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1.
PLoS One ; 7(6): e38793, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22685606

RESUMO

F(1)F(0) ATPases have been identified in most bacteria, including mycoplasmas which have very small genomes associated with a host-dependent lifestyle. In addition to the typical operon of eight genes encoding genuine F(1)F(0) ATPase (Type 1), we identified related clusters of seven genes in many mycoplasma species. Four of the encoded proteins have predicted structures similar to the α, ß, γ and ε subunits of F(1) ATPases and could form an F(1)-like ATPase. The other three proteins display no similarity to any other known proteins. Two of these proteins are probably located in the membrane, as they have three and twelve predicted transmembrane helices. Phylogenomic studies identified two types of F(1)-like ATPase clusters, Type 2 and Type 3, characterized by a rapid evolution of sequences with the conservation of structural features. Clusters encoding Type 2 and Type 3 ATPases were assumed to originate from the Hominis group of mycoplasmas. We suggest that Type 3 ATPase clusters may spread to other phylogenetic groups by horizontal gene transfer between mycoplasmas in the same host, based on phylogeny and genomic context. Functional analyses in the ruminant pathogen Mycoplasma mycoides subsp. mycoides showed that the Type 3 cluster genes were organized into an operon. Proteomic analyses demonstrated that the seven encoded proteins were produced during growth in axenic media. Mutagenesis and complementation studies demonstrated an association of the Type 3 cluster with a major ATPase activity of membrane fractions. Thus, despite their tendency toward genome reduction, mycoplasmas have evolved and exchanged specific F(1)-like ATPases with no known equivalent in other bacteria. We propose a model, in which the F(1)-like structure is associated with a hypothetical X(0) sector located in the membrane of mycoplasma cells.


Assuntos
Adenosina Trifosfatases/genética , Proteínas de Bactérias/genética , Evolução Molecular , Mycoplasma/genética , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biocatálise , Dosagem de Genes , Transferência Genética Horizontal , Genoma Bacteriano/genética , Humanos , Immunoblotting , Modelos Moleculares , Família Multigênica/genética , Mycoplasma/classificação , Mycoplasma/enzimologia , Infecções por Mycoplasma/microbiologia , Mycoplasma mycoides/enzimologia , Mycoplasma mycoides/genética , Filogenia , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Especificidade por Substrato
2.
Mol Biol Evol ; 26(12): 2849-64, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19723671

RESUMO

There is no generally accepted picture of where, when, and how the domestic dog originated. Previous studies of mitochondrial DNA (mtDNA) have failed to establish the time and precise place of origin because of lack of phylogenetic resolution in the so far studied control region (CR), and inadequate sampling. We therefore analyzed entire mitochondrial genomes for 169 dogs to obtain maximal phylogenetic resolution and the CR for 1,543 dogs across the Old World for a comprehensive picture of geographical diversity. Hereby, a detailed picture of the origins of the dog can for the first time be suggested. We obtained evidence that the dog has a single origin in time and space and an estimation of the time of origin, number of founders, and approximate region, which also gives potential clues about the human culture involved. The analyses showed that dogs universally share a common homogenous gene pool containing 10 major haplogroups. However, the full range of genetic diversity, all 10 haplogroups, was found only in southeastern Asia south of Yangtze River, and diversity decreased following a gradient across Eurasia, through seven haplogroups in Central China and five in North China and Southwest (SW)Asia, down to only four haplogroups in Europe. The mean sequence distance to ancestral haplotypes indicates an origin 5,400-16,300 years ago (ya) from at least 51 female wolf founders. These results indicate that the domestic dog originated in southern China less than 16,300 ya, from several hundred wolves. The place and time coincide approximately with the origin of rice agriculture, suggesting that the dogs may have originated among sedentary hunter-gatherers or early farmers, and the numerous founders indicate that wolf taming was an important culture trait.


Assuntos
DNA Mitocondrial/genética , Cães/genética , Filogenia , Rios , Lobos/genética , Animais , Sudeste Asiático , China , Europa (Continente) , Feminino , Pool Gênico , Genoma Mitocondrial/genética , Geografia , Haplótipos/genética , Região de Controle de Locus Gênico/genética , Dados de Sequência Molecular , Fatores de Tempo
3.
Proteomics ; 7(13): 2142-50, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17549799

RESUMO

Advances in antibody production render a growing supply of affinity reagents for immunohistochemistry (IHC), and tissue microarray (TMA) technologies facilitate simultaneous analysis of protein expression in a multitude of tissues. However, collecting validated IHC data remains a bottleneck problem, as the standard method is manual microscopical analysis. Here we present a high-throughput strategy combining IHC on a recently developed cell microarray with a novel, automated image-analysis application (TMAx). The software was evaluated on 200 digital images of IHC-stained cell spots, by comparing TMAx annotation with manual annotation performed by seven human experts. A high concordance between automated and manual annotation of staining intensity and fraction of IHC-positive cells was found. In a limited study, we also investigated the possibility to assess the correlation between mRNA and protein levels, by using TMAx output results for relative protein quantification and quantitative real-time PCR for the quantification of corresponding transcript levels. In conclusion, automated analysis of immunohistochemically stained in vitro-cultured cells in a microarray format can be used for high-throughput protein profiling, and extraction of RNA from the same cell lines provides a basis for comparing transcription and protein expression on a global scale.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Imuno-Histoquímica/métodos , Análise em Microsséries/métodos , Proteínas/análise , Anticorpos/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Expressão Gênica , Humanos , Proteínas/genética , Proteínas/imunologia , Proteômica/métodos , Reprodutibilidade dos Testes , Software
4.
Mol Cell Proteomics ; 4(12): 1920-32, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16127175

RESUMO

Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, approximately 400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.


Assuntos
Anticorpos Antineoplásicos/química , Anticorpos/química , Neoplasias/imunologia , Proteoma/imunologia , Anticorpos/isolamento & purificação , Anticorpos Antineoplásicos/isolamento & purificação , Western Blotting , Cromatografia de Afinidade , Bases de Dados de Proteínas , Epitopos/química , Etiquetas de Sequências Expressas , Humanos , Neoplasias/genética , Proteínas/imunologia , Proteoma/isolamento & purificação , Valores de Referência
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