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In this study, we performed high-resolution array comparative genomic hybridization with an array of 4153 bacterial artificial chromosome clones to assess copy number changes in 44 archival breast cancers. The tumors were flow sorted to exclude non-tumor DNA and increase our ability to detect gene copy number changes. In these tumors, losses were more frequent than gains, and gains in 1q and loss in 16q were the most frequent alterations. We compared gene copy number changes in the tumors based on histologic subtype and estrogen receptor (ER) status, i.e., ER-negative infiltrating ductal carcinoma, ER-positive infiltrating ductal carcinoma, and ER-positive infiltrating lobular carcinoma. We observed a consistent association between loss in regions of 5q and ER-negative infiltrating ductal carcinoma, as well as more frequent loss in 4p16, 8p23, 8p21, 10q25, and 17p11.2 in ER-negative infiltrating ductal carcinoma compared with ER-positive infiltrating ductal carcinoma (adjusted P values ≤ 0.05). We also observed high-level amplifications in ER-negative infiltrating ductal carcinoma in regions of 8q24 and 17q12 encompassing the c-myc and c-erbB-2 genes and apparent homozygous deletions in 3p21, 5q33, 8p23, 8p21, 9q34, 16q24, and 19q13. ER-positive infiltrating ductal carcinoma showed a higher frequency of gain in 16p13 and loss in 16q21 than ER-negative infiltrating ductal carcinoma. Correlation analysis highlighted regions of change commonly seen together in ER-negative infiltrating ductal carcinoma. ER-positive infiltrating lobular carcinoma differed from ER-positive infiltrating ductal carcinoma in the frequency of gain in 1q and loss in 11q and showed high-level amplifications in 1q32, 8p23, 11q13, and 11q14. These results indicate that array comparative genomic hybridization can identify significant differences in the genomic alterations between subtypes of breast cancer.

Supplementary Table 4 from Genomic Alterations Indicate Tumor Origin and Varied Metastatic Potential of Disseminated Cells from Prostate Cancer Patients

Supplementary Figure 1 from Array Comparative Genomic Hybridization Analysis of Genomic Alterations in Breast Cancer Subtypes

Supplementary Tables 1-6 from Comparative Analyses of Chromosome Alterations in Soft-Tissue Metastases within and across Patients with Castration-Resistant Prostate Cancer

Supplementary Table 3 from Genomic Alterations Indicate Tumor Origin and Varied Metastatic Potential of Disseminated Cells from Prostate Cancer Patients

Supplementary Methods and Materials from Genomic Alterations Indicate Tumor Origin and Varied Metastatic Potential of Disseminated Cells from Prostate Cancer Patients

Supplementary Table 2 from Genomic Alterations Indicate Tumor Origin and Varied Metastatic Potential of Disseminated Cells from Prostate Cancer Patients

Supplementary Table 5 from Genomic Alterations Indicate Tumor Origin and Varied Metastatic Potential of Disseminated Cells from Prostate Cancer Patients

Supplementary Table 2 from Array Comparative Genomic Hybridization Analysis of Genomic Alterations in Breast Cancer Subtypes

Supplementary Table 1 from Array Comparative Genomic Hybridization Analysis of Genomic Alterations in Breast Cancer Subtypes

Supplementary Figure Legends 1-3 from Comparative Analyses of Chromosome Alterations in Soft-Tissue Metastases within and across Patients with Castration-Resistant Prostate Cancer

Supplementary Table 1 from Genomic Alterations Indicate Tumor Origin and Varied Metastatic Potential of Disseminated Cells from Prostate Cancer Patients

Androgen deprivation is the mainstay of therapy for progressive prostate cancer. Despite initial and dramatic tumor inhibition, most men eventually fail therapy and die of metastatic castration-resistant (CR) disease. Here, we characterize the profound degree of genomic alteration found in CR tumors using array comparative genomic hybridization (array CGH), gene expression arrays, and fluorescence in situ hybridization (FISH). Bycluster analysis, we show that the similarity of the genomic profiles from primary and metastatic tumors is driven by the patient. Using data adjusted for this similarity, we identify numerous high-frequency alterations in the CR tumors, such as 8p loss and chromosome 7 and 8q gain. By integrating array CGH and expression array data, we reveal genes whose correlated values suggest they are relevant to prostate cancer biology. We find alterations that are significantly associated with the metastases of specific organ sites, and others with CR tumors versus the tumors of patients with localized prostate cancer not treated with androgen deprivation. Within the high-frequency sites of loss in CR metastases, we find an overrepresentation of genes involved in cellular lipid metabolism, including PTEN. Finally, using FISH, we verify the presence of a gene fusion between TMPRSS2 and ERG suggested by chromosome 21 deletions detected by array CGH. We find the fusion in 54% of our CR tumors, and 81% of the fusion-positive tumors contain cells with multiple copies of the fusion. Our investigation lays the foundation for a better understanding of and possible therapeutic targets for CR disease, the poorly responsive and final stage of prostate cancer. [Cancer Res 2009;69(19):7793–802]

We report an evolutionary analysis of the V1R gene family across 37 mammalian genomes. V1Rs comprise one of three chemosensory receptor families expressed in the vomeronasal organ, and contribute to pheromone detection. We first demonstrate that Trace Archive data can be used effectively to determine V1R family sizes and to obtain sequences of most V1R family members. Analyses of V1R sequences from trace data and genome assemblies show that species-specific expansions previously observed in only eight species were prevalent throughout mammalian evolution, resulting in “semi-private” V1R repertoires for most mammals. The largest families are found in mouse and platypus, whose V1R repertoires have been published previously, followed by mouse lemur and rabbit (∼215 and ∼160 intact V1Rs, respectively). In contrast, two bat species and dolphin possess no functional V1Rs, only pseudogenes, and suffered inactivating mutations in the vomeronasal signal transduction gene Trpc2. We show that primate V1R decline happened prior to acquisition of trichromatic vision, earlier during evolution than was previously thought. We also show that it is extremely unlikely that decline of the dog V1R repertoire occurred in response to selective pressures imposed by humans during domestication. Functional repertoire sizes in each species correlate roughly with anatomical observations of vomeronasal organ size and quality; however, no single ecological correlate explains the very diverse fates of this gene family in different mammalian genomes. V1Rs provide one of the most extreme examples observed to date of massive gene duplication in some genomes, with loss of all functional genes in other species.

