10 identifies two loci in the canine genome

Southern blot analysis revealed two different types of BAC clones for Atp7b (Fig. 1). Dual colour FISH with two of the BAC clones exhibiting different restriction patterns (N9-57 and N11-276) showed that they map to two different canine chromosomes (Fig. 3A).


Figure 3. (A) Co-hybridization of Atp7b-BAC N11-276 (red) and Atp7b-BAC N9-57 (green) shows that they map to different chromosomes. (B) Southern blot analysis of DNA of BAC clones N9-57 and N11-276 digested with BamHI (B), EcoRI/BamHI (E/B) or EcoRI (E) followed by hybridization with the ATP7B-specific exon 12 or exon 11 probe. The BAC clones N11-276 and N9-57 were originally isolated with an exon 13-15 probe.

PCR analysis of genomic DNA from both normal and affected Bedlington terriers using primers from exons 11 and 13 resulted in two products of ~350 bp and ~1.4 kb in both cases. Sequence analysis of the two products showed the larger product to be normal Atp7b sequence, homologous to human ATP7B, and the 350 bp product to be missing exon 12. These results and other sequence changes (M. Nanji and D.W. Cox, unpublished data) indicate the presence of a normal copy of Atp7b and a pseudogene in Bedlington terriers.

Hybridization analysis with both an ATP7B exon 11 and exon 12 probe showed that only BAC clone N9-57 contains exon 12 (Fig. 3B), implying that this BAC contains the genuine Atp7b gene and that BAC clone N11-276 contains an Atp7b pseudogene.

Chromosomal assignment of BAC clones

Chromosome assignment of the BAC clones was made by hybridization to elongated metaphase preparations. These assignments were then confirmed by co-hybridization with a chromosome-specific cosmid clone. The assignments were based on the idiogram of Reimann et al. (23) and were as follows: BAC N21-27 (C04107) mapped to CFA10q26; BAC N11-267 (Atp7b pseudogene) mapped to CFA4q23; BAC N9-57 (genuine Atp7b gene) mapped to CFA22q11; BAC D14-1 (CTR1) mapped to CFA11q22.2-22.5 (Fig. 2). In some metaphases BAC clone N11-267(Atp7b pseudogene) showed a weaker signal at CFA4q31.1 (data not shown).

Comparative mapping between dog and human

Sample sequencing of some 40 clones of BAC clone N21-27 revealed one subclone showing high homology to one exon of both the human MURR1 cDNA (GenBank accession no. D85433; bit score 295, E value 3e - 78) and the mouse Murr1 cDNA (GenBank accession no. D85430; bit score 208, E value 5e - 52). Human-specific primers were identified and used to assign the MURR1 gene by radiation hybrid mapping to human chromosome region 2p13-p16, close to D2S337 (lod score 2.97).

Accession numbers

The nucleotide sequence data reported in this paper have been submitted to GenBank and assigned accession nos AF087914 (IPCRTaqI) and AF113322 (canine genomic clone homologous to one exon of the human MURR1 gene).

DISCUSSION

The CT locus in Bedlington terriers was recently linked to the microsatellite marker C04107 (17). In this study we were able to map this marker to canine chromosome CFA10q26. So far, no recombination events have been reported in the literature between C04107 and CT, suggesting that the marker is extremely close to the CT locus, which must therefore also be located on canine chromosome 10.

The ATP7B gene underlying WD was an interesting candidate gene for CT in Bedlington terriers based on its function and the phenotypic resemblance between the two disorders. This study shows, however, that BAC clones corresponding to the CT locus and Atp7b map to different chromosomes (Fig. 2). Therefore, the ATP7B gene can be excluded as a candidate gene for CT in Bedlington terriers. Interestingly, however, two different Atp7b loci were identified in the canine genome (Fig. 3): a genuine gene sequence and a pseudogene. The two Atp7b loci can be distinguished by Southern blot hybridization with an exon 12 probe or by amplification with locus-specific primers. Consequently, we can assign the Atp7b gene to canine chromosome region CFA22q11 and the Atp7b pseudogene to canine chromosome region CFA4q23.

This study unambiguously shows that CT in Bedlington terriers can be excluded as an animal model for Wilson disease and also excludes the copper transporter genes CTR1 and CTR2 as candidate genes underlying CT. The canine CTR1 and CTR2 loci were mapped to canine chromosome region CFA11q22.2-22.5. As in humans, the two genes were found to be closely linked to each other, although the human genes were not isolated in the same yeast artificial chromosome (16).

