RESUMEN
Copy number variation is a defining characteristic of human subtelomeres. Human subtelomeric segmental duplication regions ('Subtelomeric Repeats') comprise about 25% of the most distal 500 kb and 80% of the most distal 100 kb in human DNA. Huge allelic disparities seen in subtelomeric DNA sequence content and organization are postulated to have an impact on the dosage of transcripts embedded within the duplicated sequences, on the transcription of genes in adjacent single copy DNA regions, and on the chromatin structures mediating telomere functions including chromosome stability. In addition to the complex duplicon substructure and huge allelic variations in extended subtelomere regions, both copy number variation and alternative sequence organizations for DNA characterize the sequences immediately adjacent to terminal (TTAGGG)n tracts ('subterminal DNA'). The structural variation in subterminal DNA is likely to have important consequences for expression of subterminal transcripts such as a newly-discovered gene family encoding actin-interacting proteins and a non-coding telomeric repeat containing RNA (TERRA) transcript family critical for telomere integrity. Major immediate challenges include discovering the full extent and nature of subtelomeric structural and copy number variation in humans, and developing methods for tracking individual allelic variants in the context of total genomic DNA.
Asunto(s)
Dosificación de Gen/genética , Telómero/genética , Secuencia de Bases , ADN Complementario/genética , Epigénesis Genética/genética , Humanos , Transcripción Genética/genéticaRESUMEN
Work towards completion of the human reference genome sequence has revealed a great deal of complexity and plasticity in human subtelomeric regions. The highly variable subtelomeric repeat regions are filled with recently shuffled genomic segments, many of which contain sequences matching transcripts and transcript fragments; the rapid duplication and combinatorial evolution of these regions has generated an extremely diverse set of subtelomeric alleles in the human species, the complexity and potential significance of which is only beginning to be understood. This review summarizes recent progress in analyzing human subtelomeric sequence assemblies and large-scale variation in human subtelomere regions.
Asunto(s)
Variación Genética/genética , Genoma Humano , Telómero/genética , Humanos , Secuencias Repetitivas Esparcidas/genética , Mapeo Físico de Cromosoma , Análisis de Secuencia de ADNRESUMEN
Telomeres are the ends of linear eukaryotic chromosomes. To ensure that no large stretches of uncharacterized DNA remain between the ends of the human working draft sequence and the ends of each chromosome, we would need to connect the sequences of the telomeres to the working draft sequence. But telomeres have an unusual DNA sequence composition and organization that makes them particularly difficult to isolate and analyse. Here we use specialized linear yeast artificial chromosome clones, each carrying a large telomere-terminal fragment of human DNA, to integrate most human telomeres with the working draft sequence. Subtelomeric sequence structure appears to vary widely, mainly as a result of large differences in subtelomeric repeat sequence abundance and organization at individual telomeres. Many subtelomeric regions appear to be gene-rich, matching both known and unknown expressed genes. This indicates that human subtelomeric regions are not simply buffers of nonfunctional 'junk DNA' next to the molecular telomere, but are instead functional parts of the expressed genome.
Asunto(s)
Genoma Humano , Telómero , Cromosomas Artificiales Bacterianos , Cromosomas Artificiales de Levadura , Proyecto Genoma Humano , HumanosRESUMEN
A 260-kb half-YAC clone derived from human chromosome 1q was mapped at high resolution using cosmid subclone fingerprint analysis and was integrated with overlapping clones from the telomeric end of a separately derived 1q44 BAC contig to create a sequence-ready map extending to the molecular telomere of 1q. Analysis of 100 kb of sample sequences from across the 260-kb region encompassed by the half-YAC revealed the presence of EST sequence matches corresponding to 12 separate Unigene clusters and to 12 separate unclustered EST sequences. Low-copy subtelomeric repeats typical of many human telomere regions are present within the distal-most 30 kb of 1q. The previously isolated and radiation hybrid-mapped markers Bda84F03, 1QTEL019, and WI11861 localized at distances approximately 32, 88, and 99 kb, respectively, from the 1q terminus. This sequence-ready map permits high-resolution integration of genetic maps with the DNA sequences directly adjacent to the tip of human chromosome 1q and will enable telomeric closure of the human chromosome 1q DNA reference sequence by connecting the molecular 1q telomere to an internal BAC contig.