Subtelomeres are concentrations of interchromosomal segmental duplications capped by telomeric repeats at the ends of chromosomes. The nature of the segments shared by different sets of human subtelomeres reflects their high rate of recent interchromosomal exchange. Here, we characterize the rearrangements incurred by the 15q subtelomere after it arose from a chromosome fission event in the common ancestor of great apes. We used FISH, sequencing of genomic clones, and PCR to map the breakpoint of this fission and track the fate of flanking sequence in human, chimpanzee, gorilla, orangutan, and macaque genomes. The ancestral locus, a cluster of olfactory receptor (OR) genes, lies internally on macaque chromosome 7. Sequence originating from this fission site is split between the terminus of 15q and the pericentromere of 14q in the great apes. Numerous structural rearrangements, including interstitial deletions and transfers of material to or from other subtelomeres, occurred subsequent to the fission, such that each species has a unique 15q structure and unique collection of ORs derived from the fission locus. The most striking rearrangement involved transfer of at least 200 kb from the fission-site region to the end of chromosome 4q, where much still resides in chimpanzee and gorilla, but not in human. This gross structural difference places the subtelomeric defect underlying facioscapulohumeral muscular dystrophy (FSHD) much closer to the telomere in human 4q than in the hybrid 4q–15q subtelomere of chimpanzee.

As much as a quarter of the human genome has been reported to vary in copy number between individuals, including regions containing about half of the members of the olfactory receptor (OR) gene family. We have undertaken a detailed study of copy-number variation of ORs to elucidate the selective and mechanistic forces acting on this gene family and the true impact of copy-number variation on human OR repertoires. We argue that the properties of copy-number variants (CNVs) and other sets of large genomic regions violate the assumptions of statistical methods that are commonly used in the assessment of gene enrichment. Using more appropriate methods, we provide evidence that OR enrichment in CNVs is not due to positive selection but is because of OR preponderance in segmentally duplicated regions, which are known to be frequently copy-number variable, and because purifying selection against CNVs is lower in OR-containing regions than in regions containing essential genes. We also combine multiplex ligation-dependent probe amplification (MLPA) and PCR to assay the copy numbers of 37 candidate CNV ORs in a panel of approximately 50 human individuals. We confirm copy-number variation of 18 ORs but find no variation in this human-diversity panel for 16 other ORs, highlighting the caveat that reported intervals often overrepresent true CNVs. The copy-number variation we describe is likely to underpin significant variation in olfactory abilities among human individuals. Finally, we show that both homology-based and homology-independent processes have played a recent role in remodeling the OR family.

The V1R gene family comprises one of two types of putative pheromone receptors expressed in the mammalian vomeronasal organ (VNO). We searched the most recent mouse, rat, dog, chimpanzee, and human genome sequence assemblies to compile a near-complete repertoire of V1R genes for each species. Dog, human, and chimpanzee have very few intact V1Rs (8, 2, and 0, respectively) compared to more than a hundred intact V1Rs in each of the rat (106) and mouse (165) genomes. We also provide the first description of the diversity of V1R pseudogenes in these species. We identify at least 165 pseudogenes in mouse, 110 in rat, 102 in chimpanzee, 115 in human, and 54 in dog. Primate and dog pseudogenes are distributed among almost all V1R subfamilies seen in rodents, indicating that the common ancestor of these species had a diverse V1R repertoire. We find that V1R genes were subject to strikingly different fates in different species and in different subfamilies. In rodents, some subfamilies remained relatively stable or underwent roughly equivalent expansion in mouse and rat; other subfamilies expanded in one species but not the other. The small number of intact V1Rs in the dog genome is unexpected given the presumption that dogs, like rodents, have a functional VNO, and a complex system of pheromone-based behaviors. We identify an intact transient receptor potential channel 2β in the dog genome, consistent with a functional VNO in dogs. The diminished V1R repertoire in dogs raises questions about the relative contributions of V1Rs versus other candidate pheromone receptor genes in the establishment of complex pheromone systems in mammals.

In this study, we performed high-resolution array comparative genomic hybridization with an array of 4153 bacterial artificial chromosome clones to assess copy number changes in 44 archival breast cancers. The tumors were flow sorted to exclude non-tumor DNA and increase our ability to detect gene copy number changes. In these tumors, losses were more frequent than gains, and gains in 1q and loss in 16q were the most frequent alterations. We compared gene copy number changes in the tumors based on histologic subtype and estrogen receptor (ER) status, i.e., ER-negative infiltrating ductal carcinoma, ER-positive infiltrating ductal carcinoma, and ER-positive infiltrating lobular carcinoma. We observed a consistent association between loss in regions of 5q and ER-negative infiltrating ductal carcinoma, as well as more frequent loss in 4p16, 8p23, 8p21, 10q25, and 17p11.2 in ER-negative infiltrating ductal carcinoma compared with ER-positive infiltrating ductal carcinoma (adjusted P values ≤ 0.05). We also observed high-level amplifications in ER-negative infiltrating ductal carcinoma in regions of 8q24 and 17q12 encompassing the c-myc and c-erbB-2 genes and apparent homozygous deletions in 3p21, 5q33, 8p23, 8p21, 9q34, 16q24, and 19q13. ER-positive infiltrating ductal carcinoma showed a higher frequency of gain in 16p13 and loss in 16q21 than ER-negative infiltrating ductal carcinoma. Correlation analysis highlighted regions of change commonly seen together in ER-negative infiltrating ductal carcinoma. ER-positive infiltrating lobular carcinoma differed from ER-positive infiltrating ductal carcinoma in the frequency of gain in 1q and loss in 11q and showed high-level amplifications in 1q32, 8p23, 11q13, and 11q14. These results indicate that array comparative genomic hybridization can identify significant differences in the genomic alterations between subtypes of breast cancer.

The mouse V1R putative pheromone receptor gene family consists of at least 137 intact genes clustered at multiple chromosomal locations in the genome. Species-specific pheromone receptor repertoires may partly explain species-specific social behavior. We conducted a genomic analysis of an orthologous pair of mouse and rat V1R gene clusters to test for species specificity in rodent pheromone systems. Mouse and rat have lineage-specific V1R repertoires in each of three major subfamilies at these loci as a result of postspeciation duplications, gene loss, and gene conversions. The onset of this diversification roughly coincides with a wave of Line1 (L1) retrotranspositions into the two loci. We propose that L1 activity has facilitated postspeciation V1R duplications and gene conversions. In addition, we find extensive homology among putative V1R promoter regions in both species. We propose a regulatory model in which promoter homogenization could ensure that V1R genes are equally competitive for a limiting transcriptional structure to account for mutually exclusive V1R expression in vomeronasal neurons.

Human cytogenetics was born in 1956 with the fundamental, but empowering, discovery that normal human cells contain 46 chromosomes. Since then, this field and our understanding of the link between chromosomal defects and disease have grown in spurts that have been fuelled by advances in cytogenetic technology. As a mature enterprise, cytogenetics now informs human genomics, disease and cancer genetics, chromosome evolution and the relationship of nuclear structure to function.

