Narrowing the position of the Treacher Collins syndrome locus to a small interval between three new microsatellite markers at 5q32-33.1.

Treacher Collins syndrome (TCOF1) is an autosomal dominant disorder of craniofacial development, the features of which include conductive hearing loss and cleft palate. The TCOF1 locus has been localized to chromosome 5q32-33.2. In the present study we have used the combined techniques of genetic linkage analysis and fluorescence in situ hybridization (FISH) to more accurately define the TCOF1 critical region. Cosmids IG90 and SPARC, which map to distal 5q, encompass two and one hypervariable microsatellite markers, respectively. The heterozygosity values of these three markers range from .72 to .81. Twenty-two unrelated TCOF1 families have been analyzed for linkage to these markers. There is strong evidence demonstrating linkage to all three markers, the strongest support for positive linkage being provided by haplotyping those markers at the locus encompassed by the cosmid IG90 (Zmax = 19.65; theta = .010). FISH to metaphase chromosomes and interphase nuclei established that IG90 lies centromeric to SPARC. This information combined with the data generated by genetic linkage analysis demonstrated that the TCOF1 locus is closely flanked proximally by IG90 and distally by SPARC.     

Refinement of a Translocation Breakpoint Associated with Blepharophimosis–Ptosis–Epicanthus Inversus Syndrome to a 280-kb Interval at Chromosome 3q23

Blepharophimosis syndrome (BPES) is an autosomal dominant disorder of craniofacial development, the features of which include blepharophimosis, ptosis, and epicanthus inversus. Although it has been suggested that BPES is genetically heterogeneous, a major locus for this condition resides at chromosome 3q23. We have previously mapped a translocation breakpoint associated with BPES to the D3S1316–D3S1615 interval. The markers in this region have subsequently been shown to lie in a different order, with the BPES locus mapping to the 1-cM D3S1576 and D3S1316 interval. In the current investigation, a physical map, consisting of 60 yeast artificial chromosome (YAC) clones and 1 bacterial artificial chromosome, that spans this region has been constructed. Ten expressed sequence tags and the cellular retinol-binding protein I locus have been mapped to the contig. YAC end isolation has led to the creation of novel STSs that have been used to reduce the size of the BPES critical region to a 280-kb interval, which has been cloned in two nonchimeric YACs.     

Detailed Comparative Mapping of Cereal Chromosome Regions Corresponding to the Ph1 Locus in Wheat

Detailed physical mapping of markers from rice chromosome 9, and from syntenous (at the genetic level) regions of other cereal genomes, has resulted in rice yeast artificial chromosome (YAC) contigs spanning parts of rice 9. This physical mapping, together with comparative genetic mapping, has demonstrated that synteny has been largely maintained between the genomes of several cereals at the level of contiged YACs. Markers located in one region of rice chromosome 9 encompassed by the YAC contigs have exhibited restriction fragment length polymorphism (RFLP) using deletion lines for the Ph1 locus. This has allowed demarcation of the region of rice chromosome 9 syntenous with the ph1b and ph1c deletions in wheat chromosome 5B. A group of probes located in wheat homoeologous group 5 and barley chromosome 5H, however, have synteny with rice chromosomes other than 9. This suggests that the usefulness of comparative trait analysis and of the rice genome as a tool to facilitate gene isolation will differ from one region to the next, and implies that the rice genome is more ancestral in structure than those of the Triticeae.     

Characterization of a YAC and cosmid contig containing markers tightly linked to the myotonic dystrophy locus on chromosome 19.

Myotonic dystrophy (DM) is caused by a defect in an unknown gene that maps to 19q13.3, flanked by the tightly linked markers ERCC1 on the proximal side and D19S51 on the distal side. We report the isolation and characterization of overlapping YAC and cosmid clones around D19S51 for the construction of a physical map around this locus. The resulting contig contains the markers D19S51 and D19S62 (another new marker tightly linked to the DM locus) and the distal breakpoint of a radiation hybrid cell line used in the physical mapping of the DM region. We have compared the restriction maps of the YACs and cosmids with that of the genome to investigate the fidelity of these clones.     

A yeast artificial chromosome contig encompassing the type 1 neurofibromatosis gene.