BAC clone N21-27 was found to contain a sequence highly homologous to the human MURR1 gene, which was used to perform comparative gene mapping between the canine and human genomes. Human chromosome 2, region p13-p16, showed conservation of synteny with CFA10q26. These results were in complete agreement with the first dog radiation hybrid map, which was recently made available via the World Wide Web (http://www-recomgen.univ-rennes1.fr/Dogs/results/table.html ). A first comparative map was constructed based on genes known to map to both CFA10q26 and HSA2p13-p216. Unfortunately, no obvious positional candidate genes exist on human 2p13-p16. Although ATOX1 (15) and metallothioneins (24) were first considered to be functional candidate genes for CT in Bedlington terriers, we can now exclude these genes since they all map to chromosomes other than human chromosome 2. Nevertheless, CT in Bedlington terriers becomes an attractive model for the human autosomal recessive copper diseases Indian childhood cirrhosis (ICC) and/or non-Indian childhood cirrhosis (NICC or ETIC) (25). Although NICC was first considered to be an allelic variant of WD, we recently excluded ATP7B as the gene underlying NICC in man (26). Furthermore, the NICC phenotype in man and the CT phenotype in dog are more similar to each other than to WD: both NICC and CT show normal serum ceruloplasmin levels and a lack of neurological symptoms or Kayser-Fleisher rings. In the search for the ICC and NICC genes, the 2p13-p16 region will be an interesting target for genetic linkage studies.

In the absence of a suitable candidate gene, the C04107-positive BAC will be a good starting point to build a contig of overlapping BAC clones covering the CT candidate region, followed by the isolation of candidate genes. The progress being made in constructing a canine linkage map (27), a panel of canine-rodent hybrid cell lines (28) and a radiation hybrid map will be instrumental in the construction of genetic and physical maps of the CT region. The Human Genome Project is rapidly generating expressed sequence tags (ESTs) and genes. We will utilize radiation hybrid mapping as a tool to perform comparative mapping for those ESTs or genes already known to map to human 2p13-16 in order to rapidly generate a transcript map of dog chromosome 10, region q26. As the specific function of the majority of these genes/ESTs is as yet unknown they are all possible candidates which may facilitate the identification of the CT gene.

MATERIALS AND METHODS

DNA probes

The Wc1.gb10 probe was isolated by digestion of the clone Wc1.gb10 with EcoRI to produce a 0.7 kb ATP7B cDNA fragment (4).

The probes ctr1pcr and ctr2pcr, corresponding to human CTR1 and CTR2, respectively, were obtained by amplification from human placenta RNA. Primers were selected from the coding regions (CTR1f, 5[prime]-GGACTCCAACAGTACCATGC-3[prime]; CTR1r, 5[prime]-GCAGAGGTACCCGTTGTAGG-3[prime]; CTR2f, 5[prime]-GAGTGTCCACAGTCCTGC-3[prime]; CTR2r, 5[prime]-GCTGAGAAGTGGGTAAGCTAG-3[prime]). The predicted sizes of the products were 448 and 376 bp, respectively. The ctr1pcr and ctr2pcr products were subcloned into the TA cloning kit (Invitrogen, Carlsbad, CA) and DNA sequence analysis by dRhodamine cycle sequencing (Perkin Elmer, Foster City, CA) was performed to confirm the identity of the probes.

Obtaining C04107 flanking sequences by inverse PCR

Flanking sequences of C04107 were obtained using the inverse polymerase chain reaction (IPCR) approach. First, C04107 polymorphic fragments (17) were subcloned into the TA cloning kit (Invitrogen) and DNA sequence analysis was performed to select primers for IPCR. For IPCR, canine genomic DNA (3 µg) was digested with each of the restriction endonucleases BamHI, CfoI and TaqI under the conditions specified by the manufacturer (Boehringer Mannheim, Mannheim, Germany).One microgram of the phenol-purified digested DNA was ligated (15 min at 37°C and overnight at room temperature) with 1 µl T4 DNA ligase (6. U/µl; Pharmacia, Uppsala, Sweden) and 10 µl 10× ligation buffer (Pharmacia) in a 100 µl reaction volume. IPCR was carried out with primers A (5[prime]-GTATGTACGTGAGTGTGATGTGG-3[prime]) and B (5[prime]-CCACCCTTCCAATTTATTTCC-3[prime]). Subsequently, a nested PCR reaction using 1 µl of the IPCR product A-B as template was carried out with primers C (5[prime]-CCTATCCTTTAGATGGGACAG-3[prime]) and D (5[prime]-CCTCTTAAATGTATAGTTGCTG-3[prime]). DNA was initially denatured at 95°C for 10 min and then subjected to 35 cycles of 94°C for 1 min, 60°C for 1 min and 72°C for 2 min, followed by a final extension at 72°C for 10 min. The IPCR products (C and D) were analysed on a 2% agarose gel and the fragments were isolated from the gel with the Qiagen Gel extraction kit (Qiagen, Valencia, CA). The three different IPCR fragments (BamHI, 250 bp; CfoI, 460 bp; TaqI, 1500 bp) were subcloned into the TA cloning kit (Invitrogen) and DNA sequence analysis by dRhodamine cycle sequencing was performed using M13 forward (-21M13F) and reverse (M13-R-2) primers.

