RESUMO
Besides serving to package nuclear DNA, histones carry information in the form of a diverse array of post-translational modifications. Methylation of histones H3 and H4 has been implicated in long-term epigenetic 'memory'. Dimethylation or trimethylation of Lys4 of histone H3 (H3 Lys4) has been found in expressible euchromatin of yeasts and mammals. In contrast, methylation of Lys9 of histone H3 (H3 Lys9) has been implicated in establishing and maintaining the largely quiescent heterochromatin of mammals, yeasts, Drosophila melanogaster and plants. We have previously shown that a DNA methylation mutant of Neurospora crassa, dim-5 (defective in methylation), has a nonsense mutation in the SET domain of an H3-specific histone methyltransferase and that substitutions of H3 Lys9 cause gross hypomethylation of DNA. Similarly, the KRYPTONITE histone methyltransferase is required for full DNA methylation in Arabidopsis thaliana. We used biochemical, genetic and immunological methods to investigate the specific mark for DNA methylation in N. crassa. Here we show that trimethylated H3 Lys9, but not dimethylated H3 Lys9, marks chromatin regions for cytosine methylation and that DIM-5 specifically creates this mark.
Assuntos
Metilação de DNA , DNA Fúngico/química , DNA Fúngico/metabolismo , Histonas/química , Histonas/metabolismo , Neurospora crassa/metabolismo , DNA Fúngico/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/genética , Lisina/química , Metilação , Metiltransferases/genética , Mutação , Neurospora crassa/genética , Proteínas Metiltransferases , Processamento de Proteína Pós-TraducionalRESUMO
When stimulated strongly, a hair cell's mechanically sensitive hair bundle may consume ATP too rapidly for replenishment by diffusion. To provide a broad view of the bundle's protein complement, including those proteins participating in energy metabolism, we used shotgun mass spectrometry methods to identify proteins of purified chicken vestibular bundles. In addition to cytoskeletal proteins, proteins involved in Ca(2+) regulation, and stress-response proteins, many of the most abundant bundle proteins that were identified by mass spectrometry were involved in ATP synthesis. After beta-actin, the cytosolic brain isoform of creatine kinase was the next most abundant bundle protein; at approximately 0.5 mM, creatine kinase is capable of maintaining high ATP levels despite 1 mM/s ATP consumption by the plasma-membrane Ca(2+)-ATPase. Consistent with this critical role in hair bundle function, the creatine kinase circuit is essential for high-sensitivity hearing as demonstrated by hearing loss in creatine kinase knockout mice.
Assuntos
Trifosfato de Adenosina/metabolismo , Galinhas/fisiologia , Creatina Quinase/metabolismo , Células Ciliadas Auditivas/metabolismo , Animais , Encéfalo/enzimologia , Creatina Quinase/genética , Citosol/enzimologia , Orelha Interna/enzimologia , Orelha Interna/metabolismo , Metabolismo Energético/fisiologia , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Células Ciliadas Auditivas/enzimologia , Audição/fisiologia , Imuno-Histoquímica , Isoenzimas/metabolismo , Espectrometria de Massas , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/classificação , Proteínas do Tecido Nervoso/metabolismo , Equilíbrio Postural/fisiologia , Rana catesbeiana , Sáculo e Utrículo/citologia , Sáculo e Utrículo/enzimologia , Sáculo e Utrículo/metabolismo , Transdução de Sinais/fisiologiaRESUMO
UNLABELLED: Integrating qualitative protein identification with quantitative protein analysis is non-trivial, given incompatibility in output formats. We present TandTRAQ, a standalone utility that integrates results from i-Tracker, an open-source iTRAQ quantitation program with the search results from X?Tandem, an open-source proteome search engine. The utility runs from the command-line and can be easily integrated into a pipeline for automation. AVAILABILITY: The TandTRAQ Perl scripts are freely available for download at http://www.ohsucancer.com/isrdev/tandtraq/