In this paper, we demonstrate the use of synthetic polyamide probes to fluorescently label heterochromatic regions on human chromosomes for discrimination in cytogenetic preparations and by flow cytometry. Polyamides bind to the minor groove of DNA in a sequence-specific manner. Unlike conventional sequence-specific DNA or RNA probes, polyamides can recognize their target sequence without the need to subject chromosomes to harsh denaturing conditions. For this study, we designed and synthesized a polyamide to target the TTCCA-motif repeated in the heterochromatic regions of chromosome 9, Y and 1. We demonstrate that the fluorescently labeled polyamide binds to its target sequence in both conventional cytogenetic preparations of metaphase chromosomes and suspended chromosomes without denaturation. Chromosomes 9 and Y can be discriminated and purified by flow sorting on the basis of polyamide binding and Hoechst 33258 staining. We generate chromosome 9- and Y-specific 'paints' from the sorted fractions. We demonstrate the utility of this technology by characterizing the sequence of an olfactory receptor gene that is duplicated on multiple chromosomes. By separating chromosome 9 from chromosomes 10-12 on the basis of polyamide fluorescence, we determine and differentiate the haplotypes of the highly similar copies of this gene on chromosomes 9 and 11.

Gene amplification is a common form of genomic instability in a wide variety of organisms and is often associated with tumor progression in mammals. One striking feature of many amplified genes is their organization as large inverted duplications (palindromes). Here, we describe a molecular mechanism for palindrome formation in mammalian cells that is also conserved in protists. We introduced a short (79 or 229 bp) inverted repeat into the genome of Chinese hamster ovary cells and showed that it promoted the formation of a large DNA palindrome after an adjacent DNA double-strand break. This finding suggests that short inverted repeats in the mammalian genome can have a critical role in the initiation of gene amplification. This specific mechanism may provide a novel target for cancer therapies.

We report a comprehensive comparative analysis of human and mouse olfactory receptor (OR) genes. The OR family is the largest mammalian gene family known. We identify approximately 93% of an estimated 1500 mouse ORs, exceeding previous estimates and the number of human ORs by 50%. Only 20% are pseudogenes, giving a functional OR repertoire in mice that is three times larger than that of human. The proteins encoded by intact human ORs are less highly conserved than those of mouse, in patterns that suggest that even some apparently intact human OR genes may encode non-functional proteins. Mouse ORs are clustered in 46 genomic locations, compared to a much more dispersed pattern in human. We find orthologous clusters at syntenic human locations for most mouse genes, indicating that most OR gene clusters predate primate-rodent divergence. However, many recent local OR duplications in both genomes obscure one-to-one orthologous relationships, thereby complicating cross-species inferences about OR-ligand interactions. Local duplications are the major force shaping the gene family. Recent interchromosomal duplications of ORs have also occurred, but much more frequently in human than in mouse. In addition to clarifying the evolutionary forces shaping this gene family, our study provides the basis for functional studies of the transcriptional regulation and ligand-binding capabilities of the OR gene family.

Subtelomeres are extraordinarily dynamic and variable regions near the ends of chromosomes. They are defined by their unusual structure: patchworks of blocks that are duplicated near the ends of multiple chromosomes. Duplications among subtelomeres have spawned small gene families, making inter-individual variation in subtelomeres a potential source of phenotypic diversity. The ectopic recombination that occurs between subtelomeres might also have a role in reconstituting telomeres in the absence of telomerase. However, the propensity for subtelomeres to interchange is a double-edged sword, as extensive subtelomeric homology can mediate deleterious rearrangements of the ends of chromosomes to cause human disease.

Chemosensory systems are among the oldest forms of communication between organisms and their environment. Throughout evolution, chemosensory receptor repertoires have undergone extensive diversification. Expansion and contraction of olfactory receptor (OR) gene families, recombination, gene conversion, translocation, and positive selection for functional change (Ben-Arie et al. 1993; Ngai et al. 1993; Glusman et al. 1996; Trask et al. 1998a; Sharon et al. 1999) are all hallmarks of a rapidly evolving olfactory subgenome. This propensity for change in OR repertoires may reflect the biological demands for adaptation to narrow, species-specific niches. The OR gene family is the largest gene family in mammalian genomes, with approximately 1000 genes arrayed in clusters at multiple chromosomal locations (Buck and Axel 1991; Trask et al. 1998b; Mombaerts 1999; Glusman et al. 2001). In mammalian olfactory systems, the internal representation of the complex odorant world is accomplished largely by virtue of one fundamental organizing principle: Each neuron that binds odorants is dedicated to a single allele of a single receptor gene (Chess et al. 1994). Thus, odor quality is encoded by discrete patterns of neuronal activity that result from the specific subset of ORs stimulated by an odorant or odorant mixture (Vassar et al. 1993, 1994). The transcriptional mechanisms responsible for ensuring that only a single OR gene is expressed per neuron are unknown. Transgenic experiments have shown that ∼3 kb surrounding an OR gene is sufficient to achieve normal expression patterns (Qasba and Reed 1998), yet comparative analyses of paralogous genes in three human and two mouse OR clusters have failed to reveal significant conservation in putative regulatory regions (Bulger et al. 2000; Sosinsky et al. 2000; Lane et al. 2001). The striking similarities between the olfactory and immune systems have provoked speculation that the two systems might use a common regulatory strategy. Both systems achieve recognition of a vast array of ligands by dedicating each ligand-binding cell to a single receptor allele, which is selectively expressed from a large genomic repertoire. In the immune system, selective receptor expression is accomplished by DNA recombination at both the TCR and immunoglobulin loci. This strategy generates receptor diversity and permits adaptability and heritability in antigen-recognizing cells. Programmed DNA editing has emerged recurrently in evolution as a viable developmental strategy for gene control (e.g., Gierl et al. 1989; Klar 1990; Muller et al. 1991; Haselkorn 1992; Prescott 1992) and is an appealing model for regulation in the olfactory system. Recombination could ensure singular gene transcription in olfactory neurons and long-term commitment of basal cells responsible for regenerating the olfactory neuroepithelium. The apparent lack of extensive promoter homology among OR genes might be explained if OR transcription requires recombination into an active locus (or loci). The observation that recombination-activating genes (RAG), key components of the recombination mechanisms of the immune system, are expressed in the olfactory neurons of two different vertebrate species (Jessen et al. 1999, 2001) lends further credence to this model. In this paper, we provide a comparative genomic analysis of the mouse and human OR clusters that are found 5′ to the TCR genes in both species. We identify orthologous relationships, characterize recent gene block duplication events, and describe paralogous ORs that appear to be subject to strong selection to be maintained as highly similar pairs. We have used 5′ RACE-PCR to identify transcription start sites and find that orthologs share extensive noncoding homology largely contained within the transcriptional unit. Our analysis reveals no strong sequence conservation, TATA-boxes, or conserved transcription initiator sites in OR promoter regions. We identify a functional TCR V-α gene segment (Vα1), which is significantly diverged from the other V-α segments and uses a recombination signal sequence (RSS) that differs markedly from consensus RSSs. Therefore, we were curious if the divergent RSS of Vα1 might be a relic of an ancestral recombination system, perhaps one used by surrounding OR genes. However, no Vα1-like RSSs are apparent near the OR genes. Thus, we find no evidence to support the hypothesis that RAG-mediated recombination plays a direct role in OR regulation.