The 193 bp single copy probe C04107ipcr was amplified from the 1500 bp TaqI IPCR product and isolated from a 2% agarose gel with the Qiagen Gel extraction kit. To amplify C04107ipcr the following primers were used: sense primer, primer A; antisense primer 5[prime]-CCTAGAAGAATACAAGCCTGAGAC-3[prime].

Isolation and restriction mapping of BAC clones

BAC clones were isolated from a total canine genomic BAC library (18) by colony hybridization with the [[alpha]-32P]dATP-labelled probes Wc1.gb10, ctr1pcr, ctr2pcr and C04107ipcr. The hybridization procedure was performed with a pool of different probes as described on the BacPac website (http://bacpac.med.buffalo.edu ). Positive colonies were picked, grown overnight in 3 ml Luria-Bertani (LB) medium with chloramphenicol (20 µg/ml) and 1 µl of the overnight bacterial culture was spotted onto a Hybond N+ filter (Amersham, Piscataway, NJ). These filters were hybridized with either [[alpha]-32P]dATP-labelled Wc1.gb10, ctr1pcr, ctr2pcr or C04107ipcr probe to ensure that the colonies contained the appropriate inserts. Six Atp7b-positive BACs (A24-234, B4-247, D16-204, D19-45, N9-57, N11-276), one C04107ipcr-positive BAC (N21-27), one CTR1-positive BAC (D14-1) and two BAC clones positive for both CTR1 and CTR2 (E2-56, K8-22) were selected for further study.

BAC DNA was isolated by the alkaline lysis method as described on the BacPac website. To confirm the identity of the BAC clones BAC DNA and genomic dog DNA were digested with EcoRI, BamHI and EcoRI/BamHI, separated on a 0.7% agarose gel, transferred to Hybond N+ (Amersham) and hybridized at 65°C with the Wc1.gb10, ctr1pcr, ctr2pcr or C04107ipcr probe (19).

Five different BAC clones were selected for FISH analysis corresponding to different loci: for Atp7b, N9-57 and N11-276; for C04107, N21-27; for CTR1 and CTR2, D14-1 and K8-22, respectively.

FISH

Routine metaphase chromosome spreads from normal canine chromosomes were prepared according to the method described by Fischer et al. (20). Total DNA of BAC clones was labelled by nick translation (BioNick labelling system; Boehringer) with either digoxigenin-11-dUTP or biotin-11-dUTP (Boehringer). Probe DNA was competed with a 100× excess of sheared genomic canine DNA. The probes were denatured at 80°C for 5 min, placed on ice for 3 min and incubated at 37°C for 30 min before hybridization. Fluorescence in situ hybridization was performed as described by Dutra et al. (21). To visualize the biotinylated probe in red (Cy3) and the digoxigeninated probe in green [fluorescein isothiocyanate (FITC)] the slides were incubated with avidin-Cy3 (1:50) or/and mouse anti-digoxigenin (1:100). Amplification of the signal was achieved by a layer of biotinylated goat anti-avidin (1:100) (Vector Laboratories, Burlingame, CA) or/and rabbit anti-mouse-FITC (1:250; Sigma, St Louis. MO), followed by a layer of avidin-Cy3 (1:50) or/and goat anti-rabbit-FITC (1:100), respectively. Chromosomes were stained with 4[prime],6-diamido-2-phenylindole·2HCl (DAPI) and actinomycin D (1:10 000) in 20 µl Vectashield (Vector Laboratories).

Images were obtained with a Leitz fluorescence microscope coupled to a CCD camera and analysed with the computer program ISIS2 (v.2.41; Metasystems, Altlussheim, Germany).

