Dosage compensation in male Drosophila relies on the X chromosome–specific recruitment of a chromatin-modifying machinery, the dosage compensation complex (DCC). The principles that assure selective targeting of the DCC are unknown. According to a prevalent model, X chromosome targeting is initiated by recruitment of the DCC core components, MSL1 and MSL2, to a ...

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The sensor kinase KdpD and the response regulator KdpE control induction of the kdpFABC operon encoding the high-affinity K+-transport system KdpFABC in response to K+ limitation or salt stress. Under K+ limiting conditions the Kdp system restores the intracellular K+ concentration, while in response to salt stress K+ is accumulated far above the normal content. ...

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The multifunctional nuclear protein positive cofactor 4 (PC4) is involved in various cellular processes including transcription, replication, and chromatin organization. Recently, PC4 has been identifi ed as a suppressor of oxidative mutagenesis in Escherichia coli and Saccharomyces cerevisiae . To investigate a potential role of PC4 in mammalian DNA repair, we ...

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Genetic and epigenetic plasticity allows tumors to evade single-targeted treatments. Here we direct Bcl2-specific short interfering RNA (siRNA) with 5¢-triphosphate ends (3p-siRNA) against melanoma. Recognition of 5¢-triphosphate by the cytosolic antiviral helicase retinoic acid–induced protein I (Rig-I, encoded by Ddx58) activated innate immune cells such as ...

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Dendritic cell (DC) vaccines have emerged as a promising strategy to induce antitumoral cytotoxic T cells for the immunotherapy of cancer. The maturation state of DC is of critical importance for the success of vaccination, but the most effective mode of maturation is still a matter of debate. Whereas immature DC carry the risk of inducing tolerance, extensive ...

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Messenger ribonucleoprotein particles (mRNPs) move randomly within nucleoplasm before they exit from the nucleus. To further understand mRNP trafficking, we have studied the intranuclear movement of a specific mRNP, the BR2 mRNP, in salivary gland cells in Chironomus tentans. Their polytene nuclei harbor giant chromosomes separated by vast regions of nucleoplasm, ...

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The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affi nity purifi cation strategy to identify specifi c interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affi nity matrices (bead proteomes). This strategy can be ...

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It is well established that all camelids have unique antibodies circulating in their blood. Unlike antibodies fromother species, these special antibodies are devoid of light chains and are composed of a heavy-chainhomodimer. These so-called heavy-chain antibodies (HCAbs) are expressed after a V–D–J rearrangement and require dedicated constant g-genes. An immune ...

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RNA interference is a powerful way to study gene function and is frequently combined with microarray analysis. Here we introduce a similar technology at the protein level by simultaneously applying Stable Isotope Labeling by Amino acids in Cell culture (SILAC) and RNA interference (RNAi) to Drosophila SL2 cells. After knockdown of ISWI, an ATP-hydrolyzing motor ...

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Imprinted genes are important in development and their allelic expression is mediated by imprinting control regions (ICRs). On their DNA-methylated allele, ICRs are marked by trimethylation at H3 Lys 9 (H3K9me3) and H4 Lys 20 (H4K20me3), similar to pericentric heterochromatin. Here, we investigate which histone methyltransferases control this methylation of ...

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Selected residues of transmembrane domain (TM) IX were previously shown to play key roles in ligand binding and transport in members of the Na+/solute symporter family. Using the Na+/proline transporter PutP as a model, a complete Cys scanning mutagenesis of TM IX (positions 324 to 351) was performed here to further investigate the functional significance of the ...

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Genetic high-throughput screens have yielded large sets of potential protein-protein interactions now to be verified and further investigated. Here we present a simple assay to directly visualize protein-protein interactions in single living cells. Using a modified lac repressor system, we tethered a fluorescent bait at a chromosomal lac operator array and ...

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The precise coordination of the different steps of DNA replication is critical for the maintenance of genome stability. We have probed the mechanisms coupling various components of the replication machinery and their response to polymerase stalling by inhibition of the DNA polymerases in living mammalian cells with aphidicolin. We observed little change in the ...

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Viral nucleic acids are recognized by specific pattern-recognition receptors of the Toll-like and RIG-I-like receptor families. Synthetic DNA and RNA oligonucleotides can activate the immune system through these receptors and potentiate Ab and CD8 cytotoxic responses to Ags. Systemic application of immunostimulatory oligonucleotides however also results in a ...

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Cytoplasmic localization and localized translation of messenger RNAs contribute to asymmetrical protein distribution. Recognition of localized mRNAs by RNA-binding proteins can occur in the cytoplasm or, alternatively, co- or post-transcriptionally in the nucleus. In budding yeast, mRNAs destined for localization are bound by the She2 protein before their nuclear ...

