RESUMO
Physical, genetic, and chemical-genetic interactions centered on the conserved chaperone Hsp90 were mapped at high resolution in yeast using systematic proteomic and genomic methods. Physical interactions were identified using genome-wide two hybrid screens combined with large-scale affinity purification of Hsp90-containing protein complexes. Genetic interactions were uncovered using synthetic genetic array technology and by a microarray-based chemical-genetic screen of a set of about 4700 viable yeast gene deletion mutants for hypersensitivity to the Hsp90 inhibitor geldanamycin. An extended network, consisting of 198 putative physical interactions and 451 putative genetic and chemical-genetic interactions, was found to connect Hsp90 to cofactors and substrates involved in a wide range of cellular functions. Two novel Hsp90 cofactors, Tah1 (YCR060W) and Pih1 (YHR034C), were also identified. These cofactors interact physically and functionally with the conserved AAA(+)-type DNA helicases Rvb1/Rvb2, which are key components of several chromatin remodeling factors, thereby linking Hsp90 to epigenetic gene regulation.
Assuntos
Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/metabolismo , Benzoquinonas , Mapeamento Cromossômico/métodos , Coenzimas/genética , Coenzimas/isolamento & purificação , Coenzimas/metabolismo , DNA Helicases , Farmacorresistência Fúngica/genética , Epigênese Genética/genética , Regulação Fúngica da Expressão Gênica/genética , Genoma Fúngico , Proteínas de Choque Térmico HSP90/química , Lactamas Macrocíclicas , Espectrometria de Massas , Chaperonas Moleculares/química , Análise de Sequência com Séries de Oligonucleotídeos , Proteômica/métodos , Quinonas/farmacologia , RNA Helicases/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição , Técnicas do Sistema de Duplo-HíbridoRESUMO
The detection of DNA using nanoparticles as labels is an interesting alternative to the standard fluorescence technique. It requires simpler detection equipment, resulting in higher stability and lower costs. Besides easier detection, metal enhancement allows a higher sensitivity of detection. The signal-response curve for labelled DNA before and after silver enhancement was studied, applying both atomic force microscope (AFM) and optical (reflection/transmission) measurements. The dynamic range and the sensitivity were determined for nanoparticle labelling with and without metal enhancement. Nanoparticle concentrations down to the fM range could be detected. The ultimate limit of detection, the identification of individual labels, is demonstrated for the optical readout. Therefore, AFM images of the particles were correlated with the optical signal of individual or clustered particles. We demonstrate that the optical signal allows the identification of single particles.
