Showing papers in "Methods in Cell Biology in 1978"
TL;DR: The chapter represents the most detailed account of preparative procedures available for mitochondria and submitochondrial particles of rat liver and presents flow diagrams that include many of the methods to obtain an overall view of a given procedure.
Abstract: Publisher Summary This chapter discusses techniques for the preparation and characterization of mitochondrial and submitochondrial fractions from liver and liver-derived tissues. The chapter represents the most detailed account of preparative procedures available for mitochondria and submitochondrial particles of rat liver. The chapter initially summarizes several methods for isolating rat liver mitochondria. Some of these methods yield type-1 mitochondria (partially purified but intact), and some yield type-2 mitochondria (highly purified). The two most common tests used to determine the quality of mitochondrial and submitochondrial preparations involve an examination of their respiratory characteristics in a small chamber equipped with an oxygen electrode, and an examination of their structural and morphological features under the electron microscope. The chapter presents flow diagrams that include many of the methods to obtain an overall view of a given procedure. The methodology involved in preparing mitochondria for evaluation in the electron microscope is summarized here, together with a few statements about what each step involves and why it is carried out.
436 citations
TL;DR: This chapter describes the technique for the resolution of histones polyacrylamide gel electrophoresis in presence of nonionic detergents, which avoids the precipitation problems and the possibility of differential loss of proteins during the transition from one buffer system to another.
Abstract: Publisher Summary This chapter describes the technique for the resolution of histones polyacrylamide gel electrophoresis in presence of nonionic detergents. Histones are difficult to resolve by classical biochemical fractionation techniques because of their similarity in size and charge, their tendency to aggregate, and the high frequency of post-transcriptional charge modification. The most widely used analytical system for histones; polyacrylamide gel electrophoresis at low pH resolves five major histone species and some of their modified forms. The resolution of the histones can be improved by the addition of nonionic detergents to the gels that results in a differential reduction in the electrophoretic mobility of different histones. This effect is because of the formation of mixed micelles between the detergent and the hydrophobic regions of protein molecules and is extremely sensitive to small differences in the hydrophobic properties of the proteins. For qualitative comparison of complex protein mixtures, the electrophoresis system can be combined with a simple and effective two-dimensional electrophoresis technique that uses the same buffer system in both directions and therefore avoids the precipitation problems and the possibility of differential loss of proteins during the transition from one buffer system to another.
309 citations
TL;DR: This chapter illustrates that crude homogenates and crude nuclei have minimal RNA synthetic activity but activity becomes evident on further purification, and in each system careful evaluation of conditions for optimal activity is required and they may well vary extensively.
Abstract: Publisher Summary This chapter describes a system that fulfills many of the requirements for the study of gene control in vitro. The system makes possible the study of the primary transcription event separate from later maturation and transport events in RNA synthesis. While the results in the chapter are described for myeloma cells, lines derived from the MPC-11 tumor, the preliminary results with chick embryos, and those of others with HeLa cells and other myeloma cells suggest that these methods may be generally applicable. There are many examples of systems of isolated nuclei where the RNA product is small. Moreover, the isolated nuclei synthesize a very small product, but the nuclear subfraction they isolate (nucleoli) synthesizes very large RNA. This chapter illustrates that crude homogenates and crude nuclei have minimal RNA synthetic activity but activity becomes evident on further purification. Thus, in each system careful evaluation of conditions for optimal activity is required and they may well vary extensively.
207 citations
TL;DR: In the chapter, cross-linking of histones in chromatin and in free solution is described as well as examples of cross- linking with dimethyl suberimidate are presented.
Abstract: Publisher Summary This chapter presents a study of histone–histone associations by chemical cross-linking. Chemical cross-linking can be used to reveal both the pattern and the degree of association of polypeptides in a multisubunit structure. Limited cross-linking results in dimmers that are formed from neighboring polypeptides. Extensive cross-linking gives a series of higher-molecular-weight products, the largest of which comprises the total number of subunits in the structure. Both types of analysis have been applied to the histones with the use of a variety of cross-linking agents, such as formaldehyde, imidoesters, tetranitromethane, ultraviolet light, and dicyclohexylcarbodiimide. The chapter focuses on the imidoesters, whose reaction with proteins is well understood. The procedures for cross-linking with imidoesters are straightforward, and success in their application to histones and chromatin is largely dependent on the resolving power of the methods used to identify the cross-linked products. Fractionation of the histones and cross-linked products is difficult because of their similar charges and molecular weights. In the chapter, cross-linking of histones in chromatin and in free solution is described as well as examples of cross-linking with dimethyl suberimidate are presented.