In mammals, the vomeronasal organ (VNO) contains chemosensory receptor cells that bind to pheromones and induce a variety of social and reproductive behaviors. It has been traditionally assumed that the human VNO (Jacobson's organ) is a vestigial structure, although recent studies have shown minor evidence for a structurally intact and possibly functional VNO. The presence and function of the human VNO remains controversial, however, as pheromones and VNO receptors have not been well characterized. In this study we screened a human Bacterial Artificial Chromosome (BAC) library with multiple primer sets designed from human cDNA sequences homologous to mouse VNO receptor genes. Utilizing these BAC sequences in addition to mouse VNO receptor sequences, we screened the High Throughput Genome Sequence (HTGS) database to find additional human putative VNO receptor genes. We report the identification of 56 BACs carrying 34 distinct putative VNO receptor gene sequences, all of which appear to be pseudogenes. Sequence analysis indicates substantial homology to mouse V1R and V2R VNO receptor families. Furthermore, chromosomal localization via FISH analysis and RH mapping reveal that the majority of the BACs are localized to telomeric and centromeric chromosomal localizations and may have arisen through duplication events. These data yield insight into the present state of pheromonal olfaction in humans and into the evolutionary history of human VNO receptors. (Sequence data from this article have been deposited with the DDBJ/EMBL/Genbank Data Libraries under accession nos. AF305393-305416.)

Olfactory receptor (OR) genes represent approximately 1% of genomic coding sequence in mammals, and these genes are clustered on multiple chromosomes in both the mouse and human genomes. We have taken a comparative genomics approach to identify features that may be involved in the dynamic evolution of this gene family and in the transcriptional control that results in a single OR gene expressed per olfactory neuron. We sequenced approximately 350 kb of the murine P2 OR cluster and used synteny, gene linkage, and phylogenetic analysis to identify and sequence approximately 111 kb of an orthologous cluster in the human genome. In total, 18 mouse and 8 human OR genes were identified, including 7 orthologs that appear to be functional in both species. Noncoding homology is evident between orthologs and generally is confined within the transcriptional unit. We find no evidence for common regulatory features shared among paralogs, and promoter regions generally do not contain strong promoter motifs. We discuss these observations, as well as OR clustering, in the context of evolutionary expansion and transcriptional regulation of OR repertoires.

A main goal of the Human Genome Project (HGP) is to provide the complete and accurate reference sequence of the euchromatic portions of all human chromosomes (Collins et al. 1998). It has been argued that this endeavor differs from previously sequenced invertebrate models not only in terms of scale but also in terms of repetitive complexity (Green 1997; Eichler 1998). Repetitive complexity leads to misassignment and misassembly of sequence. It has been suggested that segmental duplications may be particularly problematic in this regard because of their inconspicuousness, large size, and high degree of sequence similarity. The inability to identify such duplications, let alone differentiate their true position from paralogous positions, may confound sequence assembly, resulting in merging of distinct loci into the same sequence (Eichler 1998). Segmental duplications are duplicated blocks of genomic DNA typically ranging in size from 1–200 kb (IHGSC 2001). They often contain sequence features such as high-copy repeats and gene sequences with intron–exon structure. Thus, being composed of apparently normal genomic DNA, segmental duplications cannot be detected a priori; rather, most segmental duplications have to date been discovered based on experimental analyses. Over the past decade a large number of both intra- and interchromosomal segmental duplications have been observed (Wong et al. 1990; Tomlinson et al. 1994; Eichler et al. 1997; Mazzarella and Schlessinger 1997; Regnier et al. 1997; Zimonjic et al. 1997; Eichler 1998; Trask et al. 1998a; Jackson et al. 1999; Ji et al. 1999). These data suggest numerous interchromosomal exchanges during recent hominoid evolution with apparent biases into and between pericentromeric and subtelomeric regions (Eichler et al. 1997, 1999; Monfouilloux et al. 1998; Trask et al. 1998a; Jackson et al. 1999; Horvath et al. 2000a). To date, however, no systematic analysis of the genome has been performed to quantify this bias. Another unanticipated finding has been the important role segmental duplications play in disease (for review, see Ji et al. 2000; Mazzarella and Schlessinger 1998). Aberrant homologous recombination between highly similar paralogs appears to be a major mechanism for many genomic disorders such as velocardiofacial/DiGeorge, Smith-Magenis, and Prader-Willi/Angelman syndromes (Chen et al. 1997; Amos-Landgraf et al. 1999; Christian et al. 1999; Edelmann et al. 1999; Shaikh et al. 2000). A major step toward developing a final reference sequence has been the completion of the draft-sequencing phase of the HGP and its subsequent assembly. The assembly has occurred in three main steps: (1) Sequenced clones are placed into fingerprint contigs generated from the entire RPCI-11 BAC library; (2) fingerprint contigs are assigned and positioned to chromosomes using all available genetic and STS markers; and (3) the sequence within each contig is assembled by Jim Kent's Gigassembler (IHGMC 2001; IHGSC 2001). This landmark achievement has given us the ability to examine segmental duplications in a genome-wide and systematic manner. We reported an unprecedented amount (3.6%) of sequence was involved in recent segmental duplications with identity between 90%–98%. Additionally we provided examples of pericentromeric and subtelomeric regions that appear to be composed almost entirely of duplicated sequence (IHGSC 2001). However, further characterization of highly duplicated regions has yet to be accomplished. In this article, we present our methodology for the analysis of such duplications and an in-depth analysis of segmental duplications in the current working draft assembly (January 2001, oo23 assembly), paying particular attention to the quality of assignment and assembly for the duplication-rich clones and regions. Because of the estimated error rates of sequence and the potential for misassembly in the draft assembly, we consider two categories of duplications: segments with >98% nucleotide identity, and segments with 90%–98% identity. For the first time, we quantify the genome-wide enrichment of duplicated sequence in both pericentromeric and subtelomeric regions. In addition, we examine more specifically the impact of these segments on the current assembly. We find duplicated sequences are enriched in sequence contigs that have not been mapped within the current assembly. We also find that clones containing duplications are often assigned to a chromosome inconsistent with FISH and only ∼50% of the chromosomes with FISH signals from these clones have a corresponding sequence similarity by BLAST analysis. This underrepresentation may be attributable to many factors: misassignment, merging, or reduced coverage in these paralogous regions. Taken together, the clustering of duplications combined with the difficulty in positioning and assembling them, suggests that large tracts of segmental duplications, particularly those located at pericentromeres, will be refractory to currently employed assembly methods. Specialized methods will be necessary to correctly integrate these regions into the reference human genome sequence. We propose that the determination of whether an observed overlap is allelic or paralogous will facilitate the final assembly of the human genome, helping to eliminate many gaps both within paralogous as well as unique sequence regions.

