Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 17 de 17
Filtrar
Mais filtros

Base de dados
Tipo de documento
Intervalo de ano de publicação
1.
Nature ; 464(7285): 90-4, 2010 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-20173737

RESUMO

Nitrogen (N(2))-fixing marine cyanobacteria are an important source of fixed inorganic nitrogen that supports oceanic primary productivity and carbon dioxide removal from the atmosphere. A globally distributed, periodically abundant N(2)-fixing marine cyanobacterium, UCYN-A, was recently found to lack the oxygen-producing photosystem II complex of the photosynthetic apparatus, indicating a novel metabolism, but remains uncultivated. Here we show, from metabolic reconstructions inferred from the assembly of the complete UCYN-A genome using massively parallel pyrosequencing of paired-end reads, that UCYN-A has a photofermentative metabolism and is dependent on other organisms for essential compounds. We found that UCYN-A lacks a number of major metabolic pathways including the tricarboxylic acid cycle, but retains sufficient electron transport capacity to generate energy and reducing power from light. Unexpectedly, UCYN-A has a reduced genome (1.44 megabases) that is structurally similar to many chloroplasts and some bacteria, in that it contains inverted repeats of ribosomal RNA operons. The lack of biosynthetic pathways for several amino acids and purines suggests that this organism depends on other organisms, either in close association or in symbiosis, for critical nutrients. However, size fractionation experiments using natural populations have so far not provided evidence of a symbiotic association with another microorganism. The UCYN-A cyanobacterium is a paradox in evolution and adaptation to the marine environment, and is an example of the tight metabolic coupling between microorganisms in oligotrophic oceanic microbial communities.


Assuntos
Cianobactérias/genética , Cianobactérias/metabolismo , Genoma Bacteriano/genética , Fixação de Nitrogênio/fisiologia , Nitrogênio/metabolismo , Água do Mar/microbiologia , Carbono/metabolismo , Cromossomos Bacterianos/genética , Cianobactérias/classificação , Cianobactérias/citologia , Transporte de Elétrons , Genômica , Biologia Marinha , Dados de Sequência Molecular , Fixação de Nitrogênio/genética , Oceanos e Mares , Oxirredutases/genética
2.
PLoS Genet ; 7(2): e1001287, 2011 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-21304888

RESUMO

Human intervention has subjected the yeast Saccharomyces cerevisiae to multiple rounds of independent domestication and thousands of generations of artificial selection. As a result, this species comprises a genetically diverse collection of natural isolates as well as domesticated strains that are used in specific industrial applications. However the scope of genetic diversity that was captured during the domesticated evolution of the industrial representatives of this important organism remains to be determined. To begin to address this, we have produced whole-genome assemblies of six commercial strains of S. cerevisiae (four wine and two brewing strains). These represent the first genome assemblies produced from S. cerevisiae strains in their industrially-used forms and the first high-quality assemblies for S. cerevisiae strains used in brewing. By comparing these sequences to six existing high-coverage S. cerevisiae genome assemblies, clear signatures were found that defined each industrial class of yeast. This genetic variation was comprised of both single nucleotide polymorphisms and large-scale insertions and deletions, with the latter often being associated with ORF heterogeneity between strains. This included the discovery of more than twenty probable genes that had not been identified previously in the S. cerevisiae genome. Comparison of this large number of S. cerevisiae strains also enabled the characterization of a cluster of five ORFs that have integrated into the genomes of the wine and bioethanol strains on multiple occasions and at diverse genomic locations via what appears to involve the resolution of a circular DNA intermediate. This work suggests that, despite the scrutiny that has been directed at the yeast genome, there remains a significant reservoir of ORFs and novel modes of genetic transmission that may have significant phenotypic impact in this important model and industrial species.