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Fluorescence light microscopy allows multicolor visualization of cellular components with high specificity, but its utility has until recently been constrained by the intrinsic limit of spatial resolution. We applied three-dimensional structured illumination microscopy (3D-SIM) to circumvent this limit and to study the mammalian nucleus. By simultaneously imaging ...

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H4K20 methylation is a broad chromatin modification that has been linked with diverse epigenetic functions. Several enzymes target H4K20 methylation, consistent with distinct mono-, di-, and trimethylation states controlling different biological outputs. To analyze the roles of H4K20 methylation states, we generated conditional null alleles for the two Suv4-20h ...

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Heterochromatic chromosomal regions undergo large-scale reorganization and progressively aggregate, forming chromocenters. These are dynamic structures that rapidly adapt to various stimuli that influence gene expression patterns, cell cycle progression, and differentiation. Np95-ICBP90 (m- and h-UHRF1) is a histone-binding protein expressed only in proliferating ...

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Photorhabdus luminescens is a Gram-negative luminescent enterobacterium and a symbiote to soil nematodes belonging to the species Heterorhabditis bacteriophora. P.luminescens is simultaneously highly pathogenic to insects. This bacterium exhibits a complex life cycle, including one symbiotic stage characterized by colonization of the upper nematode gut, and a ...

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Intracellular mRNA localization is a common mechanism to achieve asymmetric distributions of proteins. Previous studies have revealed that in a number of cell types, differentmRNA species are localized by the same transport machinery. However, it has been unclear if these individual mRNA species are specifically sorted into separate or common ribonucleoprotein ...

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In higher eukaryotes, an unusual C-terminal domain (CTD) is crucial to the function of RNA polymerase II in transcription. The CTD consists of multiple heptapeptide repeats; differences in the number of repeats between organisms and their degree of conservation have intrigued researchers for two decades. Here, we review the evolution of the CTD at the molecular ...

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Fluorescent proteins (FP) are widely used as in vivo reporter molecules and are available in multiple colors spanning almost the entire visible light spectrum. Genetically fused to any protein target, FPs offer a powerful tool to study protein localization and dynamics. After the isolation of the prototypical green fluorescent protein (GFP) from the jellyfish ...

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Introducing point mutations into bacterial chromosomes is important for further progress in studies relying on functional genomics, systems- and synthetic biology, and for metabolic engineering. For many investigations, chromosomal systems are required rather than artificial plasmid based systems. Here we describe the introduction of a single point mutation into ...

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Antibodies against the neurite outgrowth inhibitor Nogo-A enhance axonal regeneration following spinal cord injury. However, antibodies directed against myelin components can also enhance CNS inflammation. The present study was designed to assess the efficacy of DNA vaccination for generating antibodies against Nogo-A and to study their pathogenic potential in a ...

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Chromatin serves to package, protect and organize the complex eukaryotic genomes to assure their stable inheritance over many cell generations. At the same time, chromatin must be dynamic to allow continued use of DNA during a cell’s lifetime. One important principle that endows chromatin with flexibility involves ATPdependent ‘remodeling’ factors, which alter ...

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CIPSM-Researchers Heinrich Leonhardt and Ulrich Rothbauer win the Binder-Innovation-Award for their work on the usage of fluorescent nano-antibodies to detect biological structures and processes in living cells. From left to right: Prof. Vestweber (President of the German Association of Cellbiology), Prof. Baumeister (MPI Martinsried), Dr. Ankerhold (Carl ...

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The combination of chromatin structure and the organization of chromosomes in eukaryotic nuclei affects many genome functions. Distinct functional states of genes ranging from ‘highly active’ to ‘silenced’ correlate with particular nucleosome arrangements, histone variants, histone modifications, and interactions of non-histone regulators. Transcription factors ...

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The ubiquitin-like SUMO system functions by a cyclic process of modification and demodification, and recent data suggest that the nucleolus is a site of sumoylation–desumoylation cycles.For example, the tumour suppressor ARF stimulates sumoylation of nucleolar proteins. Here, we show that the nucleolar SUMOspecific protease SENP3 is associated with nucleophosmin ...

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The linker histone H1 binds to the DNA in between adjacent nucleosomes and contributes to chromatin organization and transcriptional control. It is known that H1 carries diverse posttranslational modifications (PTMs), including phosphorylation, lysine methylation and ADP-ribosylation. Their biological functions, however, remain largely unclear. This is in part ...

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Many phenotypic changes of eukaryotic cells due to changes in gene expression depend on alterations in chromatin structure. Processes involved in the alteration of chromatin are diverse and include post-translational modifications of histone proteins, incorporation of specific histone variants, methylation of DNA and ATP-dependent chromatin remodeling. ...

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