136 citations
TL;DR: With nonmetabolizing systems, the dual-syringe apparatus is adaptable to a variety of experimental protocols-zero-trans, equilibrium exchange, and infinite-cis—which in combination make possible a thorough kinetic characterization of a transport system.
Abstract: Publisher Summary The chapter discusses techniques, which permit an operational separation of transport and metabolism. This separation can be achieved by genetic, chemical, or kinetic manipulation, or a combination thereof. The transport of various compounds across mammalian cell membranes is frequently found to occur with a rapidity which necessitates collecting data at intervals of a few seconds. By means of a dual-syringe device, suspended cells can be mixed nearly instantaneously with radioactively labeled substrate and separated from the substrate again within seconds by centrifugation into silicone oil. Depending on the cell-substrate system under investigation, initial transport velocities may be either measured directly or calculated from the time course with which equilibrium across the membrane is attained. With nonmetabolizing systems, the dual-syringe apparatus is adaptable to a variety of experimental protocols-zero-trans, equilibrium exchange, and infinite-cis—which in combination make possible a thorough kinetic characterization of a transport system.
125 citations
TL;DR: This chapter describes methods of preparing and analyzing the ribosomal proteins of S. cerevisiae and points out certain properties that are shared by the riboterrorism proteins of all eukaryotes.
Abstract: Publisher Summary The chapter studies the ribosomal proteins of Succharomyces cerevisiae because it is possible to obtain mutants which are likely to involve ribosomal proteins and focuses on the coordinated regulation of their synthesis. This chapter describes methods of preparing and analyzing the ribosomal proteins of S. cerevisiae and points out certain properties that are shared by the ribosomal proteins of all eukaryotes. Ribosomal proteins are analyzed directly on one-dimensional sodium dodecyl sulfate (SDS) gels without removing RNA or on two systems of two-dimensional polyacrylamide gel. The first is based on the Kaltschmidt–Wittman system and employs 6 M urea at pH 8.6 in the first dimension and at pH 4.5 in the second dimension. The second is a modified version of the Mets and Bogorad system and employs 8 M urea at pH 5.0 in the first dimension and sodium SDS in the second dimension. The two systems are described separately, and the results then compared. For both gel systems the importance of using proteins free of RNA cannot be overemphasized. RNA binds to ribosomal proteins even in urea solutions and causes poor resolution and decreased yields.
98 citations
TL;DR: Treatment of the tissue with purified collagenase and with mild mechanical shearing forces was found to be sufficient to dissociate the tissue into minilobules and single acini, whereas an additional step of Ca2+ chelation was finding to be essential to break down the junctional elements between cells in order to obtain separated cell.
Abstract: Publisher Summary The chapter outlines the procedure for the sequential dissociation of the exocrine pancreas into lobules, acini, and individual cells. Dispersion of the pancreas into isolated single cells involves sequential enzymic digestion of stromal collagen and basement membrane, Ca2+ chelation to disrupt cell-cell junctions (that is, desmosomes and tight junctions), and mechanical shearing to complete the separation of gap and tight junctions. Filtration and washing of the tissue digest yield a purified preparation free of cell debris, collagenase, and subcellular organelles liberated in the final steps of the procedure. The protocol for tissue dissociation described allows the investigator to control the extent of dissociation of pancreatic exocrine tissue into minilobules, single acini, and separated cells, and to analyze how the progression of dissociation affects cell structure and function. Treatment of the tissue with purified collagenase and with mild mechanical shearing forces was found to be sufficient to dissociate the tissue into minilobules and single acini, whereas an additional step of Ca2+ chelation was found to be essential to break down the junctional elements between cells in order to obtain separated cell.
84 citations
TL;DR: The chapter discusses the Lassie test—a simple test devised for qualitatively comparing the spontaneous mutation rates in two different strains of yeast, and the Leningrad test—an ingenious and accurate method for measuring the spontaneous reversion rates for alleles of ade1 and ade2 of Saccharomyce.