Assuntos
Cerveja/microbiologia , Genoma Fúngico/genética , Microbiologia Industrial , Saccharomyces cerevisiae/genética , Vinho/microbiologia , Sequência de Bases , Biologia Computacional , Evolução Molecular , Variação Genética , Mutação INDEL/genética , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Polimorfismo de Nucleotídeo Único/genética
3.
PLoS Genet ; 7(9): e1002219, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21931557

RESUMO

The Actinomycetales bacteria Rhodococcus opacus PD630 and Rhodococcus jostii RHA1 bioconvert a diverse range of organic substrates through lipid biosynthesis into large quantities of energy-rich triacylglycerols (TAGs). To describe the genetic basis of the Rhodococcus oleaginous metabolism, we sequenced and performed comparative analysis of the 9.27 Mb R. opacus PD630 genome. Metabolic-reconstruction assigned 2017 enzymatic reactions to the 8632 R. opacus PD630 genes we identified. Of these, 261 genes were implicated in the R. opacus PD630 TAGs cycle by metabolic reconstruction and gene family analysis. Rhodococcus synthesizes uncommon straight-chain odd-carbon fatty acids in high abundance and stores them as TAGs. We have identified these to be pentadecanoic, heptadecanoic, and cis-heptadecenoic acids. To identify bioconversion pathways, we screened R. opacus PD630, R. jostii RHA1, Ralstonia eutropha H16, and C. glutamicum 13032 for growth on 190 compounds. The results of the catabolic screen, phylogenetic analysis of the TAGs cycle enzymes, and metabolic product characterizations were integrated into a working model of prokaryotic oleaginy.


Assuntos
Biocombustíveis , Lipídeos/biossíntese , Redes e Vias Metabólicas/genética , Rhodococcus/genética , Triglicerídeos/biossíntese , Ácidos Graxos/genética , Ácidos Graxos/metabolismo , Genoma Bacteriano , Genômica , Filogenia , Rhodococcus/metabolismo , Triglicerídeos/genética
4.
Nat Med ; 12(7): 852-5, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16799556

RESUMO

The sensitivity of conventional DNA sequencing in tumor biopsies is limited by stromal contamination and by genetic heterogeneity within the cancer. Here, we show that microreactor-based pyrosequencing can detect rare cancer-associated sequence variations by independent and parallel sampling of multiple representatives of a given DNA fragment. This technology can thereby facilitate accurate molecular diagnosis of heterogeneous cancer specimens and enable patient selection for targeted cancer therapies.


Assuntos
Mapeamento Cromossômico/métodos , DNA de Neoplasias/genética , Mutação , Neoplasias/genética , Sequência de Bases , Humanos , Dados de Sequência Molecular , Neoplasias/diagnóstico , Sensibilidade e Especificidade
5.
FEMS Yeast Res ; 12(1): 88-96, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22136070

RESUMO

The vast majority of wine fermentations are performed principally by Saccharomyces cerevisiae. However, there are a growing number of instances in which other species of Saccharomyces play a predominant role. Interestingly, the presence of these other yeast species generally occurs via the formation of interspecific hybrids that contain genomic contributions from both S. cerevisiae and non-S. cerevisiae species. However, despite the large number of wine strains that are characterized at the genomic level, there remains limited information regarding the detailed genomic structure of hybrids used in winemaking. To address this, we describe the genome sequence of the thiol-releasing commercial wine yeast hybrid VIN7. VIN7 is shown to be an almost complete allotriploid interspecific hybrid that is comprised of a heterozygous diploid complement of S. cerevisiae chromosomes and a haploid Saccharomyces kudriavzevii genomic contribution. Both parental strains appear to be of European origin, with the S. cerevisiae parent being closely related to, but distinct from, the commercial wine yeasts QA23 and EC1118. In addition, several instances of chromosomal rearrangement between S. cerevisiae and S. kudriavzevii sequences were observed that may mark the early stages of hybrid genome consolidation.