The yeast artificial chromosome (YAC) system (Burke et al., 1987, Science 236: 806-812) allows the direct cloning of large regions of the genome. A YAC contig map of approximately 700 kb encompassing the region surrounding the type 1 neurofibromatosis (NF1) locus on 17q11.2 has been constructed. A single YAC containing the entire NF1 locus has been constructed by homologous recombination in yeast. In the process of contig construction a novel method of YAC end rescue has been developed by YAC circularization in yeast and plasmid rescue in bacteria. YACs containing homology to the NF1 region but mapping to another chromosome have also been discovered. Sequences of portions of the homologous locus indicate that this other locus is a nonprocessed pseudogene.     

Construction of a long-range YAC physical map spanning the 10-cM region between the markers D18Mit109 and D18Mit68 on mouse proximal chromosome 18.

Four yeast artificial chromosome (YAC) contigs, physically approximately 8 Mb, have been constructed spanning a 10-cM region on mouse proximal chromosome 18 and include the sites of 21 known genes, including those near the twirler (Tw) locus and the recently isolated Niemann-Pick type C1 (npc1) gene, formerly designated as the spm locus. This physical map consists of 49 YAC clones that cover roughly 15% of the chromosome. The physical order of 38 microsatellite sequence-tagged sites (STSs) could be assembled and confirmed based on their presence or absence in individual YACs, from proximal D18Mit109 through distal D18Mit68. These YACs provide an important resource for the further characterization and identification of known and unknown genes. The physical map has been integrated with our previously published genetic linkage map and showed an average genetic to physical distance of cM/Mb > 1.1.     

Analysis of the human regulators of complement activation (RCA) gene cluster with yeast artificial chromosomes (YACs).

The human regulators of complement activation gene cluster (RCA cluster) have been partially characterized with yeast artificial chromosomes (YACs). While the data confirm many points previously elucidated, the finer resolution of YAC mapping has allowed the discovery and/or localization of partial gene duplications, the determination of gene orientations, and the measurement of gaps between known genes. Here nine overlapping YACs that encompass a genomic region of 800 kb, encoding four RCA genes and three gene-like elements, are described. The encoded genes and two of the gene-like elements share the same orientation and are ordered (5' to 3') DAF, CR2, CR1, MCP-like, CR1-like, and MCP. A C4bp-like region lies upstream from DAF and is likely to correspond to one recently observed by F. Pardo-Manuel, J. Rey-Campos, A. Hillarp, B. Dahlback, and S. Rodriguez de Cordoba (1990, Proc. Natl. Acad. Sci. USA 87: 4529-4533). MCP-like, a new genetic element, was discovered and found to be homologous to the 5' portion of the MCP gene. Two large gaps of 85 kb (between CR2 and DAF) and 110 kb (between DAF and the C4bp-like element) could carry additional RCA genes. The arrangement of CR1, MCP-like, CR1-like, and MCP, in that order, strongly suggests that this region was generated by a single duplication of neighboring CR1/CR1-like and MCP/MCP-like forerunners. The RCA YACs will now serve as convenient DNA sources for the subcloning and further characterization of this region.     

Genomic Organization of the Human Heparan Sulfate-N-Deacetylase/N-Sulfotransferase Gene: Exclusion from a Causative Role in the Pathogenesis of Treacher Collins Syndrome

Heparan sulfate-N-deacetylase/N-sulfotransferase (HSST) catalyzes both theN-deacetylation and theN-sulfation of heparan sulfate. Previous studies have resulted in the isolation of the human HSST gene from within the Treacher Collins syndrome locus (TCOF1) critical region on 5q. In the present study, the genomic organization of the HSST gene has been elucidated, and the 14 exons identified have been tested for TCOF1-specific mutations. As a result of these studies, mutations within the coding sequence and adjacent splice junctions of HSST can be excluded from a causative role in the pathogenesis of Treacher Collins syndrome.     