Abstract: Publisher Summary The first method for measuring spontaneous mutation rates in microorganisms was based on variance in mutant number from clone to clone, which was caused by rare mutations early in the growth of a clone yielding. The most accurate method for measuring the spontaneous mutation rate in yeast is based on the continued growth of prototrophic revertants after a required nutrient in the medium are exhausted. A synthetic complete medium is used in which the relevant nutrient is low enough in concentration so that the titer of cells in the medium is kept well below the saturation level. This nutrient medium is called the limiting medium. For the method described in the chapter, the relevant titer of cells is usually limited to somewhere between 105 and l07 cells/ml. The titer is regulated by altering the concentration of the limiting nutrient so that a reasonable number of revertants can be scored. Special care must be taken with two items in order to ensure maximum accuracy: (1) evaporation from the compartments must be minimal, and (2) small variations in temperature can alter measurably the spontaneous mutation rate. The chapter discusses the Lassie test—a simple test devised for qualitatively comparing the spontaneous mutation rates in two different strains of yeast, and the Leningrad test—an ingenious and accurate method for measuring the spontaneous reversion rates for alleles of ade1 and ade2 of Saccharomyce. The chapter also discusses measuring the spontaneous mutation rate in mitotic cells grown in complete medium. The chapter discusses direct estimate of mutation rate from mutant frequency and the measurement of spontaneous mutation rate during meiosis.
80 citations
TL;DR: It is clear that physiologically significant amounts of proteins and nucleic acids can be transferred and the technique of red cell-mediated microinjection can be used to elucidate the roles played by various macromolecules in mammalian cells.
Abstract: Publisher Summary Red cell microinjection permits the rapid transfer of macromolecules into large numbers of mammalian cells. Routinely, l0 7 culture cells can be microinjected in several hours. A wide range of macromolecules can be transferred subject only to the limitations of red cell loading. Individual culture cells have been microinjected with l0 6 bovine serum albumin (BSA) molecules. For comparison, many chromosomal proteins are represented at l0 3 to l0 5 copies per cell and the cytoplasmic enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is found at 2 × l0 5 copies per L cell (Hughes and others, 1975). A similar comparison can be made for transfer RNA (tRNA) and other small nucleic acids. For example, it should be possible to microinject individual culture cells with 5 × l0 7 to 10 8 tRNA molecules. This level is similar to that found in various eukaryotic cell lines. Thus it is clear that physiologically significant amounts of proteins and nucleic acids can be transferred. This ensures that the technique of red cell-mediated microinjection can be used to elucidate the roles played by various macromolecules in mammalian cells.
73 citations
TL;DR: For a more complete analysis of phosphorylation occurring at growth associated sites, the more extensive procedure addressed in this chapter must be employed.
Abstract: Publisher Summary This chapter discusses the methods for the assessment of site-specific histone phosphorylation and their application in several in vivo and in vitro systems. However, for a more complete analysis of phosphorylation occurring at growth associated sites, the more extensive procedure addressed in this chapter must be employed. The procedure is also applicable to the study of H1 histone phosphorylation in vivo. In nongrowing tissues, such as adult rat liver, the phosphopeptide analysis serves in identifying ser 37 phosphorylation and in distinguishing it from radioactive contaminants normally present in H1 isolated by trichloroacetic acid extraction, as no in vivo phosphorylation of ser 106 has so far been detected. Among the properties that can be used to distinguish protein kinases, the nature of the substrates phosphorylated is the most useful; it is also a property that is likely to be closely related to the function of the protein kinase.
71 citations
TL;DR: The two most important areas of the applications of BrdU differential staining techniques are the utilization of sister chromatid exchanges (SCEs) as an indication of DNA damage and repair and the employment of BISACK for examining cellular replication.
Abstract: Publisher Summary The chapter discusses the methodology for Bromodeoxyuridine (BrdU) differential staining, both in vitro as well as in vivo, and an examination of the many applications of this technique The chapter focuses on the BrdU labeling of mammalian chromosomes in vivo They involve multiple subcutaneous injections, multiple intraperitoneal injections, or continuous intravenous infusion of BrdU Examination of the concentrations of BrdU used in vitro for differential chromatid staining reveals extreme variation, even for the same cellular system The chapter discusses the two most important areas of the applications of BrdU differential staining techniques are the utilization of sister chromatid exchanges (SCEs) as an indication of DNA damage and repair and the employment of BISACK for examining cellular replication SCE analysis potentially detects an agent, which may not be mutagenic for bacterial systems and yet be mutagenic, and perhaps even carcinogenic, for human cells SCE analyses have been employed to examine various human genetic disorders The system for analyzing cell replication kinetics (BISACK), permit new insight into the nature of replicating cell populations both in vivo and in vitro
TL;DR: This chapter describes the technique for polyacrylamide gel electrophoretic fractionation of histones, which permits an analysis of acid-labile histone modifications and some histone-histone complexes at neutral pH.