Assuntos
Quimera/genética , Genoma Fúngico , Saccharomyces/genética , Triploidia , Vinho/microbiologia , DNA Fúngico/química , DNA Fúngico/genética , Evolução Molecular , Rearranjo Gênico , Dados de Sequência Molecular , Recombinação Genética , Saccharomyces/isolamento & purificação , Análise de Sequência de DNA
6.
BMC Genomics ; 12: 116, 2011 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-21324207

RESUMO

BACKGROUND: Knowledge of the origins, distribution, and inheritance of variation in the malaria parasite (Plasmodium falciparum) genome is crucial for understanding its evolution; however the 81% (A+T) genome poses challenges to high-throughput sequencing technologies. We explore the viability of the Roche 454 Genome Sequencer FLX (GS FLX) high throughput sequencing technology for both whole genome sequencing and fine-resolution characterization of genetic exchange in malaria parasites. RESULTS: We present a scheme to survey recombination in the haploid stage genomes of two sibling parasite clones, using whole genome pyrosequencing that includes a sliding window approach to predict recombination breakpoints. Whole genome shotgun (WGS) sequencing generated approximately 2 million reads, with an average read length of approximately 300 bp. De novo assembly using a combination of WGS and 3 kb paired end libraries resulted in contigs ≤ 34 kb. More than 8,000 of the 24,599 SNP markers identified between parents were genotyped in the progeny, resulting in a marker density of approximately 1 marker/3.3 kb and allowing for the detection of previously unrecognized crossovers (COs) and many non crossover (NCO) gene conversions throughout the genome. CONCLUSIONS: By sequencing the 23 Mb genomes of two haploid progeny clones derived from a genetic cross at more than 30× coverage, we captured high resolution information on COs, NCOs and genetic variation within the progeny genomes. This study is the first to resequence progeny clones to examine fine structure of COs and NCOs in malaria parasites.


Assuntos
Mapeamento Cromossômico , Conversão Gênica , Genoma de Protozoário , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Plasmodium falciparum/genética , Alelos , Pontos de Quebra do Cromossomo , Cruzamentos Genéticos , DNA de Protozoário/genética , Dosagem de Genes , Biblioteca Genômica , Genótipo , Repetições de Microssatélites , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA/métodos
7.
PLoS Pathog ; 5(6): e1000466, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19503607

RESUMO

Recent steep declines in honey bee health have severely impacted the beekeeping industry, presenting new risks for agricultural commodities that depend on insect pollination. Honey bee declines could reflect increased pressures from parasites and pathogens. The incidence of the microsporidian pathogen Nosema ceranae has increased significantly in the past decade. Here we present a draft assembly (7.86 MB) of the N. ceranae genome derived from pyrosequence data, including initial gene models and genomic comparisons with other members of this highly derived fungal lineage. N. ceranae has a strongly AT-biased genome (74% A+T) and a diversity of repetitive elements, complicating the assembly. Of 2,614 predicted protein-coding sequences, we conservatively estimate that 1,366 have homologs in the microsporidian Encephalitozoon cuniculi, the most closely related published genome sequence. We identify genes conserved among microsporidia that lack clear homology outside this group, which are of special interest as potential virulence factors in this group of obligate parasites. A substantial fraction of the diminutive N. ceranae proteome consists of novel and transposable-element proteins. For a majority of well-supported gene models, a conserved sense-strand motif can be found within 15 bases upstream of the start codon; a previously uncharacterized version of this motif is also present in E. cuniculi. These comparisons provide insight into the architecture, regulation, and evolution of microsporidian genomes, and will drive investigations into honey bee-Nosema interactions.


Assuntos
Abelhas/microbiologia , Genes Fúngicos , Genoma Fúngico , Nosema/genética , Animais , Sequência de Bases , Códon/genética , Códon/metabolismo , Sequência Conservada , Interpretação Estatística de Dados , Encephalitozoon cuniculi/genética , Modelos Genéticos , Dados de Sequência Molecular , Nosema/patogenicidade , Elementos Reguladores de Transcrição/genética , Sequências Repetitivas de Ácido Nucleico/genética , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Esporos Fúngicos/genética
8.
BMC Genomics ; 9: 404, 2008 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-18755037

