An integrated deletion and physical map encompassing l71Rl, a chromosome 7 locus required for peri-implantation survival in the mouse

l71Rl, a locus that maps just proximal to the pink-eyed dilution (p) locus in mouse chromosome 7, was initially identified as being required for early post-implantation survival. We define further the null phenotype of l71Rl as peri-implantation lethal, with homozygous mutant embryos degenerating between embryonic day 4.5 (E4.5) and E5. 5. We constructed an integrated deletion/physical map covering a 1. 82-Mb chromosomal segment extending proximally from p. This map defines the minimum critical interval for l71Rl as an 80- to 300-kb region. This sequence-ready deletion/physical map should enable the cloning and characterization of the l71Rl gene(s).     

A physical map of the mouse shaker-2 region contains many of the genes commonly deleted in Smith-Magenis syndrome (del17p11.2p11.2)

We report the construction of a physical map of the region of mouse chromosome 11 that encompasses shaker-2 (sh2), a model for the human nonsyndromic deafness DFNB3. DFNB3 maps within the common deletion region of Smith-Magenis syndrome (SMS), del(17)(p11.2p11.2). Eleven of the genes mapping within the SMS common deletion region have murine homologs on the sh2 physical map. The gene order in this region is not perfectly conserved between mouse and human, a finding to be considered as we engineer a mouse model of Smith-Magenis syndrome.     

Molecular cloning and mapping of the ecotropic leukemia provirus Emv-23 provides molecular access to the albino-deletion complex in mouse chromosome 7

Genetic analysis of radiation-induced deletion mutations involving the chromosome 7 albino (c) locus has expanded the functional map of this 6 to 11-cM region of the mouse genome. To generate one of many points of molecular access necessary for intensifying the analysis of the genes and phenotypes associated with this particular complex of deletions, we have cloned an endogenous ecotropic leukemia provirus (Emv-23), known to be closely linked to c, along with its flanking chromosome 7 sequences. A unique-sequence probe (23.3), derived from a region immediately 5' to the proviral integration site, was found to map less than 0.5 cM from c in a standard backcross analysis. Southern blot analysis of DNAs from animals carrying homozygous or overlapping albino deletions demonstrated that the 23.3 probe was deleted in several relatively small c-region deletions. The deletion mapping of the 23.3 probe places the Emv-23 locus between c and Mod-2, just proximal to a region important for male fertility and juvenile fitness. Mapping of this locus also provides a refinement of the genetic/deletion map for several mutations within this deletion complex.     

Physical map of the wheat high-grain protein content gene Gpc-B1 and development of a high-throughput molecular marker

Grain protein content (GPC) is important for human nutrition and has a strong influence on pasta and bread quality. A quantitative trait locus, derived from a Triticum turgidum ssp. dicoccoides accession (DIC), with an average increase in GPC of 14 g kg(-1) was mapped on chromosome 6BS. Using the wheat-rice colinearity, a high-density map of the wheat region was developed and the quantitative trait locus was mapped as a simple Mendelian locus designated Gpc-B1. A physical map of approx. 250 kb of the Gpc-B1 region was developed using a tetraploid wheat bacterial artificial chromosome library. The constructed physical map included the two Gpc-B1 flanking markers and one potential candidate gene from the colinear rice region completely linked to Gpc-B1. The relationship between physical and genetic distances and the feasibility of isolating genes by positional cloning in wheat are discussed. A high-throughput codominant marker, Xuhw89, was developed. A 4-bp deletion present in the DIC allele was absent in a collection of 117 cultivated tetraploid and hexaploid wheat germplasm, suggesting that this marker will be useful to incorporate the high GPC allele from the DIC accession studied here into commercial wheat varieties.     

Further refinement of the location for autosomal dominant retinitis pigmentosa on chromosome 7p (RP9).