Abstract: Publisher Summary This chapter describes the technique for polyacrylamide gel electrophoretic fractionation of histones. Histones are small, basic proteins associated with the nuclear DNA of eukaryotes. They possess no known enzymic activity, however are present in large amounts in the nucleus. The most convenient means of assaying for histones is polyacrylamide gel electrophoresis. The basicity of histones means that they are positively charged at low pH; and routine electrophoretic analysis is often done at this pH that affords a separation on the basis of charge, molecular weight, and shape. Electrophoresis in the presence of sodium dodecyl sulfate (SDS) at high pH provides a separation on the basis of size. Electrophoresis at neutral pH permits an analysis of acid-labile histone modifications and some histone-histone complexes. Two-dimensional gels may be required to separate histones from some species, and to analyze the products from the reaction of histones with reversible cross-linking reagents. The advantages and limitations of the gel systems are also discussed.
TL;DR: Methods used for isolation and characterization of chromatin subunits are discussed and isolation of core particle subunits and other nucleoprotein fragments is illustrated in the chapter.
Abstract: Publisher Summary This chapter discusses methods used for isolation and characterization of chromatin subunits. Most reported physical studies of chromatin subunits have used more or less refined preparations of core particles. The particles are easily prepared in quantity because of their nuclease resistance and can be resolved to a fairly high degree of homogeneity. Studies of the entire repeating unit containing 200 base pairs of DNA have been inhibited by the fact that these structures are observed as intact entities only in the initial stages of digestion, and even then exhibit a rather broad heterogeneity in DNA size. This heterogeneity presumably results from the possibility that the first cleavage within spacer regions can occur at random over a range of sites and may be compounded by heterogeneity of spacer lengths. The techniques described in the chapter are employed for the isolation of core particles. If digestion conditions are carefully controlled, they can also be used for the preparation of small quantities of repeating units. Nuclease digestion of nuclei or chromatin with staphylococcal nuclease is discussed and isolation of core particle subunits and other nucleoprotein fragments is illustrated in the chapter.
TL;DR: The chapter discusses the characterization of the 5′- and 3′-structures unique to mRNA, and its translation in an extract of wheat germ.
Abstract: Publisher Summary The chapter focuses on the preparation and analysis of messenger RNA (mRNA) from Saccharomyces cerevisiae. The isolation in undegraded form from yeast cells and polysomes are discussed. The chapter discusses the characterization of the 5′- and 3′-structures unique to mRNA, and its translation in an extract of wheat germ. In S. cerevisiae, most of the mRNA molecules contain relatively homogeneous polyriboadenylic acid (poly A) sequences of about 50 residues covalently linked to the 3′-end. These molecules are readily separated from total RNA by affinity chromatography on oligo-dT-cellulose columns. Columns are prepared and run at room temperature. The affinity chromatography on oligo-dT-cellulose separates the bulk of the poly-A-containing molecules from the total RNA and thus is highly suitable for preparing large quantities of poly-A-containing RNA. More complete purification of yeast mRNA are obtained using poly U immobilized on glass-fiber filters. There are no other methyl groups in yeast mRNA. These structures are resistant to nucleases and phosphatases and therefore can be easily isolated and analyzed. Wheat germ extract actively translates yeast mRNA.
TL;DR: This technique provides an assay system in which the polymerases function on their endogenous templates and should be useful in determining whether agents that affect nucleic acid synthesis in intact cells exert their effects by direct action on the polymerase or on the template.
