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1.
Cell Stress Chaperones ; 15(5): 753-9, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20373063

RESUMO

The Hsp90 molecular chaperone has been implicated as a contributor to evolution in several organisms by revealing cryptic variation that can yield dramatic phenotypes when the chaperone is diverted from its normal functions by environmental stress. In addition, as a cancer drug target, Hsp90 inhibition has been documented to sensitize cells to DNA-damaging agents, suggesting a function for Hsp90 in DNA repair. Here we explore the potential role of Hsp90 in modulating the stability of nucleotide repeats, which in a number of species, including humans, exert subtle and quantitative consequences for protein function, morphological and behavioral traits, and disease. We report that impairment of Hsp90 in human cells induces contractions of CAG repeat tracks by tenfold. Inhibition of the recombinase Rad51, a downstream target of Hsp90, induces a comparable increase in repeat instability, suggesting that Hsp90-enabled homologous recombination normally functions to stabilize CAG repeat tracts. By contrast, Hsp90 inhibition does not increase the rate of gene-inactivating point mutations. The capacity of Hsp90 to modulate repeat-tract lengths suggests that the chaperone, in addition to exposing cryptic variation, might facilitate the expression of new phenotypes through induction of novel genetic variation.


Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Repetições de Trinucleotídeos/genética , Linhagem Celular , Proteínas de Choque Térmico HSP90/genética , Humanos , Immunoblotting , Instabilidade de Microssatélites , RNA Interferente Pequeno , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo
2.
Proc Natl Acad Sci U S A ; 106(24): 9607-12, 2009 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-19482946

RESUMO

Expanded triplet repeats have been identified as the genetic basis for a growing number of neurological and skeletal disorders. To examine the contribution of double-strand break repair to CAG x CTG repeat instability in mammalian systems, we developed zinc finger nucleases (ZFNs) that recognize and cleave CAG repeat sequences. Engineered ZFNs use a tandem array of zinc fingers, fused to the FokI DNA cleavage domain, to direct double-strand breaks (DSBs) in a site-specific manner. We first determined that the ZFNs cleave CAG repeats in vitro. Then, using our previously described tissue culture assay for identifying modifiers of CAG repeat instability, we found that transfection of ZFN-expression vectors induced up to a 15-fold increase in changes to the CAG repeat in human and rodent cell lines, and that longer repeats were much more sensitive to cleavage than shorter ones. Analysis of individual colonies arising after treatment revealed a spectrum of events consistent with ZFN-induced DSBs and dominated by repeat contractions. We also found that expressing a dominant-negative form of RAD51 in combination with a ZFN, dramatically reduced the effect of the nuclease, suggesting that DSB-induced repeat instability is mediated, in part, through homology directed repair. These studies identify a ZFN as a useful reagent for characterizing the effects of DSBs on CAG repeats in cells.


Assuntos
Dano ao DNA , Instabilidade Genômica , Repetições de Trinucleotídeos , Dedos de Zinco , Animais , Sequência de Bases , Células CHO , Cricetinae , Cricetulus , DNA , Humanos , Mutação
3.
Vision Res ; 47(27): 3394-407, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18022666

RESUMO

Bardet-Biedl syndrome (BBS) is an oligogenic syndrome whose manifestations include retinal degeneration, renal abnormalities, obesity and polydactylia. Evidence suggests that the main etiopathophysiology of this syndrome is impaired intraflagellar transport (IFT). In this study, we study the Bbs4-null mouse and investigate photoreceptor structure and function after loss of this gene. We find that Bbs4-null mice have defects in the transport of phototransduction proteins from the inner segments to the outer segments, before signs of cell death. Additionally, we show defects in synaptic transmission from the photoreceptors to secondary neurons of the visual system, demonstrating multiple functions for BBS4 in photoreceptors.


Assuntos
Síndrome de Bardet-Biedl/fisiopatologia , Proteínas Associadas aos Microtúbulos/metabolismo , Células Fotorreceptoras/metabolismo , Animais , Apoptose , Síndrome de Bardet-Biedl/metabolismo , Síndrome de Bardet-Biedl/patologia , Proteínas de Transporte/metabolismo , Eletrorretinografia , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Proteínas Associadas aos Microtúbulos/análise , Proteínas Associadas aos Microtúbulos/genética , Modelos Animais , Células Fotorreceptoras/química , Células Fotorreceptoras/ultraestrutura , Transporte Proteico , Opsinas de Bastonetes/metabolismo , Transmissão Sináptica , Visão Ocular
4.
Vision Res ; 45(28): 3445-53, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16139321

RESUMO

We have developed an imaging approach to monitor changes in gene structure in photoreceptors. We review here, the strategy and recent progress. Knock-in mice bearing a human rhodopsin-EGFP fusion gene potentially allow detection of a single molecular event: correction of a single copy of a gene within an entire retina. These mice can also be used for imaging rhodopsin distribution, membrane structure, and trafficking in normal mice or in disease states, using confocal or multiphoton fluorescence imaging techniques. They represent tools for studying molecular triggers of photoreceptor development, for following stem cell populations, and for evaluating retinal transplantation experiments.


Assuntos
Terapia Genética/métodos , Células Fotorreceptoras/metabolismo , Retina/patologia , Degeneração Retiniana/genética , Rodopsina/genética , Animais , Animais Geneticamente Modificados , Estudos de Viabilidade , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Degeneração Retiniana/patologia
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