Asunto(s)
Cromosomas Humanos Par 1/genética , Mapeo Contig , Análisis de Secuencia de ADN , Telómero/genética , Cromosomas Artificiales Bacterianos , Cromosomas Artificiales de Levadura , Cósmidos , Dermatoglifia del ADN , Etiquetas de Secuencia Expresada , Humanos , Datos de Secuencia MolecularRESUMEN
Telomere-specific clones are a valuable resource for the characterization of chromosomal rearrangements. We previously reported a first-generation set of human telomere probes consisting of 34 genomic clones, which were a known distance from the end of the chromosome ( approximately 300 kb), and 7 clones corresponding to the most distal markers on the integrated genetic/physical map (1p, 5p, 6p, 9p, 12p, 15q, and 20q). Subsequently, this resource has been optimized and completed: the size of the genomic clones has been expanded to a target size of 100-200 kb, which is optimal for use in genome-scanning methodologies, and additional probes for the remaining seven telomeres have been identified. For each clone we give an associated mapped sequence-tagged site and provide distances from the telomere estimated using a combination of fiberFISH, interphase FISH, sequence analysis, and radiation-hybrid mapping. This updated set of telomeric clones is an invaluable resource for clinical diagnosis and represents an important contribution to genetic and physical mapping efforts aimed at telomeric regions.
Asunto(s)
Cromosomas Humanos/genética , Sondas de ADN/genética , Telómero/genética , Cromosomas Artificiales de Levadura/genética , Cromosomas Humanos/química , Clonación Molecular , Marcadores Genéticos/genética , Humanos , Células Híbridas , Hibridación Fluorescente in Situ , Interfase , Mapeo Físico de Cromosoma , Polimorfismo Genético/genética , Análisis de Secuencia de ADN , Lugares Marcados de Secuencia , Telómero/químicaRESUMEN
Genome-wide physical and genetic mapping efforts have not yet fully addressed the problem of closure at the telomeric ends of human chromosomes. Targeted efforts at cloning human and mouse telomeres have succeeded in identifying unique sequences at most telomeres, but gap sizes between these telomere clones and the distal markers on integrated genetic/physical maps remain largely unknown. As telomeric regions are known to be the most gene-rich regions of the human genome, filling these gaps should have a high priority in completion of the Human Genome Project. We reported previously a first generation set of unique sequence probes for human telomeric regions. Of 41 human telomere regions, 33 were represented by unique clones with a known distance (1 Mb, thus defining the physical mapping task for filling telomeric gaps.
Asunto(s)
Telómero/genética , Telómero/ultraestructura , Mapeo Cromosómico/métodos , Cromosomas Bacterianos/genética , Cromosomas Humanos Par 12 , Cromosomas Humanos Par 15 , Cromosomas Humanos Par 16 , Cromosomas Humanos Par 9 , Bases de Datos Factuales , Biblioteca de Genes , Humanos , Hibridación Fluorescente in SituRESUMEN
A half-YAC clone derived from human chromosome 17p was mapped at high resolution using cosmid subclone fingerprint analysis. Colinearity of the half-YAC with the telomeric human genomic DNA fragment was ascertained by RecA-assisted restriction endonuclease cleavage mapping. Previously isolated and radiation hybrid-mapped markers TEL17P37, TEL17P49, and TEL17P80 mapped 30-60 kb from the 17p terminus. This sequence-ready map permits high-resolution integration of genetic maps with the DNA sequences directly adjacent to the tip of human chromosome 17p, and will provide the cloned DNA required for ascertaining the nucleotide sequence of this subtelomeric region.
Asunto(s)
Mapeo Cromosómico , Cromosomas Humanos Par 17 , Telómero/genética , Cromosomas Artificiales de Levadura , Mapeo Contig , Cósmidos , Endonucleasas/metabolismo , Humanos , Modelos Genéticos , Datos de Secuencia Molecular , Rec A Recombinasas/metabolismoRESUMEN
A contiguous physical map was constructed from the Harvey ras-1 (HRAS1) gene to the 11p telomere. The contig spans approximately 500 kb and is minimally composed of a telomere-containing YAC and P1 and cosmid clones. Included in the contig are 11 sequence-tagged sites derived from P1 and cosmid ends. Three genes were placed on the contig in the following order: telomere-ribonuclease/angiogenin inhibitor (RNH)-Harvey ras-1 (HRAS1)-HRAS1-related complex (HRC). Two novel tetranucleotide repeats (heterozygosity of 66 and 68%) and a complex CA repeat (heterozygosity of 78%) were isolated and characterized.