Abstract: Publisher Summary Permeable cell systems are developed in bacteria and disrupted or permeable cell systems are developed in eukaryotes to allow exogenously supplied deoxynucleoside triphosphates and other reaction components to be supplied directly to the replication complex functioning on its intrinsic DNA template. The technique described for rendering eukaryotic cells permeable to nucleotides has several advantages in the study of nucleic acid synthesis under near physiological conditions. The technique itself is simple; the cells remain in a monodisperse suspension and are easy to pipet quantitatively. The cells are freely permeable to phosphorylated compounds which gain rapid access to the nucleus. Nucleotides are incorporated into DNA without intermediate breakdown and rephosphorylation. DNA synthesis in permeable cells is semiconservative, the products are high-molecularweight DNA intermediates, and DNA synthesis occurs as extensions of replication sites that were active in vivo . This technique provides an assay system in which the polymerases function on their endogenous templates. It should be useful in determining whether agents that affect nucleic acid synthesis in intact cells exert their effects by direct action on the polymerase or on the template. The system should also be useful for studies of the intermediates in nucleic acid synthesis, and finally for studies of physiological changes in the activities of polymerases that occur with metabolic manipulation of the cells. As the addition of Triton X-100 renders the cells permeable to exogenous proteins, this technique may also be useful in complementation studies of eukaryotic cells with genetic defects in DNA synthesis.
TL;DR: The chapter focuses on the intactness of the macromolecular structure of the nucleolus to retain in vivo function in isolated nucleoli and nucleolar chromatin constitute a good system for the study of gene regulation in eukaryotic cells.
Abstract: Publisher Summary This chapter describes the methods for isolation of nucleoli from various cell types and for purification of chromatin from isolated nucleoli. The nucleolus is known to be the site of ribosomal RNA (rRNA) synthesis and ribosome assembly. Association of histones and nonhistone proteins with the nucleolar DNA provides an opportunity to study the regulatory function of these proteins in a DNA-protein complex, designated as chromatin that is actively synthesizing one species of RNA. The chapter focuses on the intactness of the macromolecular structure of the nucleolus to retain in vivo function in isolated nucleoli. Isolation of nucleoli consists of three steps: (1) isolation of nuclei from whole homogenate, (2) disruption of nuclei, and (3) purification of nucleoli from the disintegrated nuclear material. Combined with advanced procedures for purification of eukaryotic RNA polymerases from various sources and with the improved techniques of analysis of histone and nonhistone proteins, isolated nucleoli and nucleolar chromatin constitute a good system for the study of gene regulation in eukaryotic cells.
TL;DR: Examination of the isolated nuclei by high-resolution electron microscopy is the best method of assessing the integrity of the nuclei and the purity of the preparation.
Abstract: Publisher Summary This chapter describes a method for the isolation of nuclei from animal cells in culture. The objective of the method for isolating nuclei is to obtain a preparation that consists solely of nuclear material and in which the nuclei are morphologically identical to those in undisrupted cells, with contents qualitatively and quantitatively identical to those of nuclei in vivo . The chapter discusses the factors of importance in nuclear isolation procedures and the criteria that used to assess the integrity and purity of the isolated nuclei. The chapter describes two methods for isolating nuclei as preliminary steps in the isolation of chromatin and chromatin constituents. Nuclear isolation procedures essentially consist of two steps: (1) the lysis of the cells in a suitable medium, and (2) the separation of the nuclei from the unbroken cells and cell debris. Examination of the isolated nuclei by high-resolution electron microscopy is the best method of assessing the integrity of the nuclei and the purity of the preparation. The methods for isolating nuclei include isolation with (1) citric acid, and (2) sucrose and detergent. The chapter also compares the products obtained from these methods.
TL;DR: The procedure described in this chapter allows the purification of three distinct histone kinases, each of which catalyzes the phosphorylation of H1 histone at a specific site or group of sites, and each preparation is substantially free of the other hist one kinases and is low in protease and phosphatase activity.
Abstract: Publisher Summary This chapter discusses isolation of histone kinases. The procedure described in this chapter allows the purification of three distinct histone kinases, each of which catalyzes the phosphorylation of H1 histone at a specific site or group of sites. The resulting enzymes are not purified to homogeneity. However, each preparation is substantially free of the other histone kinases and is low in protease and phosphatase activity. The purified enzymes are suitable for the preparation of substantial quantities (batches of 50 mg are quite practical) of H1 histone phosphorylated in specific sites for sequencing studies, for physical studies of H1–DNA interactions and for use as substrates for the detection and characterization of histone phosphatases. This enzyme is purified to homogeneity in a number of laboratories. The procedure described in the chapter is modified from an earlier method and provides a useful partially purified preparation.
TL;DR: This chapter describes methods for the isolation of these ribonucleoprotein (RNP) particles from eukaryotic cells and discusses some properties of heterogeneous nuclear RNA (hnRNA) particles isolated from HeLa cell nuclei.