RESUMO

BACKGROUND: With a whole genome duplication event and wealth of biological data, salmonids are excellent model organisms for studying evolutionary processes, fates of duplicated genes and genetic and physiological processes associated with complex behavioral phenotypes. It is surprising therefore, that no salmonid genome has been sequenced. Atlantic salmon (Salmo salar) is a good representative salmonid for sequencing given its importance in aquaculture and the genomic resources available. However, the size and complexity of the genome combined with the lack of a sequenced reference genome from a closely related fish makes assembly challenging. Given the cost and time limitations of Sanger sequencing as well as recent improvements to next generation sequencing technologies, we examined the feasibility of using the Genome Sequencer (GS) FLX pyrosequencing system to obtain the sequence of a salmonid genome. Eight pooled BACs belonging to a minimum tiling path covering approximately 1 Mb of the Atlantic salmon genome were sequenced by GS FLX shotgun and Long Paired End sequencing and compared with a ninth BAC sequenced by Sanger sequencing of a shotgun library. RESULTS: An initial assembly using only GS FLX shotgun sequences (average read length 248.5 bp) with approximately 30x coverage allowed gene identification, but was incomplete even when 126 Sanger-generated BAC-end sequences (approximately 0.09x coverage) were incorporated. The addition of paired end sequencing reads (additional approximately 26x coverage) produced a final assembly comprising 175 contigs assembled into four scaffolds with 171 gaps. Sanger sequencing of the ninth BAC (approximately 10.5x coverage) produced nine contigs and two scaffolds. The number of scaffolds produced by the GS FLX assembly was comparable to Sanger-generated sequencing; however, the number of gaps was much higher in the GS FLX assembly. CONCLUSION: These results represent the first use of GS FLX paired end reads for de novo sequence assembly. Our data demonstrated that this improved the GS FLX assemblies; however, with respect to de novo sequencing of complex genomes, the GS FLX technology is limited to gene mining and establishing a set of ordered sequence contigs. Currently, for a salmonid reference sequence, it appears that a substantial portion of sequencing should be done using Sanger technology.


Assuntos
Genômica/métodos , Salmo salar/genética , Análise de Sequência de DNA/métodos , Animais , Cromossomos Artificiais Bacterianos/genética , Evolução Molecular , Duplicação Gênica , Biblioteca Gênica , Genoma , Genômica/instrumentação , Genômica/estatística & dados numéricos , Salmo salar/classificação , Salmonidae/classificação , Salmonidae/genética , Análise de Sequência de DNA/instrumentação , Análise de Sequência de DNA/estatística & dados numéricos
9.
Hum Genet ; 124(2): 161-70, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18704501

RESUMO

Recently, genome-wide association studies have identified loci across a segment of chromosome 8q24 (128,100,000-128,700,000) associated with the risk of breast, colon and prostate cancers. At least three regions of 8q24 have been independently associated with prostate cancer risk; the most centromeric of which appears to be population specific. Haplotypes in two contiguous but independent loci, marked by rs6983267 and rs1447295, have been identified in the Cancer Genetic Markers of Susceptibility project ( http://cgems.cancer.gov ), which genotyped more than 5,000 prostate cancer cases and 5,000 controls of European origin. The rs6983267 locus is also strongly associated with colorectal cancer. To ascertain a comprehensive catalog of common single-nucleotide polymorphisms (SNPs) across the two regions, we conducted a resequence analysis of 136 kb (chr8: 128,473,000-128,609,802) using the Roche/454 next-generation sequencing technology in 39 prostate cancer cases and 40 controls of European origin. We have characterized a comprehensive catalog of common (MAF > 1%) SNPs within this region, including 442 novel SNPs and have determined the pattern of linkage disequilibrium across the region. Our study has generated a detailed map of genetic variation across the region, which should be useful for choosing SNPs for fine mapping of association signals in 8q24 and investigations of the functional consequences of select common variants.


Assuntos
Cromossomos Humanos Par 8 , Neoplasias do Colo/genética , Neoplasias da Próstata/genética , Estudos de Casos e Controles , Feminino , Frequência do Gene , Humanos , Desequilíbrio de Ligação , Masculino , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA/métodos
10.
Drug Discov Today ; 9(18): 795-802, 2004 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-15364067

RESUMO

The convergence of genomic technologies and the development of drugs designed against specific molecular targets provides many opportunities for using bioinformatics to bridge the gap between biological knowledge and clinical therapy. Identifying genes that have properties similar to known targets is conceptually straightforward. Additionally, genes can be linked to cancer via recurrent genomic or genetic abnormalities. Finally, by integrating large and disparate datasets, gene-level distinctions can be made between the different biological states that the data represents. These bioinformatics approaches and their associated methodologies, which can be applied across a range of technologies, facilitate the rapid identification of new target leads for further experimental validation.


