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Despite remarkable progress in genome science, we are still far from a clear understanding of how genomic DNA is packaged without entanglement into a nucleus, how genes are wrapped up in chromatin, how chromatin structure is faithfully inherited from mother to daughter cells, and how the differential expression of genes is enabled in a given cell type. Exploring and answering these questions constitutes one of the next frontiers in the 21st century. We are just beginning to appreciate how Multifarious DNA structures provide additional structural and functional dimensions to chromatin organization and gene expression. DNA Conformation and Transcription is the first book that compiles the fruits of the studies that have been performed to date to solve the riddle ‘written’ in DNA conformation ("conformation code"). This book provides a comprehensive overview of the field by covering history of the field, up-to-date topics, clarifications of present day research, and future perspective of what is still to be discovered. Thus, it serves as an invaluable source of information on the "conformation code".
Our understanding of the mechanisms regulating gene expression, which determine the patterns of growth and development in all living organisms, ultimately involves the elucidation of the detailed and dy namic interactions of proteins with nucleic acids -both DNA and RNA. Until recently the commonly presented view of the DNA double helix as visualized on the covers of many textbooks and journals - was as a monotonous static straight rod incapable in its own right of directing the processes necessary for the conservation and selective reading of genetic information. This view, although perhaps extreme, was reinforced by the necessary linearity of genetic maps. The reality is that the biological functions of both DNA and RNA are dependent on complex, and sometimes transient, three-dimensional nucleoprotein structures in which genetically distant elements are brought into close spatial proximity. It is in such structures that the enzymatic manipulation of DNA in the essential biological processes as DNA replication, transcription and recombination are effected - the complexes are the mediators of the 'DNA transactions' of Hatch Echols.
Since the publication of the first edition five years ago, a wide range of new methodologies have been developed to facilitate studies on both isolated parts of the genome and the genome as a whole. This new edition has been updated and expanded so that it provides a comprehensive guide to the methods currently available to characterize the function and activity of an individual transcription factor. All the original chapters have been fully updated or rewritten and additional chapters cover the use of in vitro transcription assays, analysis of chromatin structure, use of the genomic binding site assay and analysis of transcription factor modifications. As with the previous edition, the book starts with a series of chapters concerned with characterizing the proteins binding to a specific DNA sequence and then a chapter on more detailed characterization of the protein itself. The next two chapters describe the isolation of cDNA clones encoding a transcription factor using oligonucleotides predicted from protein sequence and screening of a cDNA expression library. Chapter 6 deals with identification of transcription factors based on sequence homology analysis by both experimental screening and database searches. Chapter 7 is a new chapter that describes methods of identifying the target genes of a previously uncharacterized factor. The next chapters deal with analysis of transcription factor function. Chapter 8 deals with general techniques, and then the following chapters cover the specialized techniques of in vitro transcription assays using transcriptionally active nuclear extracts derived from rat brain, and analysis of the effect of transcription factors on chromatin structure. The final chapter describes methods for detecting the phosphorylation and glycosylation state of transcription factors.
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 TM Biology series.
The Regulation of DNA Replication and Transcription explores basic processes of DNA replication and transcription in an effort to identify the mechanisms responsible for the release of genetic information and its role in the regulation of cellular events. Concerned with discovering the fundamental concept that might integrate and explain the wide range of existing lines of evidence, the author reports and interprets the results of experiments conducted in an impressive range of biological systems. Focused on complex mechanisms at the biochemical level, these studies allow analysis of the pathways involved when cells, organs and animal systems react to various trigger molecules derived from both living cells and exogenous sources. These include hormones, RNA, RNA fragments, alkaloids, actinomycin D, and phorbol esters, as well as chemical carcinogens and drugs. Comnining the results of these studies with his own extensive work in this field, the author is able to formulate a uniquely integrative biochemical model for the gene expression, demonstrating that both biological and chemically synthesized molecules can trigger the differential release of information from the DNA and thus influence cell transformation. Apart from its academic significance, the model offers high potential assistance in the search for ways to induce or control the expression of certain genes and, moreover, to promote differentiation of given cells in vitro as well as in situ.