A 1-Mb BAC/PAC-based physical map of the autosomal recessive polycystic kidney disease gene (PKHD1) region on chromosome 6

The PKHD1 (polycystic kidney and hepatic disease 1) gene responsible for autosomal recessive polycystic kidney disease has been mapped to 6p21.1-p12 to an approximately 1-cM interval flanked by the markers D6S1714/D6S243 and D6S1024. We have developed a sequence-ready BAC/PAC-based contig map of this region as the next step for the positional cloning of PKHD1. This contig comprising 52 clones spanning approximately 1 Mb was established by content mapping of 44 BAC/PAC-end-derived STSs, 3 known genetic markers, 5 YAC-end-derived STSs, 3 random STSs, 1 previously mapped gene, and 1 EST. The average depth per marker is 6.3 clones, and the average STS density is 20 kb. The genomic clone overlaps were confirmed by restriction fragment fingerprint analysis. A high-resolution BAC/PAC-based contig map is essential to the ultimate goal of identifying the PKHD1 gene.     

A clone contig of 12q24.3 encompassing the distal hereditary motor neuropathy type II gene

We previously assigned the disease locus for autosomal dominant hereditary motor neuropathy type II (distal HMN II) within a 13-cM interval at chromosome 12q24.3. We constructed a physical map of the distal HMN II region based on yeast artificial chromosomes (YACs), P1 artificial chromosomes (PACs), and bacterial artificial chromosomes (BACs) using an STS content mapping approach. The contig contains 26 YAC, 15 PAC, and 60 BAC clones and covers a physical distance of approximately 5 Mb. A total of 99 STS markers, including 25 known STSs and STRs, 49 new STSs generated from clone end-fragments, 20 ESTs, and 5 known genes, were located on the contig. This physical map provides a valuable resource for mapping genes and markers located within the distal HMN II region and facilitates the positional cloning of the distal HMN II gene.     

A physical map of the 20q12-q13.1 region associated with type 2 diabetes

Several recent genetic studies have suggested linkage of Type 2 diabetes (non-insulin-dependent diabetes mellitus) susceptibility to a region of chromosome 20q12-q13.1. To facilitate the identification and cloning of a diabetes susceptibility gene(s) in this region, we have constructed correlated radiation hybrid and YAC/BAC contig physical maps of the region. A high-resolution radiation hybrid map encompassing 9.5 Mb between the PLC and the CEBPB genes was constructed using 68 markers: 25 polymorphic markers, 15 known genes, 21 ESTs, and 7 random genomic sequences. The physical order of the polymorphic markers within this radiation hybrid map is consistent with published genetic maps. A YAC/BAC contig that gives continuous coverage between PLC and CEBPB was also constructed. This contig was constructed from 24 YACs, 34 BACs, and 1 P1 phage clone onto which 71 markers were mapped: 23 polymorphic markers, 12 genes, 24 ESTs, and 12 random genomic sequences. The radiation hybrid map and YAC/BAC physical map enable precise mapping of newly identified transcribed sequences and polymorphic markers that will aid in linkage and linkage disequilibrium studies and facilitate identification and cloning of candidate Type 2 diabetes susceptibility genes residing in 20q12-q13.1.     

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.     

A sequence-ready BAC/PAC contig and partial transcript map of approximately 1.5 Mb in human chromosome 17q25 comprising multiple disease genes

Hereditary neuralgic amyotrophy (HNA) is an autosomal dominant recurrent neuropathy mapped to a 4-cM interval on chromosome 17q25 between the short tandem repeat (STR) markers D17S1603 and D17S802. Chromosome 17q25 in general and the 4-cM HNA region in particular are also implicated in the pathogenesis of a number of tumors (tylosis with esophageal cancer, sporadic breast and ovarian tumors) and harbor a psoriasis susceptibility locus. Initial attempts to construct a yeast artificial chromosome contig failed. Therefore, we have now constructed a complete P1 artificial chromosome (PAC) and bacterial artificial chromosome (BAC) contig of the region flanked by the STR markers D17S1603 and D17S802. The contig contains 22 PAC and 64 BAC clones and covers a physical distance of approximately 1. 5 Mb. A total of 83 sequence-tagged site (STS) markers (10 known STSs and STRs, 56 STSs generated from clone end-fragments, 12 expressed sequence tags, and 5 known genes) were mapped on the contig, resulting in an extremely dense physical map with approximately 1 STS per 20 kb. This sequence-ready PAC and BAC contig will be pivotal for the positional cloning of the HNA gene as well as other disease genes mapping to this region.     

