The CMT2D locus: refined genetic position and construction of a bacterial clone-based physical map.

Charcot-Marie-Tooth (CMT) disease is a progressive neuropathy of the peripheral nervous system, typically characterized by muscle weakness of the distal limbs. CMT is noted for its genetic heterogeneity, with four distinct loci already identified for the axonal form of the disease (CMT2). In 1996, linkage analysis of a single large family revealed the presence of a CMT2 locus on chromosome 7p14 (designated CMT2D). Additional families have been linked subsequently to the same genomic region, including one with distal spinal muscular atrophy (dSMA) and one with mixed features of dSMA and CMT2; symptoms in both of these latter families closely resemble those seen in the original CMT2D family. There is thus a distinct possibility that CMT2 and dSMA encountered in these families reflect allelic heterogeneity at a single chromosome 7 locus. In the study reported here, we have performed more detailed linkage analysis of the original CMT2D family based on new knowledge of the physical locations of various genetic markers. The region containing the CMT2D gene, as defined by the original family, overlaps with those defined by at least two other families with CMT2 and/or dSMA symptoms. Both yeast artificial chromosome (YAC) and bacterial clone-based [bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC)] contig maps spanning approximately 3.4 Mb have been assembled across the combined CMT2D critical region, with the latter providing suitable clones for systematic sequencing of the interval. Preliminary analyses have already revealed at least 28 candidate genes and expressed-sequence tags (ESTs). The mapping information reported here in conjunction with the evolving sequence data should expedite the identification of the CMT2D/dSMA gene or genes.

A physical map of human chromosome 7: an integrated YAC contig map with average STS spacing of 79 kb.

The construction of highly integrated and annotated physical maps of human chromosomes represents a critical goal of the ongoing Human Genome Project. Our laboratory has focused on developing a physical map of human chromosome 7, a approximately 170-Mb segment of DNA that corresponds to an estimated 5% of the human genome. Using a yeast artificial chromosome (YAC)-based sequence-tagged site (STS)-content mapping strategy, 2150 chromosome 7-specific STSs have been established and mapped to a collection of YACs highly enriched for chromosome 7 DNA. The STSs correspond to sequences generated from a variety of DNA sources, with particular emphasis placed on YAC insert ends, genetic markers, and genes. The YACs include a set of relatively nonchimeric clones from a human-hamster hybrid cell line as well as clones isolated from total genomic libraries. For map integration, we have localized 260 STSs corresponding to Genethon genetic markers and 259 STSs corresponding to markers orders by radiation hybrid (RH) mapping on our YAC contigs. Analysis of the data with the program SEGMAP results in the assembly of 22 contigs that are "anchored" on the Genethon genetic map, the RH map, and/or the cytogenetic map. These 22 contigs are ordered relative to one another, are (in all but 3 cases) oriented relative to the centromere and telomeres, and contain > 98% of the mapped STSs. The largest anchored YAC contig, accounting for most of 7p, contains 634 STSs and 1260 YACs. An additional 14 contigs, accounting for approximately 1.5% of the mapped STSs, are assembled but remain unanchored on either the genetic or RH map. Therefore, these 14 "orphan" contigs are not ordered relative to other contigs. In our contig maps, adjacent STSs are connected by two or more YACs in > 95% of cases. With 2150 mapped STSs, our map provides an average STS spacing of approximately 79 kb. The physical map we report here exceeds the goal of 100-kb average STS spacing and should provide an excellent framework for systematic sequencing of the chromosome.

The human reelin gene: isolation, sequencing, and mapping on chromosome 7.

