Gene Transcription

Understanding the mechanisms and control of gene transcription is said to be one of the central goals for a large proportion of molecular biologists currently involved in research. This book concentrates on RNA polymerase II transcription of eukaryotic protein-coding systems, but many of the approaches and techniques described apply equally well to the study of systems involving RNA polymerases I and III. This book covers the major current procedures and approaches in this field, including not only the analysis of transcription "in vitro" and "in vivo", but also the identification, purification and characterization of transcription factors and their interaction with specific DNA target sites. It is written by experts and specifically designed for all researchers intent on studying eukaryotic gene expression. Its mix of background information, advice on experimental approaches, detailed practical protocols, typical results, help with troubleshooting and reference appendices make it a guide for the newcomer to the field, and a source of new approaches and techniques for more experienced researchers. David Hames is one of the series editors of the "Practical Approach" series. Other titles in the "PA" series include "Transcription and Translation", "Nucleic Acid Hybridization", "Gel Electrophoresis of Proteins" and "Gel Electrophoresis of Nucleic Acids".

Table of Contents:
Part 1 Assay of gene transcription "in vitro", Philip J. Mason et al: northern transfer; RNA slot blots; analysis of specific RNA molecules by polymerase chain reaction (PCR); S1 nuclease mapping; RNase mapping; primer extension; "in situ" hybridization. Part 2 Transcription of exogenous genes in mammalian cells, Kevin Docherty and Andrew R. Clark: introduction of exogenous DNA into mammalian cells in culture; transient and stable expression; vectors for introducing DNA into mammalian cells in culture; the characterization of transcriptional regulatory sequences; mutagenesis of reporter gene constructs. Part 3 "In vitro" transcription with nuclear extracts differentiated tissues, Felipe Sierra et al: general practical considerations; preparation of transcriptionally-active nuclear extracts from solid rat tissues; analysis of "in vitro" transcripts; factors affecting the efficiency of "in vitro" transcription; trouble-shooting. Part 4 Transcriptional analysis using transgenic animals, Niall Dillon and Frank Grosveld: introduction - transgenic mice; generation of transgenic mice; setting up a transgenic operation for the analysis of gene function; isolation of DNA for micro-injection; integration of injected genes; generation of transgenic mice using embryonic stem cells; breeding of transgenic animals; design of transgenic experiments; other transgenic systems. Part 5 Identification and characterization of eukaryotic transcription factors, Stephen P. Jackson: DNA binding assays for identifying transcription factors in crude cell extracts; identifying the DNA-binding polypeptide; purification of sequence-specific transcription factors using DNA binding site affinity chromatography; determining the purity and activity of a purified transcription factor; problems and trouble-shooting; isolation of cDNA clones encoding transcription factors; post-translational modification of transcription factors. Part 6 Analysis of protein-DNA interactions, Michael J. Garabedian et al: the mobility shift DNA-binding assay for protein-DNA interactions; assays for sites of protein-DNA contact - footprinting techniques; methylation interference assay; random mutagenesis of DNA sequences; purification of DNA-binding proteins. Part 7 Cloning and fuctional analysis of heterologous eukaryotic transcription factors in yeast, Daniel M. Becker and John D. Fikes: expression cloning of heterologous transcription factors in yeast; analysis of heterologous transcription factors in yeast. Appendices: transcription controls - cis-elements and trans-factors, Joseph Locker; fractionation of nucleic acids by gel electrophoresis, B.D. Hames and S.J. Higgins; addresses of suppliers.