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Posttranslational Lipid Modification of Rho Family Small GTPases

The Rho family comprises a major branch of the Ras superfamily of small GTPases. A majority of Rho GTPases are synthesized as inactive, cytosolic proteins. They then undergo posttranslational modification by isoprenoid or fatty acid lipids, and together with additional carboxyl-ter ...

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Assessing Ubiquitylation of Rho GTPases in Mammalian Cells

Rho GTPases including RhoA, Cdc42, and Rac1 are master regulators of cell cytoskeleton dynamic, thus controlling essential cellular processes notably cell polarity, migration and cytokinesis. These GTPases undergo a spatiotemporal regulation primarily controlled by cell ...

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How to Analyze Bacterial Toxins Targeting Rho GTPases

Bacterial pathogens developed several strategies to overcome defense systems of eukaryotic hosts. Within the infection process, they need to attach to and cross through epithelial layers, escape from the innate and adaptive immune response, and find a physiological niche to survive. ...

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Biochemical Assays to Characterize Rho GTPases

Rho GTPases act as tightly regulated molecular switches governing a large variety of critical cellular functions. Their activity is controlled by two different biochemical reactions, the GDP/GTP exchange and the GTP hydrolysis. These very slow reactions require catalysis in cells by ...

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Rho GTPases: Deciphering the Evolutionary History of a Complex Protein Family

Rho GTPases constitute a significant subgroup of the eukaryotic Ras superfamily of small GTPases implicated in the regulation of diverse cellular processes, such as the dynamics of the actin cytoskeleton, establishment, and maintenance of cell polarity and membrane trafficking. W ...

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Historical Overview of Rho GTPases

In 1985, the first members of the Rho GTPase family were identified. Over the next 10 years, rapid progress was made in understanding Rho GTPase signalling. Multiple Rho GTPases were discovered in a wide range of eukaryotes, and shown to regulate a diverse range of cellular processes, including cyt ...

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Promoter-Associated Noncoding RNA from the CCND1 Promoter

More than 90% of the human genome have been found to be transcribed and most of the transcripts are noncoding (nc) RNAs (Willingham et al., Science 309:1570–1573, 2005; ENCODE-consortium, Science 306:636–640, 2004; Carninci et al., Science 309:1559–1563, 2005; Bertone et al., Science 306:2242–22 ...

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Detection and Characterization of Transcription Termination

In most eukaryotes, the generation of the 3′ end and transcription termination are initiated by cleavage of the pre-mRNA upstream of the polyadenylation site. This cleavage initiates 5′–3′ degradation of the 3′ end cleavage product by the exoribonuclease Rat1p leading to the dissociation of ...

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Quantitative Analysis of Transcription Elongation by RNA Polymerase I In Vitro

The elongation step in transcription has gained attention for its roles in regulation of eukaryotic gene expression and for its influence on RNA processing. Sophisticated genetic analyses have identified factors and/or conditions that may affect transcription elongation rate or ...

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Analysis of Co-transcriptional RNA Processing by RNA-ChIP Assay

It was initially assumed that RNA biogenesis and processing were two independent processes with transcripts undergoing splicing only after being completely synthesized and released from the DNA template. However, transcription and splicing are tightly linked and increasing e ...

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Genome Wide Full-Length Transcript Analysis Using 5 and 3 Paired-End-Tag Next Generation Sequencing (RNA-PET)

RNA-PET is a paired end tag (PET) sequencing method for full-length mRNA transcripts analysis using the next generation sequencer platforms such as Illumina GA and SOLiD. Unlike RNA-Seq method that sequences randomly sheared shotgun RNA short fragments, RNA-PET captures and sequences ...

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In Vivo Run-On Assays to Monitor Nascent Precursor RNA Transcripts

Biochemical methods have provided mechanistic insights into the different transcription phases during which the RNA polymerase is assembled at gene promoter and becomes engaged in the elongation of nascent transcripts. Evidence that transcription takes place in specific reg ...

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Array-Based Nuclear Run-On Analysis

There is extensive evidence that posttranscriptional mechanisms of gene regulation, such as mRNA turnover, critically affect the patterns of expressed mRNAs. Conventional microarray analysis measures steady-state messenger RNA (mRNA) levels, which represents the dynamic ...

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Analysis of mRNA Abundance and Stability by Ribonuclease Protection Assay

Gene expression is a multi-step process, which proceeds from DNA through RNA to protein. The tight regulation of this process is essential for overall cellular integrity and physiological homeostasis. Regulation of the messenger RNA (mRNA) levels has emerged as a crucial event in the modul ...

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Methods for Analyzing Histone Citrullination in Chromatin Structure and Gene Regulation

Histone posttranslational modifications play significant roles in regulating chromatin structure and gene expression. One of the histone modifications, histone citrullination, is catalyzed by an enzyme called peptidylarginine deiminase 4 (PAD4, also called PADI4), which ...

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Affi nity Purifi cation of MLL3/MLL4 Histone H3K4 Methyltransferase Complex

Methylation on histone H3 lysine 4 (H3K4) correlates with actively transcribed genes. In mammalian cells, there exist multiple Set1-like histone H3K4 methyltransferase complexes, which have overlapping but distinct subunit compositions. Developing methods to isolate each of ...

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Reconstitution of Active and Stoichiometric Multisubunit Lysine Acetyltransferase Complexes in Insect Cells

Protein lysine acetyltransferases (KATs) catalyze acetylation of the ε-amino group on a specific lysine residue, and this posttranslational modification is important for regulating the function and activities of thousands of proteins in diverse organisms from bacteria to hum ...

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Purifi cation of Multiprotein Histone Acetyltransferase Complexes

The reversible acetylation of specific lysine residues on core histones regulates gene transcription in eukaryotes. Since the discovery of GCN5 as the first transcription-regulating histone acetyltransferase (HAT), a variety of HATs have now been identified and shown to acetyla ...

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Evaluation of Histone-Modifying Enzymes in Stem Cell Populations

The histone demethylases are a relatively novel family of histone-modifying enzymes. Their gene expression suggests that each of the subfamily members may have a discrete role in cell function. The KDM5 family of H3K4 histone demethylases has four members. Each family member has a distinct c ...

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Mapping ProteinDNA and ProteinProtein Interactions of ATP-Dependent Chromatin Remodelers

Chromatin plays a key regulatory role in several DNA-dependent processes as it regulates DNA access to different protein factors. Several multisubunit protein complexes interact, modify, or mobilize nucleo-somes: the basic unit of chromatin, from its original location in an ATP-dep ...

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