The human genome is by far the largest genome to be sequenced, and its size and complexity present many challenges for sequence assembly. The International Human Genome Sequencing Consortium constructed a map of the whole genome to enable the selection of clones for sequencing and for the accurate assembly of the genome sequence. Here we report the construction of the whole-genome bacterial artificial chromosome (BAC) map and its integration with previous landmark maps and information from mapping efforts focused on specific chromosomal regions. We also describe the integration of sequence data with the map.

The olfactory sensory system allows animals to recognize odorants and respond with appropriate behavior. In mammals, the sense of smell is mediated by at least two anatomically distinct sensory organs: the main olfactory epithelium (MOE) and the vomeronasal organ (VNO) (Bargmann 1997). Ablation of the VNO in rodents interferes with the perception of pheromones, chemicals communicated between animals of the same species that convey information primarily regarding reproductive and social status (Wysocki and Lepri 1991). In mammals, the olfactory receptors (ORs) of the MOE are encoded by a large family of ∼500–1000 receptor genes belonging to the superfamily of seven-transmembrane-domain G-protein-coupled receptors. Two multigene families of G-protein receptors, named V1Rs and V2Rs, have been recently identified in VNO neurons of the rat (Dulac and Axel 1995; Herrada and Dulac 1997; Ryba and Tirindelli 1997) and mouse (Matsunami and Buck 1997). V1Rs are restricted to Gαi2-expressing neurons, and V2Rs are restricted to Gα0-expressing neurons in the VNO. On the basis of their structure and tissue distribution, these receptors are likely mediators of the response to pheromones. V1R and V2R constitute novel families of seven-transmembrane-domain receptors with no sequence similarity with the ORs. The rat V1R family is estimated to contain 30–40 genes that share 50%–90% sequence identity but show no significant homology to other known proteins. So far, no pseudogene has been found among the seven V1R sequences examined from the rat genome (Dulac and Axel 1995). The V2Rs contain a large extracellular N-terminal domain and share similarity to extracellular calcium-sensing receptors and metabotropic glutamate receptors. The rat genome is estimated to contain ∼100 V2R-like genes, including a large number of pseudogenes (Herrada and Dulac 1997; Matsunami and Buck 1997). It is not known if humans possess a functional VNO-mediated sensory system. Even the existence of a human VNO has been controversial. There is anatomical evidence for a foetal VNO in humans, but the VNO is typically described as an atrophied organ of vestigial function in the adult (Tirindelli et al. 1998). However, several groups have recently reported finding a structurally intact VNO (Garcia-Velasco and Mondragon 1991; Moran et al. 1991; Stensaas et al. 1991). Human pheromones have not been clearly identified. A recent study concluded that synchronization of menstrual cycles among women living together involves chemical signals, but it has not yet been demonstrated that this communication is mediated through the VNO (McClintock 1971; Stern and McClintock 1998). We have taken a genomic-based approach to explore the possibility that humans possess functional V1R pheromone receptors. We have determined the sequence and chromosomal locations of a number of human V1R-like sequences. Our results shed some light on the evolution of this gene family and on this form of chemical communication in humans.

In pairwise end sequencing, sequences are determined from both ends of random subclones derived from a DNA target. Sufficiently similar overlapping end sequences are identified and grouped into contigs. When a clone's paired end sequences fall in different contigs, the contigs are connected together to form scaffolds. Increasingly, the goals of pairwise strategies are large and highly repetitive genomic targets. Here, we consider large-scale pairwise strategies that employ mixtures of subclone sizes. We explore the properties of scaffold formation within a hybrid theory/simulation mathematical model of a genomic target that contains many repeat families. Using this model, we evaluate problems that may arise, such as falsely linked end sequences (due either to random matches or to homologous repeats) and scaffolds that terminate without extending the full length of the target. We illustrate our model with an exploration of a strategy for sequencing the human genome. Our results show that, for a strategy that generates 10-fold sequence coverage derived from the ends of clones ranging in length from 2 to 150 kb, using an appropriate rule for detecting overlaps, we expect few false links while obtaining a single scaffold extending the length of each chromosome.

DNA sequence amplification is one of the most frequent manifestations of genomic instability in human tumors. We have shown previously that amplification of the dihydrofolate reductase (DHFR) gene in Chinese hamster cells is initiated by chromosome breaks, followed by bridge-breakage-fusion cycles that generate large intrachromosomal repeats; these are ultimately trimmed by an unknown process to smaller, more homogenous units manifested as homogenously staining chromosome regions (HSRs). However, in most human tumor cells, amplified DNA sequences are borne on unstable, extrachromosomal double minutes (DMs), which suggests the operation of a different amplification mechanism. In this study, we have isolated a large number of independent methotrexate-resistant human cell lines, all of which contained DHFR-bearing DMs. Surprisingly, all but one of these also had suffered partial or complete loss of one of the parental DHFR-bearing chromosomes. Cells in a few populations displayed what could be transient intermediates in the amplification process, including an initial HSR, its subsequent breakage, the appearance of DHFR-containing fragments, and, finally, DMs. Our studies suggest that HSRs and DMs both are initiated by chromosome breaks, but that cell types differ in how the extra sequences ultimately are processed and/or maintained.

Ionizing radiation produces many chromosome aberrations. A rich variety of aberration types can now be seen with the technique of chromosome painting. Apart from being important in medicine and public health, radiation-produced aberrations act as colourful molecular clues to damage-processing mechanisms and, because juxtaposition of different parts of the genome is involved, to interphase nuclear organization. Recent studies using chromosome painting have helped to identify DNA double-strand-break repair and misrepair pathways, to determine the extent of chromosome territories and motions, and to characterize different aberration patterns left behind by different kinds of radiation.