Abstract: Publisher Summary This chapter describes methods for the isolation of these ribonucleoprotein (RNP) particles from eukaryotic cells and discusses some properties of heterogeneous nuclear RNA (hnRNA) particles isolated from HeLa cell nuclei. RNP particles containing hnRNA have been identified both cytologically and biochemically in eukaryotic cell nuclei. The method developed for isolation of HeLa hnRNP particles involves the isolation of nuclei followed by their mechanical disruption and purification of hnRNP particles by sucrose gradient centrifugation. The method of disrupting HeLa cell nuclei permits the high recovery of heterogeneous nuclear RNA in the presence of large amounts of nucleohistone. An understanding of the specificity of RNA-protein interactions in native hnRNP particles is impeded by the nucleotide sequence complexity of hnRNA and by the analytical complexity of the proteins. The specific association of protein with poly (A)-rich hnRNA sequences is demonstrated.
TL;DR: While antibodies to histones should be very useful reagents, progress in their application requires an appreciation of the greater difficulties in the induction and assay of these antibodies that occur with most other protein antigen systems.
Abstract: Publisher Summary This chapter focuses on serological analyses of histones. Serological analyses of histones have several applications in studies of the structure and functional organization of these proteins. Antibodies can provide unequivocal identification of individual histones in the absence of specific functional assays. Serological reactions can also detect slight alterations in structure, allowing comparisons of histones isolated from various sources and comparisons of the free soluble proteins with the forms in which they occur in chromatin. With immunofluorescence, antibodies can be used to study histones in chromosomes and whole nuclei, and with electron microscopy they can be used to study isolated chromatin and its subunits. While antibodies to histones should be very useful reagents, progress in their application requires an appreciation of the greater difficulties in the induction and assay of these antibodies that occur with most other protein antigen systems. The chapter evaluates some of these problems as well as surveys methods that have been successful. The technique of microcomplement fixation is described and sources of sera and procedures for immunization are discussed. The chapter also presents an overview of serological assays.
TL;DR: This chapter describes the isolation of proteins of nuclear ribonucleoprotein (RNP) subcomplexes, which comprise a significant fraction of the total chromatin nonhistone proteins.
Abstract: Publisher Summary This chapter describes the isolation of proteins of nuclear ribonucleoprotein (RNP) subcomplexes. Heterogeneous nuclear RNA (hnRNA) and some messenger RNA (mRNA) molecules contain internal base methylation at the N 6 -position of adenosine. The substrate for the processing of hnRNA by cleavage, polyadenylation, capping, methylation, and selection for transport to the cytoplasm is not a naked RNA molecule, but the RNP fibril. The nascent hnRNA in chromatin spreads appears in shortened RNP fibrils still attached to the chromatin template. Ribonucleoprotein complexes containing rapidly labeled RNA have also been isolated from purified nuclei and characterized biochemically. Proteins characteristic of RNP comprise a significant fraction of the total chromatin nonhistone proteins. A number of procedures release RNP from purified nuclei. Nuclear rupture by sonication or lysis with detergents can be used; however, contamination by preribosomal RNP and chromatin fragments may obscure subsequent analysis. High salt lysis combined with DNase treatment eliminate the entrapment of RNP in the chromatin gel giving larger yields than by sonication; however, released DNA-binding proteins that are maintained in solution by high salt, may interact with RNP. Gentle extraction procedures with buffered salt solutions, leaving nuclei intact and avoiding exposure to high salt, should minimize rearrangements of nuclear proteins; however they suffer the disadvantage of being lengthy, resulting in considerable cleavage of hnRNA.
TL;DR: This chapter discusses the production of antibodies against chromosomal proteins of Drosophila and their use in an indirect immunofluorescent assay to determine the in situ distribution of these proteins.
Abstract: Publisher Summary This chapter focuses on immunofluorescent techniques in the analysis of chromosomal proteins. Progress in the analysis of the nonhistone chromosomal proteins (NHC proteins) has been slow because of the complexity of this class of proteins and the fact that many of the major NHC proteins apparently play structural roles for which no direct assay is available. The use of specific antibody techniques appears likely to assist in resolving these problems. Specific antibodies against chromosomal proteins can be used to determine the distribution of these proteins in situ using indirect staining techniques. The in situ distribution patterns obtained can suggest as well as test hypotheses concerning structural and active roles of these proteins. Specific antibodies can also be used for rapid purification of components from complex mixtures and as probes in enzyme assays. The chapter discusses the production of antibodies against chromosomal proteins of Drosophila and their use in an indirect immunofluorescent assay to determine the in situ distribution of these proteins. The chapter discusses the preparation of antigens and antisera and the characterization of antiserum by indirect immunofluorescent staining of sodium dodecyl sulfate (SDS)-polyacrylamide gels.
