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Nucleosomes and the chromatin structures they assemble provide the architectural foundation for the transcription process. We describe the use of reconstituted chromatin templates assembled using purified histones and HMGs to investigate the significance of chromatin structure for transcription. Our structural assays include sucrose gradient fractionation, nucleoprotein gel analysis, micrococcal nuclease...
The use of the scanning force microscope (SFM) to visualize and analyze chromatin fiber structures is presented. Protocols to prepare chromatin fibers for SFM imaging of fibers in air and in buffer are first discussed. Next, the conditions for acquiring high-quality SFM images such as optimal instrumental parameters, appropriate deposition substrates, and adequate procedures of sample deposition are...
A number of important nuclear processes including replication, recombination, repair, and transcription involve the interaction of soluble nuclear proteins with DNA assembled as chromatin. Recent progress in a number of experimental systems has focused attention on the influence chromatin structure may exert on gene regulation in eukaryotes. With the advent of new technologies for the analysis of...
Electron microscopy, with its ability to image DNA and nucleosomes, can provide a key visual link in the understanding of chromatin conformation. We discuss applications of EM to current chromatin research with emphasis on strategies that eliminate many of the potential problems associated with conventional EM preparative techniques. Cryo-electron microscopy (cryo-EM) of isolated chromatin, whereby...
Our current level of understanding of chromatin structure was to a large extent achieved with the help of DNA–protein cross-linking. The versatile inventory of cross-linking techniques allows the identification of the contacts between DNA and proteins with a single nucleotide–single amino acid precision, to detect minor components of the complex nucleoprotein systems, to reveal the interactions of...
Nucleoprotein gel electrophoresis, as well as its use in chromatin research, is reviewed from its early application in the characterization of native nucleosome composition to current uses in analyzing transcription factor–nucleosome complexes and in visualizing multiple nucleosome positioning. Despite our incomplete understanding of the principles behind the separation of particles in native polyacrylamide...
Analytical ultracentrifugation and agarose gel electrophoresis each can be used to accurately quantify changes in structure that accompany chromatin folding in solution. Analytical ultracentrifugation directly measures the extent of compaction of each species present in a chromatin sample under a wide range of solution conditions. Agarose gel electrophoresis yields information about changes in the...
Several promoters have been shown to have sequence specifically positioned nucleosomes that determine the architecture of the promoter. DNA binding proteins that regulate gene expression are in many cases known to bind to their cognate DNA segments organized within such positioned nucleosomes. It has become increasingly evident that the cooperation of chromatin and transcription factors results in...
Acetylation of specific lysine residues in the N-terminal domains of core histones is a biochemical marker of active genes. To determine the spatial and temporal distribution of this reversible posttranslational modification, affinity-purified polyclonal antibodies recognizing the epitope ϵ-acetyllysine have been used in immunoselection procedures with mononucleosomes and salt-soluble chromatin fragments...
Increasingly, biochemical analyses of processes that occur within eukaryotic nuclei such as transcription and replication require the construction of specific chromatin substrates. Nucleosome complexes reconstitutedin vitrohave been key elements in a variety of recent studies of polymerase progression andtrans-acting factor binding activities. Reconstituted complexes can be easily constructed from...
A two-dimensional electrophoresis for fine separation of histones is described in detail. The method is relatively simple and gives very reproducible results. In the first dimension the histones are separated by their charge in acid–urea gels, while in the second dimension the separation is based on both the charge and the differential affinity of histones to Triton in acid–urea–Triton gels. In this...
The biochemical analysis of chromatin structure and function is greatly facilitated by the availability of cell-free systems that assemble chromatin under physiological conditions. One such system that has shown great potential is derived from extracts of earlyDrosophilaembryos. These embryos contain large maternal stocks of chromatin constituents, such as histones and assembly factors. Chromatin...
Use of the rat as host for the study of cancer has become popular for several reasons. The larger body size compared to mice is especially convenient for lines of experiments involving surgical manipulation, transplantation, or biochemical purification of molecules of interest. Immune response to cancer is also studied in rat models, and this article focuses on the methodological aspects ofin vivoandin...
Strategies have been developed to characterize tumor antigens recognized by cytolytic T lymphocytes (CTL). We use a genetic approach based on the transfection of HLA genes and cDNA libraries in COS cells to isolate the gene producing the antigenic peptide. The tumor-specific expression of this gene can be evaluated by cDNA synthesis and quantitative PCR amplification. Transfection of fragments of...
In preclinical models, tumor cells genetically altered to secrete cytokines or express costimulatory molecules can generate systemic antitumor immunity. In some studies, these tumor vaccines have been shown to eradicate micrometastases. These results have led to the initiation of numerous phase I clinical trials employing either genetically modified or allogenic tumor vaccines. This article addresses...
This article describes the methods used for the purification of heat shock proteins gp96, hsp90, and hsp70 from murine and human tissues and cell lines. The heat shock proteins purified by the methods described are associated noncovalently with an array of cellular peptides. The use of heat shock protein–peptide complexes for eliciting prophylactic and therapeutic immunity to cancers and infectious...
The use of recombinant and synthetic vaccines in the treatment of cancer has recently been explored using model tumor associated antigens (TAA), many of which do not model the immunological state of affairs in which the TAA is expressed by normal tissues. One potentially useful model Ag is β-galactosidase (β-gal). Because the activity of this enzyme is so easily detectable, this gene has been inserted...
Plasma membranes isolated from tumor cells retain biologically active class I MHC proteins on their surfaces. CD8 + T-cell activation by membrane antigen is much more effective when the small membrane vesicles (<1-μm diameter) are displayed on a surface with dimensions approaching those of a cell (5-μm diameter). Previous work had shown that tumor membrane antigen incorporated onto silica...
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