The BAC-end, or sequence-tagged-connector (STC), approach was proposed in 1996 as a new strategy for large-scale sequencing (Venter et al. 1996). The BAC-end sequencing strategy offers a potential solution to the growing disparity between the availability of sequence-ready maps of minimally overlapping clones and the community’s high-throughput sequencing capacity. In this scheme (Fig. ​(Fig.1),1), 400–500 bp of sequence is determined at the ends of the inserts of a large collection of BAC clones. As a result, sequence tags are randomly scattered across the genome. These BAC-end sequences, referred to as STCs, can be used to identify a minimum tiling path of BACs by computational procedures. Any “nucleation” sequence (the sequence of an entire BAC) can be compared electronically to the database of STCs to identify the next clones to be sequenced to maximally extend the contig. Groups at The Institute for Genomic Research (TIGR) and the University of Washington are currently collecting 900,000 STCs, representing the ends of >450,000 BACs. These clones are also being fingerprinted with a single restriction-enzyme digestion. To increase the likelihood that all regions of the genome are represented in this collection, the BACs are derived from at least two libraries created by digestion of the genome with different restriction enzymes. Figure 1 The STC resource and the STC sequencing strategy, as proposed by Venter et al. (1996). At its completion, this effort will characterize sequences at an unprecedented density—a sequence read every 3.3 kb on average—across the human genome. At this STC density, any nucleation BAC (with an average insert size of 150 kb) will contain ∼45 STCs. The 45 BACs containing these STCs will be oriented 5′ to 3′ and aligned across the nucleation sequence by computer. The BACs minimally overlapping the 5′ and 3′ ends of the nucleation sequence are candidates for the next sequence extension. On average, 22 clones extend 3′ of the nucleation sequence and 22 clones extend 5′; hence, the average overlap in a particular direction will be ∼7 kb. Because the pairs of end sequences are used to identify and orient overlapping BACs, the STC strategy is akin to other implementations of double-ended strategies for sequence assembly and scaffolding (e.g., Edwards et al. 1990; Edwards and Caskey 1991; Chen et al. 1993; Smith et al. 1994; Richards et al. 1994; Roach et al. 1995; Weber and Myers 1997). The STC strategy combines a means to identify a BAC tiling path with a random shotgun approach to sequencing. The BAC tiling path permits compartmentalization of the sequencing and thus overcomes the drawbacks of whole-genome random shotgunning described by Green (1997). Thus, by using this centralized STC resource, contiguous human sequence could be obtained in distributed laboratories without the need for separate dedicated “mapping factories” (e.g., Wong et al. 1997). One issue regarding this strategy is its cost. Intuitively, with more STCs scattered in the genome, one can identify clones with smaller average overlap with previously sequenced regions. This minimization of overlap leads to fewer clones completely sequenced and, hence, lower overall cost. But how do we trade-off the cost of establishing the STC resource of increasing density against the cost of sequencing an entire clone? A second issue is the impact of diverse repeats in the genome on the success of the strategy. The human genome is riddled with repeated sequence elements, ranging from the small, but very frequent, Alu elements to a variety of large, low-copy duplications. Clearly, STCs entirely contained within a repeat element can connect noncontiguous regions of the genome. Additional precautions, such as monitoring discrepancies among the restriction-digest fingerprints of overlapping BACs or checking chromosomal locations by fluorescence in situ hybridization (FISH), must be implemented to break these false connections. In this paper we develop a tractable mathematical model to derive properties of the STC sequencing strategy. This work complements analytical and simulation models presented previously on related problems of large-scale mapping and sequencing (Roach et al. 1995; Myers and Weber 1997; Siegel et al. 1998b). Using this model, various parameters—such as the number of end sequences determined, the average insert size, the costs of various steps in the procedure—can be varied to assess the effect on overall costs or success of the approach. Success can be measured in terms of problem clones, from which continuation is not possible, owing either to the lack of identified matches in the database or to the possibility that a falsely matching STC will lead to sequencing of a nonoverlapping clone. Here is an outline of the model. The target, for example, a model of the human genome, consists of bases independently chosen from a given nucleotide distribution. Clones of fixed length are assumed to be located independently and uniformly at random over the target. STCs at each clone end are sequenced with a specified error rate. A different, much lower error rate is used for completely sequenced clones, because this sequence is typically assembled from 8- to 10-fold overlapping sequence reads (i.e., a redundant shotgun approach), and each base is determined as the highest quality read from these overlapping sequences. A variety of repeat families is defined in the model. Each family is defined by its number of copies, the segment size, and percent similarity. We use a tractable, conservative decision rule for declaring a match between an STC and a sequenced clone. This decision rule is based on the number of matching bases found when comparing an STC aligned to a subregion of a sequenced clone. The problem-clone rate is derived, and the overall sequencing cost is estimated. Library parameters may then be changed to study their effect on the problem rate and on the overall cost of sequencing the target. The model can identify the optimum-cost parameters for which the incremental cost of increasing the STC resource reaches the point of diminishing returns, matching the corresponding decrease in the average cost of clone sequencing owing to smaller overlaps. Box 1 presents our notation and assumptions in detail followed by an outline of the calculations for the expected number of problem clones (indicating the extent to which difficulties will be encountered while selecting successive clones to be sequenced in their entirety) and for the expected overlap for true STC extensions (indicating the overall extra cost owing to redundant sequencing, because with larger expected overlap, more clones will need to be sequenced to cover the target). Results for sequencing on the scale of the human genome then follow, using a variety of library sizes, clone lengths, and decision-rule criteria to identify low-cost strategies.

The 18q - syndrome is representative of a group of terminal deficiency or macrodeletion syndromes characterized by mental retardation and congenital malformations. To gain insight into the mechanism of chromosomal loss and stabilization in these disorders, we cloned a putative terminal deletion breakpoint from an 18q- syndrome patient. The 18q21.3 breakpoint occurred between two nearly identical serine protease inhibitor (serpin) genes, SCCA1 and SCCA2. Although cytogenetic studies suggested that this chromosomal aberration was formed by a simple terminal deletion, DNA sequence analysis, pulsed-field gel electrophoresis and fluorescence in situ hybridization showed that the breakpoint was contiguous with a 35 bp filler sequence followed by a satellite III DNA-containing telomeric fragment of 475-1000 kb. This type of satellite III DNA sequence was not detected on the normal chromosome 18, but was highly homologous with types of satellite III DNA sequences normally located on the short arms (p11) of the acrocentric chromosomes and other heterochromatic regions. This DNA sequence analysis suggested that the terminal deficiency in this 18q - syndrome patient arose via illegitimate (non-homologous) recombination. Moreover, these data raise the possibility that a subset of chromosomal aberrations appearing cytogenetically and molecularly as simple terminal truncations or deletions are caused by small (

