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Nuclear Recruitment Assay as a Tool to Validate Transcription Factor Interactions in Mammalian Cells
Identification and verification of novel transcription factor interactions is an inherent step in the discovery of molecular mechanisms driving gene transcription and regulation. Co-immunoprecipitation and GST-pull down are often key techniques in the verification process. Despite wide applicability, their use may sometimes be restricted. We provide a detailed protocol for an intracellular immunofluorescence technique that may be used as an alternative or complimentary study for transcription factor interaction verification. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - February 25, 2013 Category: Genetics & Stem Cells Source Type: news

Fluorescence Cross-correlation Spectroscopy (FCCS) to Observe Dimerization of Transcription Factors in Living Cells
Fluorescence cross-correlation spectroscopy (FCCS) is an established spectroscopic method to observe the interaction between the different fluorescent molecules. Using FCCS, researchers can assess the interaction of target molecules in the aqueous condition, and can apply the technique in cultured cells. Here, we describe the method of FCCS to demonstrate direct observation of dimerization between transcription factors in a living cell. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - February 25, 2013 Category: Genetics & Stem Cells Source Type: news

Promoter Independent Abortive Transcription Assays Unravel Functional Interactions Between TFIIB and RNA Polymerase
TFIIB-like general transcription factors are required for transcription initiation by all eukaryotic and archaeal RNA polymerases (RNAPs). TFIIB facilitates both recruitment and post-recruitment steps of initiation; in particular, TFIIB stimulates abortive initiation. X-ray crystallography of TFIIB-RNAP II complexes shows that the TFIIB linker region penetrates the RNAP active center, yet the impact of this arrangement on RNAP activity and underlying mechanisms remains elusive. Promoter-independent abortive initiation assays exploit the intrinsic ability of RNAP enzymes to initiate transcription from nicked DNA templates a...
Source: Springer protocols feed by Genetics/Genomics - February 25, 2013 Category: Genetics & Stem Cells Source Type: news

Using FRET to Monitor Protein-Induced DNA Bending: The TBP-TATA Complex as a Model System
Proteins that bind to DNA can elicit changes in DNA conformation, such as bending and looping, which are important signals for later events such as transcription. TATA-binding protein (TBP) is one example of a protein that elicits a conformational change in DNA; TBP binds and sharply bends its recognition sequence, which is thought to facilitate the recruitment of other protein factors. Here we describe the use of fluorescence resonance energy transfer (FRET) to evaluate DNA bending using TBP as a model system. FRET is a useful technique to measure changes in DNA conformation due to protein binding because small changes in...
Source: Springer protocols feed by Genetics/Genomics - February 25, 2013 Category: Genetics & Stem Cells Source Type: news

Chromatin Assembly and In Vitro Transcription Analyses for Evaluation of Individual Protein Activities in Multicomponent Transcriptional Complexes
Eukaryotic DNA and core histones form the fundamental repeating units of chromatin. Condensed c­hromatin, which has higher-order structures, prevents transcriptional complexes from accessing their target genes. Epigenetic regulation, including structural changes of chromatin, histone modification, and DNA methylation, strictly controls the pattern of gene expression and silencing. Recent studies have revealed that histone acetylation plays a crucial role in relaxing chromatin structure for initiation of transcription. Crosstalk between DNA-binding transcription factors and histone acetyltransferases (HATs) serves as a ...
Source: Springer protocols feed by Genetics/Genomics - February 25, 2013 Category: Genetics & Stem Cells Source Type: news

Quantitative NanoProteomics Approach for Protein Complex (QNanoPX) Using Gold Nanoparticle-Based DNA Probe
Affinity purification by pulldown methods using target-bound gel beads provides a powerful approach for purifying endogenous protein complexes. Such methods can be improved by using nanoparticle-based probe, coupled with immunoblot analysis or quantitative proteomics method using stable isotope labeling via liquid chromatography-mass spectrometry (LC-MS). Here, we describe sample preparation and a pulldown method using gold nanoparticle-based DNA probe for characterizing the transcriptional complex of estrogen response element (ERE). The described protocol includes the fabrication of gold nanoparticle-based probe, nuclear ...
Source: Springer protocols feed by Genetics/Genomics - February 25, 2013 Category: Genetics & Stem Cells Source Type: news

Combination of Native and Denaturing PAGE for the Detection of Protein Binding Regions in Long Fragments of Genomic DNA
In traditional electrophoresis mobility shift assay (EMSA) a single 32P-labeled double-stranded DNA oligonucleotide or a restriction fragment bound to a protein is separated from the unbound DNA by polyacrylamide gel electrophoresis (PAGE) under nondenaturing conditions. An extension of this method uses a population of DNA restriction fragments derived from long genomic regions for the identification of fragments containing protein binding regions. Although the method allows simultaneous analysis of large fragments, it is relatively laborious and can be used to detect only fragments containing high affinity protein binding...
Source: Springer protocols feed by Genetics/Genomics - February 25, 2013 Category: Genetics & Stem Cells Source Type: news

