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.     

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.     

Physical mapping and cloning of the proximal segment of the myotonic dystrophy gene region.

The myotonic dystrophy (DM) region has been recently shown to be bracketed by two key recombinant events. One recombinant occurs in a Dutch DM family, which maps the DM locus distal to the ERCC1 gene and D19S115 (pE0.8). The other recombinant event is in a French Canadian DM family, which maps DM proximal to D19S51 (p134c). To further resolve this region, we initiated a chromosome walk in a telomeric direction from pE0.8, a proximal marker tightly linked to DM, toward the genetic locus. An Alu-PCR approach to chromosome walking in a cosmid library from flow-sorted chromosome 19 was used to isolate DM region cosmids. This effort has resulted in the cloning of a 350-kb genomic contig of human chromosome 19q13.3. New genetic and physical mapping information has been generated using the newly cloned markers from this study. As a result of this new mapping information, the minimal area that is to contain the DM gene has been redefined. Approximately 200 kb of sequence between pE0.8 and the closest proximal marker to DM, pKEX0.8, that would have otherwise been screened for DM candidate genes, has been eliminated as containing the DM gene.     

A 2-Mb sequence-ready contig map and a novel immunoglobulin superfamily gene IGSF4 in the LOH region of chromosome 11q23.2

Human chromosome 11q23.2 has been proposed to contain a tumor suppressor gene(s) whose deletion has been associated with cancer of the lung and breast and with neuroblastoma. To analyze the genomic structure and to isolate a candidate tumor suppressor gene from this region, we constructed a 2-Mb sequence-ready contig map using bacteriophage P1 (P1), bacterial artificial chromosome (BAC), and P1-derived artificial chromosome (PAC). The map comprises a contig of 24 overlapping P1, BAC, and PAC clones. To isolate gene fragments from the region, we performed direct cDNA library screening, exon trapping, EST mapping, and genomic sequencing using the P1, BAC, and PAC clones. Sequence analysis of 5 clones, which spans 23% (458,738 bp) of the region, and extensive gene scanning along the entire region revealed that the region is extraordinarily scarce in genes, but we identified one ubiquitously expressed novel gene and one testis-specific gene fragment. The novel gene, which we call IGSF4 (immunoglobulin superfamily 4), is transcribed into a 1.6- or 4.4-kb RNA encoding a 442-amino-acid protein. It shares strong homology with mouse IGSF-B12 and cell adhesion molecules NCAM1 and NCAM2 within their Ig-like C2-type domains. The IGSF4 gene, a novel gene that is shown to be located in the common loss of heterozygosity region, possesses a number of interesting features and may be good candidate for a tumor suppressor gene.     

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 BAC library of <Emphasis Type="Italic"> Rosa rugosa </Emphasis>Thunb. and assembly of a contig spanning<Emphasis Type="Italic"> Rdr1</Emphasis>, a gene that confers resistance to blackspot

A BAC library to serve as a general tool for the physical mapping and positional cloning of rose genes has been constructed from Rosa rugosa DNA. With 27,264 clones the library contains 5.2 genome equivalents. The library was used to assemble a contig of BAC clones spanning Rdr1, a locus that confers resistance to blackspot. For this purpose fine-scale mapping of the target locus was achieved by bulked segregant analysis using 816 AFLP primer combinations. The target region around Rdr1 comprises about 400 kb and is covered by a minimum of six BAC clones. Furthermore, the detection of at least five resistance gene analogs of the TIR-NBS-LRR family on the contig indicates the presence of a cluster of resistance genes around Rdr1. These results will not only allow the isolation and identification of Rdr1 in the near future, but also provide the tools for the physical mapping and positional cloning of other horticulturally interesting genes in roses.     

The Mouse Region Syntenic for Human Spinal Muscular Atrophy Lies within theLgn1Critical Interval and Contains Multiple Copies ofNaipExon 5

Spinal muscular atrophy (SMA) is a relatively common, autosomal recessively inherited neurodegenerative disorder that maps to human chromosome 5q13. This region of the human genome has an intricate genomic structure that has complicated the evaluation of SMA candidate genes. We have chosen to study the mouse region syntenic for human SMA in the hope that the homologous mouse interval would contain the same genes as human 5q13 on a simpler genomic background. Here, we report the mapping of such a region to mouse chromosome 13 and to the critical interval forLgn1,a mouse locus responsible for modulating the intracellular replication and pathogenicity of the bacteriumLegionella pneumophila.We have generated a mouse YAC contig across theLgn1/Smainterval and have mapped the two flanking gene markers for the human SMA locus, MAP1B and CCNB1, onto this contig. In addition, we have localized the two SMA candidate genes, SMN and NAIP, to theLgn1critical region, making these two genes candidates for theLgn1phenotype. Upon subcloning of the YAC contig into P1s and BACs, we have detected a large, low copy number repeat that contains at least one copy ofNaipexon 5. Identification of theLgn1gene will either provide a novel function for SMN or NAIP or reveal the existence of another, yet uncharacterized gene in the SMA critical region. Mutations in such a gene might help to explain some of the phenotypic variability among the human SMAs.     

Comparative mapping on the mouse X Chromosome defines a myotubular myopathy equivalent region

The gene for X-linked myotubular myopathy (MTM1) has been localized to a 300-kb critical region in human Xq28 between IDS and GABRA3. As part of an effort to clone this gene, we developed a YAC contig on the mouse X Chromosome (Chr) which includes loci homologous to those within the human MTM1 critical region. The murine contig consists of 18 YACs and spans 2.5–3.0 Mb. We have aligned the human and murine physical maps by isolating conserved mouse genomic fragments, including CpG islands and trapped exons. We believe that the simultaneous isolation of genes from both mouse and human and continued comparative mapping will prove helpful in the eventual identification of MTM1 and other genes in the region. Received: 9 February 1996 / Accepted: 30 March 1996