The human immunoglobulin κ locus on yeast artificial chromosomes (YACs)

The human immunoglobulin κ locus is a duplicated structure. Contigs of 600 kb with 40 Vκ genes and 440 kb with 36 Vκ genes had been established for the Cκ proximal (p) and distal (d) copies, respectively. In addition the human genome contains more than 24 dispersed Vκ genes, called orphons. In the present study, 22 κ-locus derived YACs were analyzed in detail, while 30 orphon-derived YACs were characterized only with respect to some parameters. The κ-locus derived YACs allowed three gaps to be closed which previously could not be bridged by cosmid and phage λ cloning. At the 5′ side, the p contig was extended in the YACs by 50 kb and the d contig by 16 kb. At the 3′side, the d contig was extended by 11.5 kb. Beyond the 3′ end of the d contig a new Vκ gene was found, which is located, according to pulsed field gel electrophoresis (PFGE) experiments, at a distance of at least 140 kb from the last Vκ gene of the contig. This Vκ gene, which was termed Z0, occurred on three YACs, albeit at distances smaller than 140 kb; this was probably due to deletions in the YACs caused by abundant repetitive sequences at the borders of the locus. According to its sequence and to the restriction map of its surroundings, Z0 is an orphon gene of the so-called Z family, of which several members are known to be dispersed throughout the genome. The possibility that Z0 has been the parent of the other Z orphons is discussed.     

Characterization of a YAC Contig Spanning the Pseudoautosomal Region

Due to its unique biology of partial sex linkage and high recombination rates, the pseudoautosomal region (PAR1) on both X and Y chromosomes has attracted considerable interest. In addition, an extremely high level of YAC instability has been observed in this region. We have derived 82 YAC clones from six different YAC libraries mapping to this 2.6-Mb region. Of these a subset of 22 YACs was analyzed in detail. YAC contigs were assembled using 67 pseudoautosomal probes, of which 64 were unambiguously ordered. All markers are well distributed over the entire region, including the middle part of the region, which has previously been found difficult to contig. Two gaps of less than 50 kb within the genomic locus of CSF2RA and around XE7 remain, which could not be covered with YACs, cosmids, or phages. This YAC contig anchored on the physical map of PAR1 represents one of the best characterized large regions of the human genome with a map completion greater than 90% at 100-kb resolution and has permitted the accurate localization of all known genes within this region.     

Fine mapping and cloning of the breakpoint associated with Menkes syndrome in a female patient.

The gene responsible for Menkes syndrome has been assigned to Xq13 by a combination of comparative mapping and linkage analysis. A previous report has mapped the translocation breakpoint associated with the disease in a female patient to an interval delimited by PGK1 and a group of six more proximal Xq13 markers, including DXS56. We have characterized a number of PGK1- or DXS56-positive YACs, from which we have generated six new markers. One of them identifies a small overlap region between a PGK1-positive YAC and three DXS56-positive YACs, distal to the Menkes breakpoint. A 560-kb region covered by a DXS56-positive YAC has been restriction-mapped and subcloned, disclosing a 187-kb MluI fragment astride the breakpoint. A probe mapping distal to the rearrangement in the same interval reveals altered PGFE fragments in a hybrid constructed from the translocation patient's DNA. We describe the development of a cosmid contig extending 150 kb from a nearby CpG island across the breakpoint. This contig includes four adjacent clones displaying cross-specific hybridization.     

Chromosomal Localization in Mouse and Human of the Vasoactive Intestinal Peptide Receptor Type 2 Gene: A Possible Contributor to the Holoprosencephaly 3 Phenotype

The neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) have been shown to act on a wide range of tissue and cell types, both in the central nervous system and in the periphery. Two distinct receptors for VIP, the VIP receptor type 1 (VIPR1) and the VIP receptor type 2 (VIPR2), have recently been cloned, each of which binds PACAP and VIP with equal affinity. We report here the chromosomal mapping of the human and mouse VIPR2 genes by fluorescencein situhybridization. The VIPR2 gene maps to the human chromosomal region 7q36.3 and to the F2 region of mouse chromosome 12. Our localization of the human gene places it in the region where the locus for the craniofacial defect holoprosencephaly type 3 (HPE3) maps. Further mapping experiments, carried out on cell lines derived from patients with HPE or HPE microforms and associated 7q deletions, have led us to redefine the distal extent of the HPE3 minimal critical region, originally characterized by Gurrieriet al.(1993,Nature Genet.3: 247–251.) The VIPR2 gene lies within this new HPE3 minimal critical region. Our results suggest that deletion of the VIPR2 gene is not the sole factor responsible for the HPE3 phenotype. However, it is possible that monosomy at the VIPR2 locus may contribute to the phenotype observed in many cases of HPE3.     

