Transcription Factor Protocols, Softcover reprint of hardcover 1st ed. 2000 Methods in Molecular Biology Series, Vol. 130

The effort to sequence the human genome is now moving toward a c- clusion. As all of the protein coding sequences are described, an increasing emphasis will be placed on understanding gene function and regulation. One important aspect of this analysis is the study of how transcription factors re- late transcriptional initiation by RNA polymerase II, which is responsible for transcribing nuclear genes encoding messenger RNAs. The initiation of Class II transcription is dependent upon transcription factors binding to DNA e- ments that include the core or basal promoter elements, proximal promoter elements, and distal enhancer elements. General initiation factors are involved in positioning RNA polymerase II on the core promoter, but the complex - teraction of these proteins and transcriptional activators binding to DNA e- ments outside the core promoter regulate the rate of transcriptional initiation. This initiation process appears to be a crucial step in the modulation of mRNA levels in response to developmental and environmental signals. Transcription Factor Protocols provides step-by-step procedures for key techniques that have been developed to study DNA sequences and the protein factors that regulate the transcription of protein encoding genes. This volume is aimed at providing researchers in the field with the well-detailed protocols that have been the hallmark of previous volumes of the Methods in Molecular ? Biology series.

Isolation of Target Gene Promoter/Enhancer Sequences by Whole Genome PCR Method.- In Vivo Footprinting Using UV Light and Ligation-Mediated PCR.- Identification of DNaseI Hypersensitive Sites Within Nuclei.- Analysis of In Vivo Methylation.- Detection of Transcription Factor Partners with a Yeast One Hybrid Screen.- Inverse PCR (IPCR) for Obtaining Promoter Sequence.- PCR-Directed Linker Scanning Mutagenesis.- Transfection Technologies.- The Use of Particle-Mediated Gene Transfer for the Study of Promoter Activity in Somatic Tissues.- Optimizing Electroporation Conditions for the Transformation of Mammalian Cells.- Calcium Phosphate Transfection of Mammalian Cultured Cells.- DEAE-Dextran Transfection of Mammalian Cultured Cells.- Liposome-Mediated Transfection of Mammalian Cells.- Assays for Transcriptional Activity Based on the Luciferase Reporter Gene.- Transient Transfection of Schneider Cells in the Study of Transcription Factors.- Triplex-Forming Oligonucleotides and Their Use in the Analysis of Gene Transcription.- Expression and Purification of Histidine-Tagged Transcription Factors.- Generation of Transcription Factors in Rabbit Reticulocyte Lysate Depleted of Endogenous DNA-Binding Protein.- Electrophoretic Mobility Shift Assays.- In Vitro Promoter Analysis Using Nuclear Extracts and G-Free Cassette Vectors.- In Vitro Transcription Using Competitor Oligonucleotides to Deplete Specific Transcription Factors.- Computer Software of Eukaryotic Promoter Analysis.

Includes supplementary material: sn.pub/extras