Assuntos
Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Neoplasias/genética , Análise de Variância , Humanos , Neoplasias/metabolismo , Técnicas de Amplificação de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Sequência/métodos
11.
Nat Biotechnol ; 32(7): 656-62, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24908277

RESUMO

Cultivated citrus are selections from, or hybrids of, wild progenitor species whose identities and contributions to citrus domestication remain controversial. Here we sequence and compare citrus genomes--a high-quality reference haploid clementine genome and mandarin, pummelo, sweet-orange and sour-orange genomes--and show that cultivated types derive from two progenitor species. Although cultivated pummelos represent selections from one progenitor species, Citrus maxima, cultivated mandarins are introgressions of C. maxima into the ancestral mandarin species Citrus reticulata. The most widely cultivated citrus, sweet orange, is the offspring of previously admixed individuals, but sour orange is an F1 hybrid of pure C. maxima and C. reticulata parents, thus implying that wild mandarins were part of the early breeding germplasm. A Chinese wild 'mandarin' diverges substantially from C. reticulata, thus suggesting the possibility of other unrecognized wild citrus species. Understanding citrus phylogeny through genome analysis clarifies taxonomic relationships and facilitates sequence-directed genetic improvement.


Assuntos
Cruzamento , Citrus/classificação , Citrus/genética , Sequência Conservada/genética , Produtos Agrícolas/genética , Variação Genética/genética , Genoma de Planta/genética , Sequência de Bases , Evolução Molecular , Dados de Sequência Molecular , Análise de Sequência de DNA , Especificidade da Espécie
12.
Trop Plant Biol ; 5(1): 88-94, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22523606

RESUMO

The starchy swollen roots of cassava provide an essential food source for nearly a billion people, as well as possibilities for bioenergy, yet improvements to nutritional content and resistance to threatening diseases are currently impeded. A 454-based whole genome shotgun sequence has been assembled, which covers 69% of the predicted genome size and 96% of protein-coding gene space, with genome finishing underway. The predicted 30,666 genes and 3,485 alternate splice forms are supported by 1.4 M expressed sequence tags (ESTs). Maps based on simple sequence repeat (SSR)-, and EST-derived single nucleotide polymorphisms (SNPs) already exist. Thanks to the genome sequence, a high-density linkage map is currently being developed from a cross between two diverse cassava cultivars: one susceptible to cassava brown streak disease; the other resistant. An efficient genotyping-by-sequencing (GBS) approach is being developed to catalog SNPs both within the mapping population and among diverse African farmer-preferred varieties of cassava. These resources will accelerate marker-assisted breeding programs, allowing improvements in disease-resistance and nutrition, and will help us understand the genetic basis for disease resistance.

13.
Genome Biol ; 13(4): R30, 2012 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-22537947

RESUMO

BACKGROUND: Recent years have shown a marked increase in the use of next-generation sequencing technologies for quantification of gene expression (RNA sequencing, RNA-Seq). The expression level of a gene is a function of both its rate of transcription and RNA decay, and the influence of mRNA decay rates on gene expression in genome-wide studies of Gram-positive bacteria is under-investigated. RESULTS: In this work, we employed RNA-Seq in a genome-wide determination of mRNA half-lives in the Gram-positive bacterium Bacillus cereus. By utilizing a newly developed normalization protocol, RNA-Seq was used successfully to determine global mRNA decay rates at the single nucleotide level. The analysis revealed positional degradation patterns, with mRNAs being degraded from both ends of the molecule, indicating that both 5' to 3' and 3' to 5' directions of RNA decay are present in B. cereus. Other operons showed segmental degradation patterns where specific ORFs within polycistrons were degraded at variable rates, underlining the importance of RNA processing in gene regulation. We determined the half-lives for more than 2,700 ORFs in B. cereus ATCC 10987, ranging from less than one minute to more than fifteen minutes, and showed that mRNA decay rate correlates globally with mRNA expression level, GC content, and functional class of the ORF. CONCLUSIONS: To our knowledge, this study presents the first global analysis of mRNA decay in a bacterium at single nucleotide resolution. We provide a proof of principle for using RNA-Seq in bacterial mRNA decay analysis, revealing RNA processing patterns at the single nucleotide level.