The mouse reelin gene (Reln) encodes a novel protein that, when mutated, results in the characteristic reeler phenotype. A key component of this phenotype is the extensive disruption of the organization of many brain structures. Reelin is believed to be an extracellular protein that controls neural cell positioning during brain development. The reelin gene is conserved in many vertebrate species, including humans. To study the role of the reelin homolog in human brain development, we have isolated and characterized the human gene (RELN). Like its murine counterpart, RELN is large, encoding an mRNA of approximately 12 kb. Overlapping cDNA clones containing the entire open reading frame were isolated and sequenced, revealing that the predicted mouse and human proteins are similar in size (388 kD) and that the amino acid and nucleotide sequences are 94.2% and 87.2% identical, respectively. Northern hybridization analyses revealed that RELN is expressed in fetal and postnatal brain as well as liver. The expression of RELN in postnatal human brain was high in the cerebellum. RELN was mapped to human chromosome 7q22, based on both fluorescence in situ hybridization studies and localization within a well-positioned yeast artificial chromosome (YAC) contig. The YAC contig also contains a number of gentic markers. Together, these studies provide the sequence information and genetic tools for performing more detailed analyses of RELN in an attempt to define its role in human brain development and possibly in human disease.

A collection of 1814 human chromosome 7-specific STSs.

An established goal of the ongoing Human Genome Project is the development and mapping of sequence-tagged sites (STSs) every 100 kb, on average, across all human chromosomes. En route to constructing such a physical map of human chromosome 7, we have generated 1814 chromosome 7-specific STSs. The corresponding PCR assays were designed by the use of DNA sequence determined in our laboratory (79%) or generated elsewhere (21%) and were demonstrated to be suitable for screening yeast artificial chromosome (YAC) libraries. This collection provides the requisite landmarks for constructing a physical map of chromosome 7 at < 100-kb average spacing of STSs.

Mapping the RP10 locus for autosomal dominant retinitis pigmentosa on 7q: refined genetic positioning and localization within a well-defined YAC contig.

Retinitis pigmentosa is a genetically heterogeneous disease that has autosomal dominant, autosomal recessive and X-linked forms. Autosomal dominant retinitis pigmentosa (adRP) has thus far been associated with eight distinct loci, including the rhodopsin and peripherin/RDS genes as well as unidentified genes on chromosomes 7p, 7q, 8q, 17p, 17q, and 19q. The RP10 locus for adRP on chromosome 7q was first mapped in a Spanish family; later, an unrelated American family was identified that also showed linkage to 7q. By combining the linkage results from both families, we are able to assign the disease gene to a 5-cM interval on 7q. Based on extensive physical mapping of this region, the genetic interval is now fully contained within a approximately 5-Mb segment on a well-defined YAC contig. These studies significantly reduce the size of the RP10 critical region, exclude a number of possible candidate genes, and provide the necessary cloned DNA for the positional cloning of the RP10 gene.

The human obese (OB) gene: RNA expression pattern and mapping on the physical, cytogenetic, and genetic maps of chromosome 7.

The recently identified mouse obese (ob) gene apparently encodes a secreted protein that may function in the signaling pathway of adipose tissue. Mutations in the mouse ob gene are associated with the early development of gross obesity. A detailed knowledge concerning the RNA expression pattern and precise genomic location of the human homolog, the OB gene, would facilitate examination of the role of this gene in the inheritance of human obesity. Northern blot analysis revealed that OB RNA is present at a high level in adipose tissue but at much lower levels in placenta and heart. OB RNA is undetectable in a wide range of other tissues. Comparative mapping of mouse and human DNA indicated that the ob gene is located within a region of mouse chromosome 6 that is homologous to a portion of human chromosome 7q. We mapped the human OB gene on a yeast artificial chromosome (YAC) contig from chromosome 7q31.3 that contains 43 clones and 19 sequence-tagged sites (STSs). Among the 19 STSs are eight corresponding to microsatellite-type genetic markers, including seven (CA)n repeat-type Genethon markers. Because of their close physical proximity to the human OB gene, these eight genetic markers represent valuable tools for analyzing families with evidence of hereditary obesity and for investigating the possible association between OB mutations and human obesity.