Chromosomal assignment

To assign each of the five BAC clones to their chromosome of origin, elongated metaphase chromosome preparations were produced by methotrexate synchronization/thymidine release of mitogenically stimulated canine lymphocytes. Conventional harvesting procedures of colcemid arrest and hypotonic treatments, followed by fixation in methanol:glacial acetic acid (3:1) were used. Slides were dehydrated through an ethanol series (70, 90 and 100%) and aged for 1 week. Chromosome preparations were denatured in 70% formamide, 2× SSC for 2 min at 65°C, passed through an ethanol series and air dried before use. BAC DNAs were labelled with either biotin-16-dUTP or digoxigenin-11-dUTP using nick translation and 2.5 ng/ml pre-annealed with 1 mg/ml sonicated genomic dog DNA at 37°C for 30 min in a final volume of 10 µl. Hybridization was performed for 16-18 h at 37°C in a humidified chamber. Post-hybridization stringency washes were performed as described by Breen et al. (22). The chromosomes were counterstained in DAPI (80 ng/ml) prior to being mounted with Vectashield (Vector Laboratories) and sealed with a coverslip. Images were captured using a fluorescence microscope (Axiophot; Zeiss, Jena, Germany) equipped with an FITC/Texas Red/DAPI filter set and a cooled CCD camera (KAF1400; Photometrics, Tucson, AZ), both driven by SmartCapture software (Vysis, Downers Grove, USA). Identification of the corresponding chromosome was made using image enhanced DAPI banding and the precise band location identified by reference to the idiogram in Reimann et al. (23). For confirmation of the chromosomal assignments, each BAC clone was then re-hybridized under the same conditions as above but in the presence of 5 ng/µl of a chromosome-specific cosmid clone (M. Breen and N.G. Holmes, in preparation) labelled with an alternative hapten allowing dual colour detection.

PCR of Bedlington terrier genomic DNA and sequence analysis of the regions encoding the channel/transmembrane 6 (corresponding to exons 11-13 in human ATP7B)

Total genomic DNA from normal and affected Bedlington terriers was analysed by PCR, using 20 µl volumes containing 50 mM KCl, 10 mM Tris, pH 8.0, 50 ng of each primer (sense, 5[prime]-CCACGTGGGCAACGATACCA-3[prime]; antisense, 5[prime]-GCCATCTCCAGAGGCTTGC-3[prime]), 10 mg/ml BSA, 1.5 mM MgCl2, 200 mM each dCTP, dGTP, dTTP and dATP and 5 U AmpliTaq (Perkin Elmer). Amplification was performed in a thermal controller (PTC-100-96V; MJ Research, Watertown, USA) for 35 cycles of 30 s denaturation at 94°C, 30 s annealing at 55°C and 90 s extension at 72°C. PCR products were purified with a Qiaquick spin column (Qiagen), cycle sequenced (Thermosequanase; Amersham) using each of the PCR primers and electrophoresed through 8% denaturing polyacrylamide gels.

Comparative mapping

BAC clone N21-27 was randomly subcloned by BamHI/HindIII digestion into Bluescript SK (Stratagene, La Jolla, CA). DNA sequence analysis was performed by dRhodamine cycle sequencing using M13 forward (-21M13F) and reverse (M13-R-2) primers. Transcribed sequences were identified by a Basic BLAST search (http:///www3.ncbi.nlm.nih.gov ) and GRAIL2 analysis (http://avalon.epm.ornl.gov/Grail-bin/ ). Subclone 11R was found to contain one exon homologous to the MURR1 gene. To screen the Genebridge 4 radiation hybrid panel (Research Genetics, Huntsville, USA) two MURR1 human-specific primers were used to amplify a 169 bp fragment (sense primer, 5[prime]-GACATGGATTTCAACCAGCTG-3[prime]; antisense primer, 5[prime]-GGCCCCGAAGCCCGCTATTC-3[prime]). The radiation hybrid panel was analysed according to the manufacturer’s specifications.

ACKNOWLEDGEMENTS

This research was supported by grants from the International Copper Association (to C.W.), the Natural Sciences and Research Council of Canada (to D.W.C.) and the Guide Dogs for the Blind Association (to M.B. and M.M.B.).

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*To whom correspondence should be addressed at Center for Medical Genetics, WKZ, PO Box 85090, 3508 AB Utrecht, The Netherlands. Tel: +31 30 250 3800; Fax: +31 30 250 3801; Email: t.n.wijmenga@med.uu.nl

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