A form of autosomal dominant retinitis pigmentosa (adRP) mapping to chromosome 7p was recently reported by this laboratory, in a single large family from southeastern England. Further sampling of the family and the use a number of genetic markers from 7p have facilitated the construction of a series of multipoint linkage maps of the region with the most likely disease gene location. From this and haplotype data, the locus can now be placed between the markers D7S484 and D7S526, in an interval estimated to be 1.6-4 cM. Genetic distances between the markers previously reported to be linked to this region and those described in the recent whole-genome poly-CA map were estimated from data in this and other families. These data should assist in the construction of a physical map of the region and will help to identify candidate genes for the 7p adRP locus.     

Molecular Analysis of 36 Mutations at the Mouse Pink-Eyed Dilution (P) Locus

Thirty-six radiation- or chemically induced homozygous-lethal mutations at the p locus in mouse chromosome 7 have been analyzed at 17 loci defined by molecular probes to determine the types of lesions, numbers of p-region markers deleted or rearranged, regions of overlap of deletion mutations, and genetic distances between loci. A linear deletion map of the [Myod1, Ldh3]-[Snrpn, Znf127] region has been constructed from the molecular analyses of the p-locus deletions. The utility of these deletions as tools for the isolation and characterization of the genes specifying the neurological, reproductive, and developmental phenotypes genetically mapped to this region will grow as more detailed molecular analyses continue.     

A physical map, including a BAC/PAC clone contig, of the Williams-Beuren syndrome--deletion region at 7q11.23

Williams-Beuren syndrome (WBS) is a developmental disorder caused by haploinsufficiency for genes in a 2-cM region of chromosome band 7q11.23. With the exception of vascular stenoses due to deletion of the elastin gene, the various features of WBS have not yet been attributed to specific genes. Although >/=16 genes have been identified within the WBS deletion, completion of a physical map of the region has been difficult because of the large duplicated regions flanking the deletion. We present a physical map of the WBS deletion and flanking regions, based on assembly of a bacterial artificial chromosome/P1-derived artificial chromosome contig, analysis of high-throughput genome-sequence data, and long-range restriction mapping of genomic and cloned DNA by pulsed-field gel electrophoresis. Our map encompasses 3 Mb, including 1.6 Mb within the deletion. Two large duplicons, flanking the deletion, of >/=320 kb contain unique sequence elements from the internal border regions of the deletion, such as sequences from GTF2I (telomeric) and FKBP6 (centromeric). A third copy of this duplicon exists in inverted orientation distal to the telomeric flanking one. These duplicons show stronger sequence conservation with regard to each other than to the presumptive ancestral loci within the common deletion region. Sequence elements originating from beyond 7q11.23 are also present in these duplicons. Although the duplicons are not present in mice, the order of the single-copy genes in the conserved syntenic region of mouse chromosome 5 is inverted relative to the human map. A model is presented for a mechanism of WBS-deletion formation, based on the orientation of duplicons' components relative to each other and to the ancestral elements within the deletion region.     

Contiguous localization of the genes encoding human insulin-like growth factor binding proteins 1 (IGBP1) and 3 (IGBP3) on chromosome 7.

In extracellular fluids the insulin-like growth factors (IGFs) are bound to specific binding proteins (IGBPs). The genes for two members of this protein family have been mapped, the IGBP1 gene to human chromosomal region 7p14-p12 and the IGBP2 gene to region 2q33-q34. In this study, somatic cell hybrid analysis indicated that IGBP3 is also located on chromosome 7. Pulsed-field gel electrophoresis was used to demonstrate the close physical linkage between IGBP1 and IGBP3. Overlapping cosmid clones encompassing these genes were isolated, and restriction endonuclease mapping showed that the genes are arranged in a tail-to-tail fashion separated by 20 kb of DNA. Further characterization of the IGBP1 DNA sequence disclosed a duplication of the intron 3-exon 4 junction within the third intron. In addition, we report RFLPs for ApaLI and TaqI in the IGBP1 locus.     