Electrophoretic Mobility-Shift and Super-Shift Assays for Studies and Characterization of Protein–DNA Complexes
Gene expression is in part regulated by transcription factors that bind specific sequence motifs in genomic DNA. Transcription factors cooperate with the basal machinery to upregulate or downregulate transcription. Experimental data have revealed the importance of interactions among members of distinct families of transcription factors to form complexes that regulate gene expression. Thus, a full characterization of protein–DNA complexes is essential to understanding of gene regulation in a more complex cellular environment. Electrophoretic mobility shift assay (EMSA) is a powerful technique to resolve nucleic acid&n...
Source: Springer protocols feed by Genetics/Genomics - February 25, 2013 Category: Genetics & Stem Cells Source Type: news

Identifying Specific Protein–DNA Interactions Using SILAC-Based Quantitative Proteomics
A comprehensive identification of protein–DNA interactions that drive processes such as transcription and replication, both in prokaryotic and eukaryotic organisms, remains a major technical challenge. In this chapter, we present a SILAC-based DNA affinity purification method that can be used to identify specific interactions between proteins and functional DNA elements in an unbiased manner. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - February 25, 2013 Category: Genetics & Stem Cells Source Type: news

A Modified Yeast One-Hybrid System for Genome-Wide Identification of Transcription Factor Binding Sites
The yeast one-hybrid system is a powerful genetic method to identify DNA–protein interactions, but there is a major limitation inherent to the system. Namely, frequency of false positives generated by yeast endogenous transcription factors has been thought to be higher than that of true positives by orders of magnitude. However, our modification efficiently can eliminate the false positives. When compared to the other methods for the analysis of DNA–protein interactions on a genome-wide scale, a modified yeast one-hybrid system offers several advantages including low initial and running cost, large-scale output...
Source: Springer protocols feed by Genetics/Genomics - February 25, 2013 Category: Genetics & Stem Cells Source Type: news

Mycosis Fungoides and Sézary Syndrome
The development of array comparative genomic hybridization (aCGH) techniques has allowed to characterize more precisely several human neoplasms with the aim of providing prognostic markers and targets for directed therapeutic intervention. Recently, several studies applying aCGH technique have been reported in which an exhaustive genetic characterization of mycosis fungoides (MF) and Sézary syndrome (SS) has been performed. Regarding MF, a genomic profile characterized by the gains of 7q, 17q, and 8q and losses in 9p, 13q, 17p, and 10q has been described. In SS, the most common abnormalities are gains in 8q and 17q ...
Source: Springer protocols feed by Genetics/Genomics - February 15, 2013 Category: Genetics & Stem Cells Source Type: news

Array CGH Reveals Clonal Evolution of Adult T-Cell Leukemia/Lymphoma
Adult T-cell leukemia/lymphoma (ATLL) is the neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1). We performed oligoarray comparative genomic hybridization (CGH) against paired samples comprising peripheral blood (PB) and lymph node (LN) samples from patients with acute-type ATLL. Results disproved the established theory that true monoclonal proliferation, such as identical clonal expansion in all respects, occurred in acute-type ATLL, and our findings revealed that acute-type ATLL contains multiple subclones with differing genomic aberrations. Oligoarray CGH technology has been developed not only for high-resol...
Source: Springer protocols feed by Genetics/Genomics - February 15, 2013 Category: Genetics & Stem Cells Source Type: news

Analysis of Acquired Genomic Copy Number Aberrations and Regions of Loss of Heterozygosity in Acute Myelogenous Leukemia Genomes Using Affymetrix SNP 6. Arrays and Supporting Software Tools
The application of SNP array technology to the analysis of cancer genomes has greatly advanced our knowledge of the incidence and functional consequences of acquired genomic copy number aberrations (aCNA) and LOH in various malignancies. The major challenges of using SNP arrays are accurately identifying acquired genomic DNA aberrations in the raw array data with very high sensitivity and specificity and meaningfully assessing the associations between these aberrations and biological characteristics or patient outcomes. Critical to the success and valid interpretation of data derived from SNP array profiling are (1) the pu...
Source: Springer protocols feed by Genetics/Genomics - February 15, 2013 Category: Genetics & Stem Cells Source Type: news

CGH Protocols: Chronic Lymphocytic Leukemia
Array-based comparative genomic hybridization (aCGH) is a powerful assay to identify copy number abnormalities underlying the pathogenesis of cancer. aCGH has become the gold standard for whole genome copy number analysis in medium and large cohorts in clinical and research laboratories. Identifying the best workflow is critical to achieving the optimal performance for this assay. Here we describe the aCGH protocol used by our group in the study of B-chronic lymphocytic leukemia (CLL). We also describe some initial applications of aCGH in association with clinical outcome for CLL. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - February 15, 2013 Category: Genetics & Stem Cells Source Type: news

The Use of Cytogenetic Microarrays in Myelodysplastic Syndrome Characterization
Various microarray platforms, including BAC, oligonucleotide, and SNP arrays, have been shown to ­provide clinically useful diagnostic and prognostic information for patients with myelodysplastic syndromes (MDS). Clinically useful arrays are designed with specific purposes in mind and with attention to genomic content and probe density. All array types have been shown to detect genomic copy gains and losses, with SNP arrays having the added advantage of detecting copy neutral loss of heterozygosity (CNLOH). The finding of CNLOH has led to the identification of certain disease genes implicated in the initiation or progr...
Source: Springer protocols feed by Genetics/Genomics - February 15, 2013 Category: Genetics & Stem Cells Source Type: news