TL;DR: This chapter describes the procedures for the preparation of chromatin from animal tissues and cultured cells that depend on the principles for purification of chromatis from animal cells, starting from either whole cells (direct methods) or purified nuclei.
Abstract: Publisher Summary This chapter describes the procedures for the preparation of chromatin from animal tissues and cultured cells that depend on the principles for purification of chromatin from animal cells, starting from either whole cells (direct methods) or purified nuclei. The rationales, advantages and disadvantages, and appropriate uses of these procedures are discussed. In the direct methods of chromatin isolation, the chromatin is isolated from a cell lysate rather than from purified nuclei. The chapter also outlines the methods for the chromatin isolation from nuclei. Purified chromatin preparations contain fragments associated by intermolecular interactions, or long chromatin molecules; further fragmentation (shearing) may be necessary to obtain material suitable for further manipulations. Conditions for storage of purified chromatin have been determined empirically only for certain specific purposes. There is no absolute criterion of purity for chromatin is at the present time. The absolute criteria of structural preservation cannot yet be proposed. The method of choice for chromatin preparation depends heavily on the problem to be investigated.
TL;DR: The method used for minimizing proteolysis can be employed for the isolation of nuclei and nucleoprotein from any source, with the proviso that the integrity of the nucleoproteins is assayed at every step.
Abstract: Publisher Summary This chapter outlines the procedures for minimizing protease activity during isolation of nuclei, chromatin, and the histones. Preparation of nucleoprotein has followed one of two principles: (1) purification from disrupted whole tissue, or (2) preparation from purified nuclei. Extraction of histones with detergents is used as an analytical tool rather than on a preparative scale. The histones may be conveniently assayed on acid-urea gels. The loss of any of the major fractions and appearances of degradation products are apparent when compared to a standard of acid-extracted whole histone from calf thymus. A sodium dodecyle sulfate (SDS)-polyacrylamide gel system is used to assay for proteolysis of the nonhistone proteins. The method used for minimizing proteolysis can be employed for the isolation of nuclei and nucleoprotein from any source, with the proviso that the integrity of the nucleoproteins is assayed at every step.
TL;DR: The procedure described in this chapter permits the detection of N -phosphoryl amino acids in chromosomal proteins based upon isolation of 32 P-labeled N - phosphoryl substances.
Abstract: Publisher Summary Phosphorylation of chromosomal proteins has generally been measured either after isolation of phosphorylated chromosomal proteins from the nucleus or after the action of a chromosomal protein kinase in an in vitro system. These procedures involve one or the other step carried out at an acidic pH value such that the only surviving phosphoryl linkage is found on the hydroxyl group of serine or threonine. The single most outstanding property of N -phosphorylated compounds is their extreme sensitivity to acidic pH and their relative stability under basic conditions. The procedure described in this chapter permits the detection of N -phosphoryl amino acids in chromosomal proteins based upon isolation of 32 P-labeled N -phosphoryl substances. This procedure has been used directly with rat liver preparations, rat mammary tumor carcinosarcoma, or directly from enzyme reaction mixtures.
TL;DR: The description of the strategies and methodology employed in the development of the Neurospora plasma membrane will provide a framework which can be used by others to identify, isolate, and assess the functionality of plasma membranes from other eukaryotic cells.
Abstract: Publisher Summary This chapter acquaints the reader with the Neurospora plasma membrane, a new experimental system for exploring the molecular biology of a variety of important aspects of eukaryote plasma membrane structure and function It is hoped that the description of the strategies and methodology employed in the development of this system will provide a framework which can be used by others to identify, isolate, and assess the functionality of plasma membranes from other eukaryotic cells The study of this system has engendered fundamental concepts of plasma membrane structure such as the asymmetric distribution of lipids and carbohydrates and the multiple modes of protein orientation in the plasma membrane The Neurosporu plasma membrane system described here is suitable for both structural and functional studies, as these plasma membranes can be isolated as open sheets or closed vesicles Neurospora do not exhibit cell–cell interactions, hormone response, pino- and phagocytosis, and certain other critical functions of higher eukaryotes
TL;DR: A brief description of the characteristic physical and chemical changes occurring in both DNA and the bound small molecule as a result of intercalation are presented as an introduction to the more complex phenomena which occur with chromatin.