Construction of a 1.2-Mb BAC/PAC contig of the porcine gene RYR1 region on SSC 6q1.2 and comparative analysis with HSA 19q13.13

We screened a porcine bacterial artificial chromosome (BAC) and a P1 derived artificial chromosome (PAC) library to construct a sequence-ready approximately 1.2-Mb BAC/PAC contig of the ryanodine receptor-1 gene (RYR1) region on porcine chromosome (SSC) 6q1.2. This genomic segment is of special interest because it harbors the locus for stress susceptibility in pigs and a putative quantitative trait locus for muscle growth. Detailed physical mapping of this gene-rich region allowed us to assign to this contig 17 porcine genes orthologous to known human chromosome 19 genes. Apart from the relatively well-characterized porcine gene RYR1, the other 16 genes represent novel chromosomal assignments and 14 genes have been cloned for the first time in pig. Comparative analysis of the porcine BAC/PAC contig with the human chromosome (HSA) 19q13.13 map revealed a completely conserved gene order of this segment between pig and human. A detailed porcine-human-mouse comparative map of this region was constructed.     

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.     

Low-copy repeats mediate the common 3-Mb deletion in patients with velo-cardio-facial syndrome

Velo-cardio-facial syndrome (VCFS) is the most common microdeletion syndrome in humans. It occurs with an estimated frequency of 1 in 4, 000 live births. Most cases occur sporadically, indicating that the deletion is recurrent in the population. More than 90% of patients with VCFS and a 22q11 deletion have a similar 3-Mb hemizygous deletion, suggesting that sequences at the breakpoints confer susceptibility to rearrangements. To define the region containing the chromosome breakpoints, we constructed an 8-kb-resolution physical map. We identified a low-copy repeat in the vicinity of both breakpoints. A set of genetic markers were integrated into the physical map to determine whether the deletions occur within the repeat. Haplotype analysis with genetic markers that flank the repeats showed that most patients with VCFS had deletion breakpoints in the repeat. Within the repeat is a 200-kb duplication of sequences, including a tandem repeat of genes/pseudogenes, surrounding the breakpoints. The genes in the repeat are GGT, BCRL, V7-rel, POM121-like, and GGT-rel. Physical mapping and genomic fingerprint analysis showed that the repeats are virtually identical in the 200-kb region, suggesting that the deletion is mediated by homologous recombination. Examination of two three-generation families showed that meiotic intrachromosomal recombination mediated the deletion.     

Detecting high-resolution polymorphisms in human coding loci by combining PCR and single-strand conformation polymorphism (SSCP) analysis.

A strategy is described that allows the development of polymorphic genetic markers to be characterized in individual genes. Segments of the 3' untranslated regions are amplified, and polymorphisms are detected by digestion with frequently cutting enzymes and with the detection of single-stranded conformation polymorphisms. This allows these genes, or DNA segments, to be placed on the linkage maps of human chromosomes. Polymorphisms in two genes have been identified using this approach. A HaeIII polymorphism was detected in the KIT proto-oncogene, physically assigned to chromosome 4q11-12. This polymorphism is linked to other chromosome 4p markers and is in linkage disequilibrium with a HindIII polymorphism previously described at this locus. We have also identified in the insulin-like growth factor1 receptor gene (IGF1R) a 2-bp deletion that is present at a frequency of .25 in the Caucasian population. Pedigree analysis with this insertion/deletion polymorphism placed the IGF1R gene at the end of the current linkage map of chromosome 15q, consistent with the physical assignment of 15q2526. Thus, polymorphisms in specific genes can be used to related the physical, genetic, and comparative maps of mammalian genomes and to simplify the testing of candidate genes for human diseases.     

