RESUMO
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.
Assuntos
Cromossomos Humanos Par 5/genética , Análise de Sequência de DNA , Animais , Composição de Bases , Caderinas/genética , Sequência Conservada/genética , Duplicação Gênica , Genes/genética , Doenças Genéticas Inatas/genética , Genômica , Humanos , Interleucinas/genética , Dados de Sequência Molecular , Atrofia Muscular Espinal/genética , Pan troglodytes/genética , Mapeamento Físico do Cromossomo , Pseudogenes/genética , Sintenia/genética , Vertebrados/genéticaRESUMO
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.
Assuntos
Cromossomos Humanos Par 19/genética , Genes/genética , Mapeamento Físico do Cromossomo , Processamento Alternativo/genética , Animais , Composição de Bases , Sequência Conservada/genética , Ilhas de CpG/genética , Evolução Molecular , Duplicação Gênica , Genética Médica , Humanos , Camundongos , Dados de Sequência Molecular , Família Multigênica/genética , Pseudogenes/genética , Análise de Sequência de DNARESUMO
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.