Localization on a physical map of the NKC-linked Cmv1 locus between Ly49b and the Prp gene cluster on mouse chromosome 6.

The Cmv1 locus controls NK cell-mediated resistance to infection with murine CMV. Our recent genetic analysis of backcross mice demonstrated that the NK gene complex (NKC)-linked Cmv1 locus should reside between the Ly49 and Prp gene clusters on distal mouse chromosome 6. We have aligned yeast artificial chromosome (YAC) inserts in a contig spanning the interval between the Ly49 and Prp gene clusters. This YAC contig includes 13 overlapping YAC inserts that span more than 2 megabases (Mb) in C57BL/6 (B6) mice. Since we have identified genomic clones that span the Ly49-Prp gene region, we hypothesize that at least one should contain the Cmv1 locus. To narrow the Cmv1 critical region, we developed novel NKC genetic markers and used these to genotype informative backcross and intra-NKC recombinant congenic mouse DNA samples. These data suggest that Cmv1 resides on a single YAC insert within an interval that corresponds to a physical distance of approximately 390 kb. This high resolution, integrated physical and genetic NKC map will facilitate identification of Cmv1 and other NKC-linked loci that regulate NK cell-mediated immunity.     

An integrated map of human 6q22.3-q24 including a 3-Mb high-resolution BAC/PAC contig encompassing a QTL for fetal hemoglobin

Genetic studies have previously assigned a quantitative trait locus (QTL) for hemoglobin F and F cells to a region of approximately 4 Mb between the markers D6S408 and D6S292 on chromosome 6q23. An initial yeast artificial chromosome contig of 13 clones spanning this region was generated. Further linkage analysis of an extended kindred refined the candidate interval to 1-2 cM, and key recombination events now place the QTL within a region of <800 kb. We describe a high-resolution bacterial clone contig spanning 3 Mb covering this critical region. The map consists of 223 bacterial artificial chromosome (BAC) and 100 P1 artificial chromosome (PAC) clones ordered by sequence-tagged site (STS) content and restriction fragment fingerprinting with a minimum tiling path of 22 BACs and 1 PAC. A total of 194 STSs map to this interval of 3 Mb, giving an average marker resolution of approximately one per 15 kb. About half of the markers were novel and were isolated in the present study, including three CA repeats and 13 single nucleotide polymorphisms. Altogether 24 expressed sequence tags, 6 of which are unique genes, have been mapped to the contig.     

TheCmv1Host Resistance Locus Is Closely Linked to theLy49Multigene Family within the Natural Killer Cell Gene Complex on Mouse Chromosome 6

Natural killer (NK) cells play important roles in controlling tumor cells and against a range of infectious organisms. Recent studies of mouse NK cell surface receptors, which may be involved in the specificity of NK cells, have shown that many of these molecules are encoded by theLy49andLy55(Nkrp1) multigene families that map to distal mouse chromosome 6. Also mapping to this NK cell gene complex (NKC) is the resistance locus,Cmv1,which is involved in genetically determined resistance to murine cytomegalovirus (MCMV). The aim of this study was to localizeCmv1more precisely in relation to other NKC loci by generating a high-resolution genetic map of the region. We have analyzed 1250 backcross mice comprising panels of 700 (BALB/c × C57BL/6J)F₁× BALB/c and 550 (A/J × C57BL/6J)F₁× A/J progeny. A total of 25 polymorphic genes or microsatellite markers were analyzed over a region of 10 map units fromD6Mit134toD6Mit59.TheCmv1phenotypes of mice recombinant in this interval were tested by infection with MCMV. The results obtained indicate that the functionally important NKC region is a tightly linked cluster of loci spanning at least 0.4 map units. Furthermore,Cmv1maps distal to, but very closely linked to, theLy49multigene family (<0.2 map units), suggesting that MCMV resistance may be conferred by MHC class I-specific NK cell receptors.     