A Physical Map of the Mouseshaker-2Region Contains Many of the Genes Commonly Deleted in Smith–Magenis Syndrome (del17p11.2p11.2)

We report the construction of a physical map of the region of mouse chromosome 11 that encompassesshaker-2(sh2), a model for the human nonsyndromic deafnessDFNB3. DFNB3maps within the common deletion region of Smith–Magenis syndrome (SMS), del(17)(p11.2p11.2). Eleven of the genes mapping within the SMS common deletion region have murine homologs on thesh2physical map. The gene order in this region is not perfectly conserved between mouse and human, a finding to be considered as we engineer a mouse model of Smith–Magenis syndrome.     

Molecular Genetics of the Brown (B)-Locus Region of Mouse Chromosome 4. II. Complementation Analyses of Lethal Brown Deletions

Numerous new mutations at the brown (b) locus in mouse chromosome 4 have been recovered over the years in germ-cell mutagenesis experiments performed at the Oak Ridge National Laboratory. A large series of radiation- and chemical-induced b mutations known to be chromosomal deletions, and also known to be prenatally lethal when homozygous, were analyzed by pairwise complementation crosses as well as by pseudodominance tests involving flanking loci defined by externally visible phenotypes. These crosses were designed to determine the extent of each deletion on the genetic and phenotype map of the chromosomal region surrounding the b locus; the crosses also provided basic data that assigned deletions to complementation groups and defined four new loci associated with aberrancies in normal development. Specifically, the pseudodominance tests identified deletions that include the proximally mapping whirler (wi) and the distally mapping depilated (dep) genes, thereby bracketing these loci defined by visible developmental abnormalities with landmarks (deletion breakpoints) that are easily identified on the physical map. Furthermore, the complementation crosses, which were supplemented with additional crosses that allowed determination of the gross time of lethality of selected deletions, defined four new loci required for normal development. Homozygous deletion of one of these loci (b-associated fitness, baf) results in a runting syndrome evident during postnatal development; deletion of one locus [l(4)2Rn] causes death in the late gestation/neonatal period; and deletion of either of two loci [l(4)1Rn or l(4)3Rn] results in embryonic death, most likely in pre-, peri- or postimplantation stages. The placement of these new functionally defined loci on the evolving molecular map of the b region should be useful for continuing the analysis of the roles played in development by genes in this segment of chromosome 4.     

A 2.5-Mb physical map within 3p21.1 spans the breakpoint associated with Greig cephalopolysyndactyly syndrome.

Numerous investigations suggest that one or more genes residing in the p14 to p21 region of human chromosome 3 are critical to the development of neoplastic diseases such as renal cell carcinoma and small-cell lung cancer (SCLC). This region is additionally involved in several interchromosomal translocations, one of which is associated with the developmental disorder Greig cephalopolysyndactyly syndrome. A series of five loci that map in close proximity to the Greig syndrome breakpoint [t(3;7)(p21.1;p13)] at 3p21.1 have been physically linked by pulsed-field gel analysis over a 2.5-Mb region. The probes include ACY1, cA84 (D3S92), cA199 (D3S93), pHF12-32 (D3S2), and MW-Not153 (D3S332). The Greig 3;7 translocation breakpoint was discovered between clones cA199 and MW-Not153, separated by 825 kb. Further analysis revealed comigration of a rearranged fragment detected by MW-Not153 and a chromosome 7 probe previously shown to be in close proximity to the breakpoint (CRI-R944). This latter probe also detects a rearrangement in a second Greig-associated translocation, (6;7)(q27;p13). The physical map resulting from this analysis orders the markers along the chromosome and identifies several locations for CpG islands, likely associated with genes. Although probe pEFD145.1 (D3S32) has been genetically linked to D3S2 (2 cM), physical linkage to the other five loci could not be demonstrated. One of the linked loci, D3S2, has been widely utilized in the analysis of chromosome 3p loss in several malignant diseases. Since expression of ACY1, a housekeeping gene, is specifically reduced in many cases of SCLC, knowledge of its precise chromosomal position and identification of neighboring putative gene loci should facilitate investigation into the mechanism of this reduction.