Assuntos
Bacillus cereus/genética , Regulação Bacteriana da Expressão Gênica , Nucleotídeos/genética , Estabilidade de RNA , RNA Bacteriano/genética , Composição de Bases , Sequência de Bases , Genes de RNAr , Meia-Vida , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Óperon , Biossíntese de Proteínas , RNA Ribossômico 16S/genética , Análise de Sequência de RNA/métodos , Sítio de Iniciação de Transcrição
14.
Nat Genet ; 43(2): 109-16, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21186353

RESUMO

The woodland strawberry, Fragaria vesca (2n = 2x = 14), is a versatile experimental plant system. This diminutive herbaceous perennial has a small genome (240 Mb), is amenable to genetic transformation and shares substantial sequence identity with the cultivated strawberry (Fragaria × ananassa) and other economically important rosaceous plants. Here we report the draft F. vesca genome, which was sequenced to ×39 coverage using second-generation technology, assembled de novo and then anchored to the genetic linkage map into seven pseudochromosomes. This diploid strawberry sequence lacks the large genome duplications seen in other rosids. Gene prediction modeling identified 34,809 genes, with most being supported by transcriptome mapping. Genes critical to valuable horticultural traits including flavor, nutritional value and flowering time were identified. Macrosyntenic relationships between Fragaria and Prunus predict a hypothetical ancestral Rosaceae genome that had nine chromosomes. New phylogenetic analysis of 154 protein-coding genes suggests that assignment of Populus to Malvidae, rather than Fabidae, is warranted.


Assuntos
Fragaria/genética , Genoma de Planta , Algoritmos , Cloroplastos/genética , Mapeamento Cromossômico , Perfilação da Expressão Gênica , Genes de Plantas , Ligação Genética , Hibridização in Situ Fluorescente , Funções Verossimilhança , Modelos Genéticos , Filogenia , Sequências Repetidas Terminais , Transcrição Gênica
15.
Nat Genet ; 42(10): 833-9, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20802477

RESUMO

We report a high-quality draft genome sequence of the domesticated apple (Malus × domestica). We show that a relatively recent (>50 million years ago) genome-wide duplication (GWD) has resulted in the transition from nine ancestral chromosomes to 17 chromosomes in the Pyreae. Traces of older GWDs partly support the monophyly of the ancestral paleohexaploidy of eudicots. Phylogenetic reconstruction of Pyreae and the genus Malus, relative to major Rosaceae taxa, identified the progenitor of the cultivated apple as M. sieversii. Expansion of gene families reported to be involved in fruit development may explain formation of the pome, a Pyreae-specific false fruit that develops by proliferation of the basal part of the sepals, the receptacle. In apple, a subclade of MADS-box genes, normally involved in flower and fruit development, is expanded to include 15 members, as are other gene families involved in Rosaceae-specific metabolism, such as transport and assimilation of sorbitol.


Assuntos
Duplicação Gênica , Genes de Plantas/genética , Genoma de Planta , Malus/genética , Flores/genética , Flores/crescimento & desenvolvimento , Frutas/genética , Frutas/crescimento & desenvolvimento , Ligação Genética , Estudo de Associação Genômica Ampla , Malus/crescimento & desenvolvimento , Filogenia
16.
AIDS ; 23(10): 1209-18, 2009 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-19424056