Genetic and physical mapping of the cerebellar deficient folia (cdf) locus on mouse chromosome 6

Cerebellar deficient folia (cdf) is a recessive mouse mutation causing ataxia and cerebellar cytoarchitectural abnormalities, including hypoplasia, foliation defects, and Purkinje cell ectopia. To identify the cdf gene, we have generated a high-resolution genetic map of a 3.24 +/- 0.55 cM (95% CI) region encompassing the cdf gene using 1997 F2 mice generated from a (C3H/HeSnJ-cdf/cdf x CAST/Ei)F1 intercross. Linkage analysis showed that the cdf gene cosegregates with D6Mit208, D6Mit359, and D6Mit225. A contig of five YACs, nine BACs, and three P1s was constructed across the cdf nonrecombinant region. Based on genetic and physical maps, the cdf gene was localized to the 0.28 +/- 0.23 cM (95% CI) interval between D6Mit209 and D6Ack1. These results will greatly facilitate the map-based cloning of the cdf gene, which in turn should further knowledge of the molecular mechanisms of neuronal positioning and foliation during cerebellar development.     

Rapid mapping of markers applying vectorette technology to YAC fragmentation allows easy assembly of a high-density STS bacterial clone contig spanning the markers D6S1260-D6S1918

We have generated a detailed physical map of the 6p21.3/p22.1 boundary, using a combination of yeast artificial chromosome (YAC) fragmentation and high-resolution sequence tagged site (STS) content mapping. YACs from the CEPH, St. Louis, and ICRF libraries have been used to construct a 4.5-Mb contig spanning the markers D6S306 to D6S1571. YAC insert sizes were determined by pulsed field gel electrophoresis (PFGE). Chimerism of YACs was determined by fluorescent in situ hybridization (FISH), and their integrity was determined by fingerprinting with Alu-PCR. We have identified 10 new CA repeat loci in this region as well as over 50 novel STSs, several tRNA genes, a new histone H2B gene and the phospholipase D gene. Using these new markers, we have rapidly generated a bacterial clone contig of over 250 kb, spanning the markers D6S1260 to D6S1918 (WI-3111) with STSs spaced on average every 6 kb. Received: 18 September 1997 / Accepted: 13 November 1997     

Physical and cDNA mapping in the DBH region of human chromosome 9q34

Chromosome 9q34 has been extensively studied and mapped due to the presence of known disease genes, principally tuberous sclerosis 1 (TSC1), in this region. During the course of our mapping of this region we constructed a 555-kb contig beginning approximately 50 kb proximal to the dopamine-beta-hydroxylase (DBH) gene and extending, with one small deletion, distal to the D9S114 marker. The contig consists of 11 P1 clones, four PAC clones, one BAC clone and six cosmid clones and contains 27 new nonpolymorphic STSs. We have found the region to be unstable in P1, PAC and BAC cloning vehicles and have identified several deleted genomic clones. In addition, we have isolated and mapped the 3' portions of three putative genes located within or immediately distal to the DBH gene, including one large gene that runs on the opposite strand to DBH and utilizes portions of two DBH exons. The genomic clones of the contig, cDNAs and new STSs will be useful reagents for the further study and mapping of this region.     

A BAC-based STS-content map spanning a 35-Mb region of human chromosome 1p35-p36

We have devised a mapping method for rapid assembly and ordering of bacterial artificial chromosome (BAC) clones on a radiation hybrid (RH) panel, using sequence-tagged sites (STSs) and PCR. The protocol consists of two rounds of two-dimensional screening from a limited number of BACs to correspond each to an STS. In the first round, STSs are assembled in the RH bins and ordered according to PCR signals derived from 384-well microtiter plates (MTPs) in which BAC clones have been arrayed. In the second round, individual BAC clones are isolated from the MTPs to build a contig. We applied this method to a 35-Mb region spanning human chromosome 1p35-p36 and assembled 1366 BACs in 11 contigs, the longest being about 20 Mb. The working draft sequences of the human genome have been integrated into the contigs to validate the accuracy.     

Physical and transcriptional map of a 3-Mb region of mouse chromosome 1 containing the gene for the neural tube defect mutant loop-tail (Lp)

The Lp mouse mutant provides a model for the severe human neural tube defect (NTD), cranio-rachischisis. To identify the Lp gene, a positional cloning approach has been adopted. Previously, linkage analysis in a large intraspecific backcross was used to map the Lp locus to distal mouse chromosome 1. Here we report a detailed physical map of this region. The interval surrounding Lp has been cloned in a yeast artificial chromosome (YAC) contig consisting of 63 clones spanning approximately 3.2 Mb. Fifty sequence tagged sites (STSs) have been used to construct the contig and establish marker order across the interval. Based on the high level of conserved synteny between distal mouse chromosome 1 and human 1q21-q24, many of these STSs were designed from expressed sequences identified by cross-screening human and mouse databases of expressed sequence tags. Added to other known genes in the region, a total of 29 genes were located and ordered within the contig. Seven novel polymorphisms were identified within the region, allowing refinement of the genetic map and a reduction in the size of the physical interval containing the Lp gene. The Lp interval, between D1Mit113 and Tagln2, can be spanned by two nonchimeric overlapping YACs that define a physical distance of approximately 1 Mb. Within this region, 10 potential candidate genes have been mapped. The materials and genes described here will provide a resource for the identification and further study of the mutated Lp gene that causes this severe neural tube defect and will provide candidates for other defects known to map to the homologous region on human chromosome 1q.     