Asunto(s)
Cromosomas Humanos Par 11 , Genes ras , Telómero , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Artificiales de Levadura , Repeticiones de Dinucleótido , Tamización de Portadores Genéticos , Marcadores Genéticos , Humanos , Datos de Secuencia Molecular , Polimorfismo Genético , Técnica del ADN Polimorfo Amplificado AleatorioAsunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/genética , Cromosomas Humanos Par 2 , Genes Supresores de Tumor , Proteínas del Tejido Nervioso , Proteínas Nucleares/genética , Proteínas Supresoras de Tumor , Animales , Línea Celular , Mapeo Cromosómico , Cricetinae , Humanos , Células Híbridas , Hibridación Fluorescente in Situ , Ratones , Reacción en Cadena de la Polimerasa , RoedoresRESUMEN
Large terminal fragments of human chromosomes 2p, 6p, 8q, 12q, and 18q were cloned using yeast artificial chromosomes (YACs). RecA-assisted restriction endonuclease (RARE) cleavage analysis of genomic DNA samples from II unrelated individuals using YAC-derived probes confirmed the telomeric localizations of the half-YACs studied. The cloned fragments provide telomeric closure of maps for the respective chromosome arms and will supply the reagents needed for analyzing and sequencing these distal subtelomeric regions.
Asunto(s)
Cromosomas Humanos/genética , Telómero/genética , Secuencia de Bases , Cromosomas Artificiales de Levadura , Cromosomas Humanos/ultraestructura , Cromosomas Humanos Par 12/genética , Cromosomas Humanos Par 12/ultraestructura , Cromosomas Humanos Par 18/genética , Cromosomas Humanos Par 18/ultraestructura , Cromosomas Humanos Par 2/genética , Cromosomas Humanos Par 2/ultraestructura , Cromosomas Humanos Par 6/genética , Cromosomas Humanos Par 6/ultraestructura , Cromosomas Humanos Par 8/genética , Cromosomas Humanos Par 8/ultraestructura , Clonación Molecular , Humanos , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Mapeo RestrictivoAsunto(s)
Mapeo Cromosómico , Genes de Inmunoglobulinas , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Diversidad de Anticuerpos/genética , Cromosomas Humanos Par 14 , Cromosomas Humanos Par 15 , Cromosomas Humanos Par 16 , Bases de Datos Factuales , Haplotipos/genética , Humanos , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , TelómeroRESUMEN
Mutations in the human PAX3 gene have previously been associated with two distinct diseases, Waardenburg syndrome and alveolar rhabdomyosarcoma. In this report we establish that the normal human PAX3 gene is encoded by 8 exons. Intron-exon boundary sequences were obtained for PAX3 exons 5, 6, 7, and 8 and together with previous work provide the complete genomic sequence organization for PAX3. Difficulties in obtaining overlapping genomic clone coverage of PAX3 were circumvented in part by RARE cleavage mapping, which showed that the entire PAX3 gene spans 100 kb of chromosome 2. Sequence analysis of the last intron of PAX3, which contains the previously mapped t(2;13)(q35;q14) translocation breakpoints of alveolar rhabdomyosarcoma, revealed the presence of a pair of inverted Alu repeats and a pair of inverted (GT)n-rich microsatellite repeats within a 5-kb region. This work establishes the complete structure of PAX3 and will permit high-resolution analyses of this locus for mutations associated with Waardenburg syndrome, alveolar rhabdomyosarcoma, and other phenotypes for which PAX3 may be a candidate locus.