RESUMO

OBJECTIVE: Identification of low-frequency variants is of clinical importance in the identification of preexisting drug resistance. Using 'ultra-deep' sequencing, we address the detection of potential resistance to the chemokine (C-C motif) receptor 5 (CCR5) antagonist, maraviroc, due to the pretreatment presence of low levels of chemokine (CXC motif) receptor 4 (CXCR4)-using virus. METHODS: We present a novel protocol for the phenotyping of HIV based on '454' pyrosequence data and apply this to two large data sets comprised of 104 628 (before treatment, day 1) and 191 637 (after treatment, day 11) reads from the envelope region. We study resistance in the context of the evolutionary history of the intrapatient viral population. Variation was also investigated both within and outside the V3 region, the region associated with the receptor switch. RESULTS: CXCR4-using virus can be detected at low frequency prior to maraviroc treatment ( approximately 0.5%) and at high frequency after failure of monotherapy ( approximately 81%). Inferring an evolutionary tree from the 1674 unique reads that span the V3 region confirms that the CXCR4-using population emerged from low-frequency CXCR4-using variants present before treatment. Changes in the frequency of amino acid residues used at individual sites were found in regions outside the V3 region, indicative of other potential sites associated with receptor usage. CONCLUSION: We have provided a high-resolution snapshot of intrapatient viral variation, prior and after treatment with maraviroc, and detected preexisting CXCR4-using variants present at an extremely low frequency. The evolutionary analysis demonstrates the extent of diversity present at a single time point within an infected individual and the rapid effect of drug pressure on the structure of a viral population.


Assuntos
Infecções por HIV/virologia , HIV-1/metabolismo , Receptores CXCR4/metabolismo , Cicloexanos/farmacologia , DNA Viral/genética , Farmacorresistência Viral/genética , Evolução Molecular , Inibidores da Fusão de HIV/farmacologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Maraviroc , Fenótipo , Filogenia , Receptores CXCR4/antagonistas & inibidores , Alinhamento de Sequência , Análise de Sequência de DNA/métodos , Triazóis/farmacologia
17.
J Infect Dis ; 199(5): 693-701, 2009 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-19210162

RESUMO

BACKGROUND: Minor (i.e., <20% prevalence) drug-resistant human immunodeficiency virus (HIV) variants may go undetected, yet be clinically important. OBJECTIVES: To compare the prevalence of drug-resistant variants detected with standard and ultra-deep sequencing (detection down to 1% prevalence) and to determine the impact of minor resistant variants on virologic failure (VF). METHODS: The Flexible Initial Retrovirus Suppressive Therapies (FIRST) Study (N = 1397) compared 3 initial antiretroviral therapy (ART) strategies. A random subset (n = 491) had baseline testing for drug-resistance mutations performed by use of standard sequencing methods. Ultra-deep sequencing was performed on samples that had sufficient viral content (N = 264). Proportional hazards models were used to compare rates of VF for those who did and did not have mutations identified. RESULTS: Mutations were detected by standard and ultra-deep sequencing (in 14% and 28% of participants, respectively; P < .001). Among individuals who initiated treatment with an ART regimen that combined nucleoside and nonnucleoside reverse-transcriptase inhibitors (hereafter, "NNRTI strategy"), all individuals who had an NNRTI-resistance mutation identified by ultra-deep sequencing experienced VF. When these individuals were compared with individuals who initiated treatment with the NNRTI strategy but who had no NNRTI-resistance mutations, the risk of VF was higher for those who had an NNRTI-resistance mutation detected by both methods (hazard ratio [HR], 12.40 [95% confidence interval {CI}, 3.41-45.10]) and those who had mutation(s) detected only with ultra-deep sequencing (HR, 2.50 [95% CI, 1.17-5.36]). CONCLUSIONS: Ultra-deep sequencing identified a significantly larger proportion of HIV-infected, treatment-naive persons as harboring drug-resistant viral variants. Among participants who initiated treatment with the NNRTI strategy, the risk of VF was significantly greater for participants who had low- and high-prevalence NNRTI-resistant variants.


Assuntos
Fármacos Anti-HIV/uso terapêutico , Farmacorresistência Viral , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Adulto , Doença Crônica , DNA Complementar/química , Progressão da Doença , Feminino , Variação Genética , HIV-1/genética , Humanos , Masculino , Mutação , RNA Viral/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA