RESUMEN
Dominant mutations in the alpha-B crystallin (CryAB) gene are responsible for a number of inherited human disorders, including cardiomyopathy, skeletal muscle myopathy, and cataracts. The cellular mechanisms of disease pathology for these disorders are not well understood. Among recent advances is that the disease state can be linked to a disturbance in the oxidation/reduction environment of the cell. In a mouse model, cardiomyopathy caused by the dominant CryAB(R120G) missense mutation was suppressed by mutation of the gene that encodes glucose 6-phosphate dehydrogenase (G6PD), one of the cell's primary sources of reducing equivalents in the form of NADPH. Here, we report the development of a Drosophila model for cellular dysfunction caused by this CryAB mutation. With this model, we confirmed the link between G6PD and mutant CryAB pathology by finding that reduction of G6PD expression suppressed the phenotype while overexpression enhanced it. Moreover, we find that expression of mutant CryAB in the Drosophila heart impaired cardiac function and increased heart tube dimensions, similar to the effects produced in mice and humans, and that reduction of G6PD ameliorated these effects. Finally, to determine whether CryAB pathology responds generally to NADPH levels we tested mutants or RNAi-mediated knockdowns of phosphogluconate dehydrogenase (PGD), isocitrate dehydrogenase (IDH), and malic enzyme (MEN), the other major enzymatic sources of NADPH, and we found that all are capable of suppressing CryAB(R120G) pathology, confirming the link between NADP/H metabolism and CryAB.
Asunto(s)
Drosophila melanogaster/genética , Redes y Vías Metabólicas/genética , NADP/metabolismo , Cadena B de alfa-Cristalina/genética , Animales , Cardiomiopatías/etiología , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Catarata/genética , Catarata/patología , Drosophila melanogaster/enzimología , Drosophila melanogaster/metabolismo , Glucosafosfato Deshidrogenasa/genética , Glucosafosfato Deshidrogenasa/metabolismo , Humanos , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Malato Deshidrogenasa/genética , Malato Deshidrogenasa/metabolismo , Ratones , Enfermedades Musculares/genética , Enfermedades Musculares/patología , Mutación Missense , NADP/genética , Fosfogluconato Deshidrogenasa/genética , Fosfogluconato Deshidrogenasa/metabolismo , Cadena B de alfa-Cristalina/metabolismoRESUMEN
The mechanisms that cells use to monitor telomere integrity, and the array of responses that may be induced, are not fully defined. To date there have been no studies in animals describing the ability of cells to survive and contribute to adult organs following telomere loss. We developed assays to monitor the ability of somatic cells to proliferate and differentiate after telomere loss. Here we show that p53 and Chk2 limit the growth and differentiation of cells that lose a telomere. Furthermore, our results show that two copies of the genes encoding p53 and Chk2 are required for the cell to mount a rapid wildtype response to a missing telomere. Finally, our results show that, while Chk2 functions by activating the p53-dependent apoptotic cascade, Chk2 also functions independently of p53 to limit survival. In spite of these mechanisms to eliminate cells that have lost a telomere, we find that such cells can make a substantial contribution to differentiated adult tissues.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila/genética , Genes p53/genética , Haplotipos/genética , Proteínas Serina-Treonina Quinasas/genética , Telómero/genética , Animales , Apoptosis/genética , Senescencia Celular , Quinasa de Punto de Control 2 , Proteínas de Drosophila/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Telómero/metabolismoRESUMEN
Following the advent of a gene targeting technique in Drosophila, different methods have been developed to modify the Drosophila genome. The initial demonstration of gene targeting in flies used an ends-in method, which generates a duplication of the target locus. The duplicated locus can then be efficiently reduced to a single copy by generating a double-strand break between the duplicated segments. This method has been used to knock out target genes by introducing point mutations. A derivative of this method is reported here. By using different homologous regions for the targeting and reduction steps, a complete deletion of the target gene can be generated to produce a definitive null allele. The breakpoints of the deletion can be precisely controlled. Unlike ends-out targeting, this method does not leave exogenous sequence at the deleted locus. Three endogenous genes, Sir2, Sirt2, and p53 have been successfully deleted using this method.
Asunto(s)
Drosophila melanogaster/genética , Eliminación de Gen , Marcación de Gen , Animales , Elementos Transponibles de ADN , Proteínas de Drosophila/genética , Histona Desacetilasas/genética , Sirtuinas/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
We used a recently developed method to produce mutant alleles of five endogenous Drosophila genes, including the homolog of the p53 tumor suppressor. Transgenic expression of the FLP site-specific recombinase and the I-SceI endonuclease generates extrachromosomal linear DNA molecules in vivo. These molecules undergo homologous recombination with the corresponding chromosomal locus to generate targeted alterations of the host genome. The results address several questions about the general utility of this technique. We show that genes not near telomeres can be efficiently targeted; that no knowledge of the mutant phenotype is needed for targeting; and that insertional mutations and allelic substitutions can be easily produced.