RESUMEN
Centromeres are sites for assembly of the chromosomal structures that mediate faithful segregation at mitosis and meiosis. Plant and animal centromeres are typically located in megabase-sized arrays of tandem satellite repeats, making their precise mapping difficult. However, some rice centromeres are largely embedded in nonsatellite DNA, providing an excellent model to study centromere structure and evolution. We used chromatin immunoprecipitation and 454 sequencing to define the boundaries of nine of the 12 centromeres of rice. Centromere regions from chromosomes 8 and 9 were found to share synteny, most likely reflecting an ancient genome duplication. For four centromeres, we mapped discrete subdomains of binding by the centromeric histone variant CENH3. These subdomains were depleted in both intact and nonfunctional genes relative to interspersed subdomains lacking CENH3. The intergenic location of rice centromeric chromatin resembles the situation for human neocentromeres and supports a model of the evolution of centromeres from gene-poor regions.
Asunto(s)
Centrómero/genética , Cromatina/genética , Mapeo Cromosómico , Histonas/metabolismo , Oryza/genética , Secuencia de Bases , Sitios de Unión , Centrómero/metabolismo , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Cromosomas de las Plantas , Duplicación de GenRESUMEN
Chromosome 5 is one of the largest human chromosomes and contains numerous intrachromosomal duplications, yet it has one of the lowest gene densities. This is partially explained by numerous gene-poor regions that display a remarkable degree of noncoding conservation with non-mammalian vertebrates, suggesting that they are functionally constrained. In total, we compiled 177.7 million base pairs of highly accurate finished sequence containing 923 manually curated protein-coding genes including the protocadherin and interleukin gene families. We also completely sequenced versions of the large chromosome-5-specific internal duplications. These duplications are very recent evolutionary events and probably have a mechanistic role in human physiological variation, as deletions in these regions are the cause of debilitating disorders including spinal muscular atrophy.
Asunto(s)
Cromosomas Humanos Par 5/genética , Análisis de Secuencia de ADN , Animales , Composición de Base , Cadherinas/genética , Secuencia Conservada/genética , Duplicación de Gen , Genes/genética , Enfermedades Genéticas Congénitas/genética , Genómica , Humanos , Interleucinas/genética , Datos de Secuencia Molecular , Atrofia Muscular Espinal/genética , Pan troglodytes/genética , Mapeo Físico de Cromosoma , Seudogenes/genética , Sintenía/genética , Vertebrados/genéticaRESUMEN
Chromosome 19 has the highest gene density of all human chromosomes, more than double the genome-wide average. The large clustered gene families, corresponding high G + C content, CpG islands and density of repetitive DNA indicate a chromosome rich in biological and evolutionary significance. Here we describe 55.8 million base pairs of highly accurate finished sequence representing 99.9% of the euchromatin portion of the chromosome. Manual curation of gene loci reveals 1,461 protein-coding genes and 321 pseudogenes. Among these are genes directly implicated in mendelian disorders, including familial hypercholesterolaemia and insulin-resistant diabetes. Nearly one-quarter of these genes belong to tandemly arranged families, encompassing more than 25% of the chromosome. Comparative analyses show a fascinating picture of conservation and divergence, revealing large blocks of gene orthology with rodents, scattered regions with more recent gene family expansions and deletions, and segments of coding and non-coding conservation with the distant fish species Takifugu.
Asunto(s)
Cromosomas Humanos Par 19/genética , Genes/genética , Mapeo Físico de Cromosoma , Empalme Alternativo/genética , Animales , Composición de Base , Secuencia Conservada/genética , Islas de CpG/genética , Evolución Molecular , Duplicación de Gen , Genética Médica , Humanos , Ratones , Datos de Secuencia Molecular , Familia de Multigenes/genética , Seudogenes/genética , Análisis de Secuencia de ADNRESUMEN
Amplification of source DNA is a nearly universal requirement for molecular biology applications. The primary methods currently available to researchers are limited to in vivo amplification in Escherichia coli hosts and the polymerase chain reaction. Rolling-circle DNA replication is a well-known method for synthesis of phage genomes and recently has been applied as rolling circle amplification (RCA) of specific target sequences as well as circular vectors used in cloning. Here, we demonstrate that RCA using random hexamer primers with 29 DNA polymerase can be used for strand-displacement amplification of different vector constructs containing a variety of insert sizes to produce consistently uniform template for end-sequencing reactions. We show this procedure to be especially effective in a high-throughput plasmid production sequencing process. In addition, we demonstrate that whole bacterial genomes can be effectively amplified from cells or small amounts of purified genomic DNA without apparent bias for use in downstream applications, including whole genome shotgun sequencing.