Williams syndrome (WS) is a contiguous gene deletion disorder caused by haploinsufficiency of genes at 7q11.23. We have shown that hemizygosity of elastin is responsible for one feature of WS, supravalvular aortic stenosis (SVAS). We have also implicated LIM-kinase 1 hemizygosity as a contributing factor to impaired visual-spatial constructive cognition in WS. However, the common WS deletion region has not been completely characterized, and genes for additional features of WS, including mental retardation, infantile hypercalcemia, and unique personality profile, are yet to be discovered. Here, we present a physical map encompassing 1.5 Mb DNA that is commonly deleted in individuals with WS. Fluorescence in situ hybridization analysis of 200 WS individuals shows that WS individuals have the consistent deletion interval. In addition, we identify three novel genes from the common deletion region: WS-betaTRP, WS-bHLH, and BCL7B. WS-betaTRP has four putative beta-transducin (WD40) repeats, and WS-bHLH is a novel basic helix-loop-helix leucine zipper (bHLHZip) gene. BCL7B belongs to a novel family of highly conserved genes. We describe the expression profile and genomic structure for each of these genes. Hemizygous deletion of one or more of these genes may contribute to developmental defects in WS.

p53 is a tumor suppressor protein that controls cell proliferation by regulating the expression of growth control genes. In a previous study, we identified two proteins, 53BP1 and 53BP2, that are able to bind to wild type but not to mutant p53 via the DNA-binding domain of p53. We isolated cDNAs expressing a full-length human 53BP1 clone, which predicts a protein of 1972 residues that can be detected in the H358 human lung carcinoma cell line. The 53BP1 and 53BP2 genes were mapped to chromosomes 15q15-21 and 1q41-42, respectively. Immunofluorescence studies showed three types of staining patterns for 53BP1 as follows: both cytoplasmic and nuclear, homogeneous nuclear, and a nuclear dot pattern. In contrast, 53BP2 localized exclusively to the cytoplasm, and this pattern did not change upon coexpression of wild type p53. Although our previous study revealed that p53 is not able to bind simultaneously to either 53BP1 or 53BP2 and to DNA carrying a consensus binding site, both 53BP1 and 53BP2 enhanced p53-mediated transcriptional activation and induced the expression of a p53-dependent protein, suggesting that these proteins might function in signal transduction pathways to promote p53 activity.

X-linked spinal and bulbar muscular atrophy (SBMA) is caused by a CAG repeat expansion in the first exon of the androgen receptor (AR) gene. Disease-associated alleles (37-66 CAGs) change in length when transmitted from parents to offspring, with a significantly greater tendency to shift size when inherited paternally. As transgenic mice carrying human AR cDNAs with 45 and 66 CAG repeats do not display repeat instability, we attempted to model trinucleotide repeat instability by generating transgenic mice with yeast artificial chromosomes (YACs) carrying AR CAG repeat expansions in their genomic context. Studies of independent lines of AR YAC transgenic mice with CAG 45 alleles reveal intergenerational instability at an overall rate of approximately 10%. We also find that the 45 CAG repeat tracts are significantly more unstable with maternal transmission and as the transmitting mother ages. Of all the CAG/CTG repeat transgenic mice produced to date the AR YAC CAG 45 mice are unstable with the smallest trinucleotide repeat mutations, suggesting that the length threshold for repeat instability in the mouse may be lowered by including the appropriate flanking human DNA sequences. By sequence-tagged site content analysis and long range mapping we determined that one unstable transgenic line has integrated an approximately 70 kb segment of the AR locus due to fragmentation of the AR YAC. Identification of the cis -acting elements that permit CAG tract instability and the trans -acting factors that modulate repeat instability in the AR YAC CAG 45 mice may provide insights into the molecular basis of trinucleotide repeat instability in humans.

Remarkable structural and functional similarities exist between theDrosophila Toll/Cactus/Dorsal signaling pathway and the mammalian cytokine-mediated interleukin-1 receptor (IL-1R)/I-κB/NF-κB activation cascade. In addition to a role regulating dorsal-ventral polarity in the developing Drosophilaembryo, signaling through Drosophila Toll (dToll) activates the nonclonal, or innate, immune response in the adult fly. Recent evidence indicates that a human homologue of the dToll protein participates in the regulation of both innate and adaptive human immunity through the activation of NF-κB and the expression of the NF-κB–controlled genes IL-1, IL-6, and IL-8, thus affirming the evolutionary conservation of this host defense pathway. We report here the cloning of two novel human genes, TIL3 and TIL4 (Toll/IL-1R–like-3, -4) that exhibit homology to both the leucine-rich repeat extracellular domains and the IL-1R–like intracellular domains of human andDrosophila Toll. Northern analysis showed distinctly different tissue distribution patterns with TIL3 expressed predominantly in ovary, peripheral blood leukocytes, and prostate, and TIL4 expressed primarily in peripheral blood leukocytes and spleen. Chromosomal mapping by fluorescence in situ hybridization localized the TIL3 gene to chromosome 1q41-42 and TIL4 to chromosome 4q31.3-32. Functional studies showed that both TIL3 and TIL4 are able to activate NF-κB, though in a cell type–dependent fashion. Together with human Toll, TIL3 and TIL4 encode a family of genes with conserved structural and functional features involved in immune modulation.

A cDNA clone encoding the human homolog of rat Jagged1 was isolated from normal human marrow. Analyses of human stromal cell lines indicate that this gene, designated hJagged1 , is expressed by marrow stromal cells typified by the cell line HS-27a, which supports the long-term maintenance of hematopoietic progenitor cells. G-CSF–induced differentiation of 32D cells expressing Notch1 was inhibited by coculturing with HS-27a. A peptide corresponding to the Delta/Serrate/LAG-2 domain of hJagged1 and supernatants from COS cells expressing a soluble form of the extracellular portion of hJagged1 were able to mimic this effect. These observations suggest that hJagged1 may function as a ligand for Notch1 and play a role in mediating cell fate decisions during hematopoiesis.

Activation of the cascade of proteolytic caspases has been identified as the final common pathway of apoptosis in diverse biological systems. We have isolated a gene, termed MRIT , that possesses overall sequence homology to FLICE (MACH), a large prodomain caspase that links the aggregated complex of the death domain receptors of the tumor necrosis factor receptor family to downstream caspases. However, unlike FLICE, the C-terminal domain of MRIT lacks the caspase catalytic consensus sequence QAC(R/Q)G. Nonetheless MRIT activates caspase-dependent death. Using yeast two-hybrid assays, we demonstrate that MRIT associates with caspases possessing large and small prodomains (FLICE, and CPP32/YAMA), as well as with the adaptor molecule FADD. In addition, MRIT simultaneously and independently interacts with BclX L and FLICE in mammalian cells. Thus, MRIT is a mammalian protein that interacts simultaneously with both caspases and a Bcl-2 family member.