A human chromosome 7 yeast artificial chromosome (YAC) resource: construction, characterization, and screening.

The paradigm of sequence-tagged site (STS)-content mapping involves the systematic assignment of STSs to individual cloned DNA segments. To date, yeast artificial chromosomes (YACs) represent the most commonly employed cloning system for constructing STS maps of large genomic intervals, such as whole human chromosomes. For developing a complete YAC-based STS-content map of human chromosome 7, we wished to utilize a limited set of YAC clones that were highly enriched for chromosome 7 DNA. Toward that end, we have assembled a human chromosome 7 YAC resource that consists of three major components: (1) a newly constructed library derived from a human-hamster hybrid cell line containing chromosome 7 as its only human DNA; (2) a chromosome 7-enriched sublibrary derived from the CEPH mega-YAC collection by Alu-polymerase chain reaction (Alu-PCR)-based hybridization; and (3) a set of YACs isolated from several total genomic libraries by screening for > 125 chromosome 7 STSs. In particular, the hybrid cell line-derived YACs, which comprise the majority of the clones in the resource, have a relatively low chimera frequency (10-20%) based on mapping isolated insert ends to panels of human-hamster hybrid cell lines and analyzing individual clones by fluorescence in situ hybridization. An efficient strategy for polymerase chain reaction (PCR)-based screening of this YAC resource, which totals 4190 clones, has been developed and utilized to identify corresponding YACs for > 600 STSs. The results of this initial screening effort indicate that the human chromosome 7 YAC resource provides an average of 6.9 positive clones per STS, a level of redundancy that should support the assembly of large YAC contigs and the construction of a high-resolution STS-content map of the chromosome.

Integration of physical, genetic and cytogenetic maps of human chromosome 7: isolation and analysis of yeast artificial chromosome clones for 117 mapped genetic markers.

An important goal for the human genome project is to assemble fully integrated physical, genetic and cytogenetic maps for each human chromosome. Towards that end, we have isolated yeast artificial chromosome (YAC) clones containing 117 of the 119 genetic markers that constitute a recently constructed, detailed genetic map of human chromosome 7. Analysis of these clones reveals numerous examples where adjacent genetic markers have been physically connected, either in individual YACs or in multi-YAC contigs. At present, the 117 genetic markers are contained in fewer than 80 YAC contigs, with most of these contigs uniquely ordered relative to one another based on the genetic map positions of the corresponding markers. These YACs and YAC contigs are estimated to contain approximately 60-85% of the DNA from human chromosome 7. YACs representing 36 genetic markers were mapped by fluorescence in situ hybridization (FISH) to metaphase chromosomes, allowing assignment of these genetic markers to cytogenetic bands along chromosome 7 and placement of the centromere within the genetic map. Together, these studies provide genetically and cytogenetically anchored YAC clones covering the majority of chromosome 7 that will be useful both for the positional cloning of genes and as a framework for assembling a complete YAC-based physical map of the chromosome.

Physical maps of the six smallest chromosomes of Saccharomyces cerevisiae at a resolution of 2.6 kilobase pairs.

Physical maps of the six smallest chromosomes of Saccharomyces cerevisiae are presented. In order of increasing size, they are chromosomes I, VI, III, IX, V and VIII, comprising 2.49 megabase pairs of DNA. The maps are based on the analysis of an overlapping set of lambda and cosmid clones. Overlaps between adjacent clones were recognized by shared restriction fragments produced by the combined action of EcoRI and HindIII. The average spacing between mapped cleavage sites is 2.6 kb. Five of the six chromosomes were mapped from end to end without discontinuities; a single internal gap remains in the map of chromosome IX. The reported maps span an estimated 97% of the DNA on the six chromosomes; nearly all the missing segments are telomeric. The maps are fully cross-correlated with the previously published SfiI/NotI map of the yeast genome by A. J. Link and M. V. Olson. They have also been cross-correlated with the yeast genetic map at 51 loci.