Asunto(s)
Cromosomas Humanos Par 2/genética , Proteínas de Unión al ADN/genética , Rabdomiosarcoma Alveolar/genética , Factores de Transcripción , Translocación Genética , Alelos , Secuencia de Bases , Mapeo Cromosómico , ADN Satélite/genética , Exones , Femenino , Frecuencia de los Genes , Heterocigoto , Humanos , Masculino , Datos de Secuencia Molecular , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box , Secuencias Repetitivas de Ácidos Nucleicos , Análisis de Secuencia de ADNRESUMEN
The most distal 300 kb of human chromosome 21q was cloned and mapped using telomeric yeast artificial chromosomes (YACs). The region contains low-copy subtelomeric repeats at the telomeric end, chromosome 21-specific sequences more centromerically, and the S100B gene at a distance of 100-140 kb from the chromosome terminus. RecA-assisted restriction endonuclease cleavage of genomic DNA showed that the cloned fragments correspond to telomere-terminal genomic DNA, and restriction enzyme mapping of the YACs shows that the smaller clone (175 kb) corresponds exactly to the telomeric end of the larger one (300 kb). PCR assays for 21q-specific markers were used to show that COL6A1, COL6A2, and LA161 were all outside of the subtelomeric region spanned by the YACs and thus at least 300 kb from the 21q terminus. The molecular probes provide telomeric closure for existing 21q maps.
Asunto(s)
Mapeo Cromosómico , Cromosomas Humanos Par 21/genética , Animales , Secuencia de Bases , Southern Blotting , Cromosomas Artificiales de Levadura , Cricetinae , Marcadores Genéticos , Humanos , Hibridación Fluorescente in Situ , Ratones , Datos de Secuencia Molecular , Mapeo Restrictivo , Lugares Marcados de Secuencia , Telómero/genéticaRESUMEN
The terminal 240 kb of a human 2q telomere region was cloned in two overlapping yeast artificial-chromosomes (YACs). This DNA contains a region of low-copy subtelomeric repeats (within 50 kb of the 2q telomere), a segment of DNA duplicated on distal 8p23 (100 kb from the 2q telomere), and a region of single-copy DNA (230 kb from the 2q telomere). Two CpG islands are present in the DNA segment duplicated on distal 8p23. RecA-assisted restriction endonuclease cleavage of genomic DNA samples revealed a potential 55 kb chromosome length polymorphism at the 2q telomere. This work provides telomeric closure of maps for human chromosome 2q, demonstrates a novel, subtelomere-specific DNA duplication, and will permit detailed molecular and cytological studies of this human telomere region.
Asunto(s)
Cromosomas Humanos Par 2 , Secuencias Repetitivas de Ácidos Nucleicos , Telómero , Secuencia de Bases , Bandeo Cromosómico , Mapeo Cromosómico , Cromosomas Artificiales de Levadura , Cartilla de ADN , Humanos , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Mapeo RestrictivoRESUMEN
DNA from three 1q44-derived human telomeric yeast artificial chromosome clones was analyzed using physical mapping methods. The smallest clone, yRM2004 (65 kb), corresponded exactly to the distal end of the largest clone, yRM2123 (270 kb). The third clone, yRM2192, overlapped with the proximal end of yRM2123 but not the distal end, suggesting that it is most likely a deletion artifact of a clone originally derived from a 1q telomere fragment. Data from fluorescence in situ hybridization analysis, restriction mapping, and RecA-assisted restriction enzyme cleavage experiments indicate that the molecular clone yRM2123 contains a 260-kb DNA fragment colinear with a genomic telomere-terminal fragment from 1q. yRM2123 contains low-copy subtelomeric and subterminal repeats at its distal end, single-copy DNA more centromerically, and a CG-rich region with homology to mouse DNA. Markers derived from this clone will allow telomeric closure of the physical and genetic linkage maps of human chromosome 1q.
Asunto(s)
Cromosomas Humanos Par 1 , ADN/genética , Animales , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Artificiales de Levadura , Cromosomas Humanos Par 1/ultraestructura , Clonación Molecular , Cartilla de ADN/genética , Ligamiento Genético , Humanos , Hibridación Fluorescente in Situ , Ratones , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/genética , Reacción en Cadena de la Polimerasa , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Ácido Nucleico , Especificidad de la Especie , Telómero/ultraestructuraRESUMEN
Most patients who present with the 18q- syndrome have an apparent terminal deletion of the long arm of chromosome 18. For precise phenotypic mapping of this syndrome, it is important to determine whether the deletions are terminal deletions or interstitial deletions. A human telomeric YAC clone has been identified that hybridizes specifically to the telomeric end of 18q. This clone was characterized and used to analyze seven patients with 18q deletions. By FISH and Southern blotting analysis, all patients were found to lack this chromosomal region on their deleted chromosome, demonstrating that the patients do not have cryptic interstitial deletions.