Map-based cloning in crop plants: tomato as a model system II. Isolation and characterization of a set of overlapping yeast artificial chromosomes encompassing the jointless locus

A map-based cloning technique for crop plants is being developed using tomato as a model system. The target gene jointless is a recessive mutation that completely suppresses the formation of flower and fruit pedicel abscission zones. Previously, the jointless locus was mapped to a 3 cM interval between the two molecular markers TG523 and RPD158. Physical mapping of the jointless region by pulsed-field gel electrophoresis demonstrated that TG523 and RPD158 reside on a 600 kb SmaI fragment. In this study, TG523 was used as a probe to screen a tomato yeast artificial chromosome (YAC) library. Six tomato YAC (TY) clones were isolated, ranging from 220 to 380 kb in size. Genetic mapping of YAC ends demonstrated that this set of overlapping YACs encompasses the jointless locus. Two YAC ends, TY159L (L indicates left end) and TY143R (R indicates right end), cosegregate with the jointless locus. Only one of the six YACs (TY142) contained single-copy DNA sequences at both ends that could be mapped. The two ends of TY142 were mapped to either side of the jointless locus, indicating that TY142 contains a contiguous 285 kb tomato DNA fragment that probably includes the jointless locus. Physical mapping of the TY142 clone revealed that TY159L and TY143R reside on a 55 kb SalI fragment. Southern blot hybridization analysis of the DNAs of tomato lines nearly isogenic for the jointless mutation has allowed localization of the target locus to a region of less than 50 kb within the TY142 clone.Communicated by H. Saedler     

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.     

Linkage disequilibrium and physical mapping of X-linked juvenile retinoschisis.

X-linked juvenile retinoschisis (RS) is a recessively inherited disorder resulting in poor visual acuity. Affected males typically show retinal degeneration and intraretinal splitting. The prevalence of RS is 1:15,000-1:30,000. Elsewhere we have mapped the RS gene between the markers DXS43 and DXS274 in Xp22.1-p22.2. To narrow the RS region, we analyzed 31 Finnish RS families with the markers DXS418, DXS999, DXS7161, and DXS365 and a new polymorphic microsatellite marker, HYAT1. Multipoint linkage analysis allowed us to localize the RS gene between the markers DXS418 and DXS7161 (LOD score = 31.3). We have covered this region with nine YAC clones. On the basis of the sizes of the YACs, sequence-tagged site (STS) content mapping, and restriction mapping, the physical distance between DXS418 and DXS7161 is approximately 0.9 Mb. A total of five potential CpG islands could be identified. For haplotype analysis, eight additional Finnish RS families were analyzed with the markers DXS1195, DXS418, HYAT1, DXS999, DXS7161, and DXS365. On the basis of the linkage-disequilibrium data that were derived from the genetically isolated Finnish population, the critical region for RS could be narrowed to 0.2-0.3 cM, between the markers DXS418 and HYAT1.     

Characterization of a panel of somatic cell hybrids for subregional mapping along 11p and within band 11p13

Summary The short arm of chromosome 11 carries genes involved in malformation syndromes, including the aniridia/genitourinary abnormalities/mental retardation (WAGR) syndrome and the Beckwith-Wiedemann syndrome, both of which are associated with an increased risk of childhood malignancy. Evidence comes from constitutional chromosomal aberrations and from losses of heterozygosity, limited to tumor cells, involving regions 11p13 and 11p15. In order to map the genes involved more precisely, we have fused a mouse cell line with cell lines from patients with constitutional deletions or translocations. Characterization of somatic cell hybrids with 11p-specific DNA markers has allowed us to subdivide the short arm into 11 subregions, 7 of which belong to band 11p13. We have thus defined the smallest region of overlap for the Wilms' tumor locus bracketed by the closest proximal and distal breakpoints in two of these hybrids. The region associated with the Beckwith-Wiedemann syndrome spans the region flanked by two 11p15.5 markers, HRAS1 and HBB. These hybrids also represent useful tools for mapping new markers to this region of the human genome.