Alagille syndrome is an autosomal dominant disorder characterized by abnormal development of liver, heart, skeleton, eye, face and, less frequently, kidney. Analyses of many patients with cytogenetic deletions or rearrangements have mapped the gene to chromosome 20p12, although deletions are found in a relatively small proportion of patients (< 7%). We have mapped the human Jagged1 gene (JAG1), encoding a ligand for the developmentally important Notch transmembrane receptor, to the Alagille syndrome critical region within 20p12. The Notch intercellular signalling pathway has been shown to mediate cell fate decisions during development in invertebrates and vertebrates. We demonstrate four distinct coding mutations in JAG1 from four Alagille syndrome families, providing evidence that it is the causal gene for Alagille syndrome. All four mutations lie within conserved regions of the gene and cause translational frameshifts, resulting in gross alterations of the protein product Patients with cytogenetically detectable deletions including JAG1 have Alagille syndrome, supporting the hypothesis that haploinsufficiency for this gene is one of the mechanisms causing the Alagille syndrome phenotype.

The macrophage colony-stimulating factor receptor and several other hematopoietic growth factor receptors induce the tyrosine phosphorylation of a 145- to 150-kD protein in murine cells. We have previously cloned a cDNA for the murine 150-kD protein, SHIP, and found that it encodes a unique signaling intermediate that binds the SHC PTB domain through at least one tyrosine phosphorylated (NPXY) site in the carboxyl-terminal region. SHIP also contains several potential SH3 domain-binding sites, an SH2 domain for binding other tyrosine phosphorylated proteins, and an enzymatic activity that removes the phosphate from the 5 position of phosphatidylinositol 3,4,5-phosphate or from inositol 1,3,4,5-phosphate. SHIP has a negative effect on cell growth and therefore loss or modification may have profound effects on hematopoietic cell development. In this study, we have cloned a cDNA for human SHIP and examined mRNA and protein expression of SHIP and related species in bone marrow and blood cells. Flow cytometry indicates that at least 74% of immature CD34+ cells express SHIP cross-reacting protein species, whereas within the more mature population of CD33+ cells, only 10% of cells have similar expression. The majority of T cells react positively with the anti-SHIP antibodies, but significantly fewer B cells are positive. Immunoblotting detects up to seven different cross-reacting SHIP species, with peripheral blood mononuclear cells exhibiting primarily a 100-kD protein and a CD34+ acute myeloblastic leukemia expressing mainly 130-kD and 145-kD forms of SHIP. Overall, these results indicate that there is an enormous diversity in the size of SHIP or SHIP-related mRNA and protein species. Furthermore, the expression of these protein species changes according to both the developmental stage and differentiated lineage of the mature blood cell.

The large-scale structure of chromatin corresponding to G- and R-bands in human G0/G1 interphase nuclei was compared. Fluorescence in situ hybridization (FISH) was used to measure the interphase distance between 42 pairs of probes separated by 0.1–1.5Mbp. The probe pairs were derived from 21q22.2 and Xp21.3, G-band positive regions, and from 4p16.3, 6p21.3, and Xq28, R-band positive regions. Distributions of measured interphase distances in all regions approximated a Rayleigh distribution, suggesting that the chromatin follows a random-walk path over this range. A linear correlation of mean-square interphase distance and genomic separation, also indicative of random-walk folding, was observed in all regions. The slope of the correlation observed using probes from G-band regions was systematically lower than that from R-band regions. The difference in the slope between Xp21.3 and Xq28 was particularly striking and was observed in normal fibroblast cells, fixed alternatively with methanol and acetic acid or paraformaldehyde, and HeLa cells. These results demonstrate regional differences in large-scale chromosome structure during interphase, with the more openly configured chromatin corresponding to R-bands.

A yeast artificial chromosome (YAC), P1, and cosmid clone contig was constructed for the Werner syndrome (WRN) region of chromosome 8p12-p21 and used to clone a candidate gene for WRN. This region also possibly contains a familial breast cancer locus. The contig was initiated by isolating YACs for the glutathione reductase (GSR) gene and extended in either direction by walking techniques. Sequence-tagged site (STS) markers were generated from subclones of 2 GSR YACs and used to identify P1 and cosmid clones. Additional STSs were generated from P1 and cosmid clones and from potential expressed sequences identified by cDNA selection and exon amplification methods. The final contig was assembled by typing 17 YACs, 20 P1 clones, and 109 cosmids for 54 STS markers. The WRN region could be spanned by 2 nonchimeric YACs covering approximately 1.4 Mb. A P1/cosmid contig was established covering the core 700-800 kb of the WRN region. Fifteen new short tandem repeat polymorphisms and 2 biallelic polymorphic markers were identified and included as STSs in the contig. Analysis of these markers in Werner syndrome subjects demonstrates that the candidate WRN gene is in a region of linkage disequilibrium.

Split hand/split foot malformation (SHSF) has been described in several patients associated with cytogenetically visible rearrangements involving chromosome 7q. Characterisation of these patients has led to localisation of an autosomal dominant form of SHSF to 7q21-22; the locus has been designated SHFM1. We describe a patient with a complex, apparently balanced cytogenetic rearrangement, including a translocation breakpoint at 7q21.3 near the DSS1 gene. In addition to ectrodactyly of all four limbs, the patient has congenital deafness, submucous cleft palate, microcephaly, and mental retardation. This patient represents an additional case of syndromic ectrodactyly related to the SHFM1 gene region, which may be responsible for both syndromic and non-syndromic ectrodactyly.

We determined the folding of chromosomes in interphase nuclei by measuring the distance between points on the same chromosome. Over 25,000 measurements were made in G0/G1 nuclei between DNA sequences separated by 0.15-190 megabase pairs (Mbp) on three human chromosomes. The DNA sequences were specifically labeled by fluorescence in situ hybridization. The relationship between mean-square interphase distance and genomic separation has two linear phases, with a transition at approximately 2 Mbp. This biphasic relationship indicates the existence of two organizational levels at scales > 100 kbp. On one level, chromatin appears to be arranged in large loops several Mbp in size. Within each loop, chromatin is randomly folded. On the second level, specific loop-attachment sites are arranged to form a supple, backbonelike structure, which also shows characteristic random walk behavior. This random walk/giant loop model is the simplest model that fully describes the observed large-scale spatial relationships. Additional evidence for large loops comes from measurements among probes in Xq28, where interphase distance increases and then locally decreases with increasing genomic separation.