A High-Resolution PAC and BAC Map of the SCA2 Region

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

A 1.5-Mb physical map of the hidrotic ectodermal dysplasia (Clouston syndrome) gene region on human chromosome 13q11

The HED (hidrotic ectodermal dysplasia) or Clouston syndrome gene (named ED2) has been mapped to the pericentromeric region of chromosome 13 (13q11) to a 2.4-cM interval flanked by markers D13S1828 and D13S1830. We have developed a BAC/PAC-based contig map of this region. This contig, comprising 23 clones and spanning 1.5 Mb, was established by mapping of 27 BAC/PAC end-derived STSs, 11 known polymorphic markers, 2 previously mapped genes, and 14 ESTs. The genomic clone overlaps were confirmed by restriction fragment fingerprint analysis. This contig provides the basis for genomic sequencing and gene identification in the ED2 critical region. Of the 14 ESTs mapped to the contig, 6 show homology to human genes and 8 appear to be novel. Expression patterns of the genes/ESTs were tested by Northern blot and RT-PCR. Full characterization of some of these genes, as well as the novel ESTs, will be useful in assessing their involvement in the HED/Clouston syndrome.     

Molecular genetic characterization of the distal NKC recombination hotspot and putative murine CMV resistance control locus

The NK gene complex (NKC) controls murine cytomegalovirus (MCMV) immunity through Cmv1-dependent natural killer (NK) cell responses. Ly49H expression correlates with Cmv1 phenotypes in different inbred strains, is required for MCMV resistance in C57BL/6 (B6) mice, and its interaction with the MCMV encoded m157 protein leads to NK cell-mediated destruction of virus-infected cells. However, genetic mapping studies have previously indicated that Cmv1 should reside in the D6Wum9–16 NKC interval, distal to Ly49h. Since these data suggested that multiple NKC-linked loci could regulate viral immunity, a putative MCMV resistance control (Mrc) locus was pinpointed to within the D6Wum9–16 interval on a NKC-aligned bacterial artificial chromosome (BAC). Sequence analysis of BAC 151 revealed several novel G-protein coupled receptor genes, an HMG-1 remnant and many additional polymorphic microsatellites that were useful in determining the minimal genetic interval for the Mrc locus. Moreover, comparison of B6, BALB/c, A/J and recombinant Mrc alleles restricted the genetic interval to approximately 470 bp and showed that it was also a hotspot for recombination. MCMV challenge of novel NKC recombinant mice demonstrated that Mrc^B6 was not required for MCMV resistance nor could it directly complement the Ly49^BALB haplotype to rescue MCMV susceptibility. Taken together, these data show that while Mrc apparently guides recombination, Ly49H expression is sufficient for MCMV resistance in B6 mice. A direct role for Mrc^B6 in virus resistance is excluded in the novel mice.The nucleotide sequence data reported in this paper were assigned GenBank accession numbers AF462604, AY145449 and AY145450.     

Construction of a 1.5-Mb Bacterial Artificial Chromosome Contig in Xp11.23, a Region of High Gene Content

To generate sequence-ready templates for the gene-rich Xp11.23 region, we have constructed a 1.5-Mb bacterial artificial chromosome (BAC) contig spanning the interval between the DNA markers OATL1 and DXS255. The contig includes 28 BACs, ranging in size from 58 to 285 kb with an average size of 135 kb, which provide 2.5-fold coverage of the region. The BAC contig was constructed based entirely on the content of 40 DNA markers from a previously established YAC contig and 11 new markers developed from BAC-end DNA sequences, 4 of which were required to close gaps in the map. There was no evidence of rearrangement, instability, or chimerism in any of the BAC clones. The BAC cloning system appears to provide robust and total physical coverage of this gene-rich region with clones that are suitable for DNA sequencing.