RESUMO
Tagging of proteins in vivo by covalent attachment of a biotin moiety has emerged as a new prospective tool for protein detection and purification. Previously, we established a strategy for expression of in vivo biotinylated proteins in mammalian cells. It is based on coexpression of the protein of interest fused to a short biotin acceptor peptide and biotin ligase BirA cloned in the same vector. We show here that the in vivo biotinylation can be used for immunogold postembedding labeling in immunoelectron microscopy experiments. We show that immunoelectron microscopy with biotinylated nuclear proteins is compatible with a wide range of postembedding methods, facilitating combination of morphological and localization studies in a single experiment. We also show that the method works in both transient transfection and stable cell line expression protocols and can be used for colocalization studies. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.
Assuntos
Biotina/metabolismo , Proteínas Nucleares/metabolismo , Biotinilação , Carbono-Nitrogênio Ligases/genética , Linhagem Celular , Cromatina/metabolismo , Proteínas de Escherichia coli/genética , Histonas/metabolismo , Humanos , Microscopia Imunoeletrônica , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Ligação Proteica , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Inclusão do Tecido , TransfecçãoRESUMO
Chromatin is considered to be a principal carrier of epigenetic information due to the ability of alternative chromatin states to persist through generations of cell divisions and to spread on DNA. Replacement histone variants are novel candidates for epigenetic marking of chromatin. We developed a novel approach to analyze the chromatin environment of nucleosomes containing a particular replacement histone. We applied it to human H2AZ, one of the most studied alternative histones. We find that neither H2AZ itself nor other features of the H2AZ-containing nucleosome spread to the neighboring nucleosomes in vivo, arguing against a role for H2AZ as a self-perpetuating epigenetic mark.
Assuntos
Epigênese Genética , Histonas/genética , Histonas/metabolismo , Sequência de Aminoácidos , Animais , Cromatina/genética , Cromatina/metabolismo , Inativação Gênica , Variação Genética , Células HeLa , Histonas/química , Humanos , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Células NIH 3T3 , Nucleossomos/metabolismo , Processamento de Proteína Pós-Traducional , Estrutura Quaternária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de AminoácidosRESUMO
We describe a system designed to express biotinylated proteins in mammalian cells in vivo and its application to the study of protein-DNA interactions in vivo by chromatin immunoprecipitation (ChIP). The system is based on coexpression of the target protein fused to a short biotin acceptor domain together with the biotinylating enzyme BirA from Escherichia coli. The superior strength of the biotin-avidin interaction allows one to employ more stringent washing conditions in the ChIP protocol, resulting in a better signal/noise ratio.
Assuntos
Biotinilação , Carbono-Nitrogênio Ligases/química , Cromatina/química , Proteínas de Escherichia coli/química , Testes de Precipitina , Proteínas Repressoras/química , Fatores de Transcrição/química , Animais , Carbono-Nitrogênio Ligases/genética , Proteínas de Ciclo Celular/genética , Cromatina/genética , Cromatina/imunologia , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Imunoadsorventes , Proteínas Luminescentes/genética , Camundongos , Células NIH 3T3 , Proteínas Nucleares/química , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Repressoras/genética , Fator de Transcrição DP1 , Fatores de Transcrição/genética , Transdução GenéticaRESUMO
AIM: Lercanidipine is a new vasoselective dihydropyridine calcium channel blocker with a short plasma half-life, long duration of action, and demonstrated cardioprotective properties. We hypothesized that it might be effective at attenuating the adverse impact observed on the coronary compartment and myocardium in the transition phase to heart failure in the UM-X7.1 cardiomyopathic (CM) hamster. METHODS: The effects of 4-month exposure to lercanidipine 3 and 10 mg/kg (daily oral administration) were evaluated in 150-day-old CM hamsters and in age-matched normal hamsters. Coronary reactivity (reactive hyperemia to 30-s coronary occlusion) and the response to the administration of acetylcholine (100 nmol/L) and sodium nitroprusside (1 micromol/L) were assessed monthly, using the isolated perfused heart model. The left ventricular chamber dilatation index and wall thickness, myocardial fibrosis and myocardial capillary density (papillary muscle) were estimated in selected subgroups at monthly intervals. RESULTS: High-dose lercanidipine had beneficial effects on coronary dysfunctions: at month 4 of the treatment period, reactive hyperemia to short duration ischemia was improved, as was the endothelium-dependent vasodilator response (acetylcholine=68 %+/-16 % vs 11 %+/-5 % in untreated CM hamsters, P<0.05) and endothelium-independent vasodilator response (sodium nitroprusside=36 %+/-5 % vs 22 %+/-12 % in untreated CM hamsters, P<0.05). Capillary density averaged 10,879+/-474 capillaries per mm2 in papillary muscle from normal hamsters; this value did not change over time in normal hamsters and was not affected during the transition phase to heart failure in CM hamsters. Lercanidipine preserved myocardial capillary density in these conditions. Chronic exposure to lercanidipine had no impact on myocardial remodeling observed in CM hamsters. CONCLUSION: Lercanidipine had a beneficial impact on the coronary compartment in the transition phase to heart failure in a model of dilated cardiomyopathy.