Abstract: Publisher Summary This chapter focuses on intercalating agents as probes of chromatin structure. The intercalation model, proposed in the early 1960s, involves the unwinding of the deoxyribose sugar phosphate chains and elongation of the DNA molecule to produce an approximately 3.4 A-thick hydrophobic space to accommodate the insertion of planar aromatic molecules between base pairs. Lerman provided extensive experimental support for the intercalation model. A number of planar aromatic molecules have been shown to form intercalated complexes with DNA; these include the acridine dyes, antiparasitic drugs, antitumor agents, and a variety of small molecules. The structure of some typical intercalating molecules is shown in the chapter. A brief description of the characteristic physical and chemical changes occurring in both DNA and the bound small molecule as a result of intercalation are presented as an introduction to the more complex phenomena which occur with chromatin. Effects of intercalation on the physical properties of DNA are discussed. Proton magnetic resonance techniques are also described in the chapter.
TL;DR: Preparation of isolated chromatin suspension for electron microscopy and spreading of chromatin and chromosomes on a hypophase are discussed in the chapter.
Abstract: Publisher Summary This chapter discusses some methods for preparation of chromatin and chromosomes for electron microscopy. Analysis of chromatin structure in situ must be done in sections of cells. Because chromatin fibers are usually closely packed and complexly folded, thin sections of well-fixed cells are not very informative. The situation is somewhat improved by swelling the chromatin in hypotonic fixatives. The fibers are then separated from each other and measurement of fiber thickness is greatly facilitated. In the usual thin sections, it is impossible to get information on the spatial arrangement of chromatin fibers. High-voltage electron microscopes (one million eV) have now become available for biomedical research providing a resolution of 20–50 A with sections up to several micrometers thick. As section thickness increases, longer segments of chromatin fibers are contained in a section and the three-dimensional arrangement becomes more evident, especially in stereoscopic photographs. The practical limit to section thickness is in the overlap of structures that makes analysis difficult. The chapter also presents an overview of hypotonic fixation. Preparation of isolated chromatin suspension for electron microscopy and spreading of chromatin and chromosomes on a hypophase are discussed in the chapter.
TL;DR: This chapter discusses use of laser microbeam for determining chromatin structure and function and the special ancillary techniques that have been developed for subsequent analysis of the laser microirradiated cells.
Abstract: Publisher Summary This chapter discusses use of laser microbeam for determining chromatin structure and function. The laser is a device that can provide electromagnetic radiation from the ultraviolet to the infrared region of the spectrum. The unique features of laser have led to its use in several areas of genetic research: (1) stimulated Raman spectroscopy, (2) diffraction-limited fluorescent microscopy, and (3) selective laser microbeam irradiation. In Raman spectroscopy, the intense monochromatic light is used to stimulate the emission of Raman spectra. The second area of chromatin research where the laser is being applied is in fluorescent analysis of chromosomes. The third area of laser application to chromatin research is laser microbeam irradiation. Studies have been conducted in which the major approach has been to focus a laser beam down to an effective spot size of 0.25–1 pm on selected individual chromosomes of mitotic tissue culture cells. This technique has been used to study problems on (1) the organization of chromosomes, (2) gene location and function, (3) chromosome stability, and (4) mitotic mechanisms. The chapter focuses on the methods and experiments and the special ancillary techniques that have been developed for subsequent analysis of the laser microirradiated cells.
TL;DR: Methods for the manual enucleation of Xenopus oocytes are described and are applicable to problems relating specifically to oocytes and are also useful for studying more general aspects of nuclear functions, especially when the experiments require minimum exposure to nonphysiological conditions.
Abstract: Publisher Summary This chapter describes methods for the manual enucleation of Xenopus oocytes. Manual isolation is the only means to obtain the amphibian oocyte nuclei. However, the technique is not only useful for experiments relating to this specific cell type but is also appropriate for more general studies. Nuclear isolation techniques for mature Xenopus laevis oocytes are described, however, with minor modifications, they should be applicable to other mature amphibian oocytes. The procedures are applicable to problems relating specifically to oocytes and are also useful for studying more general aspects of nuclear functions, especially when the experiments require minimum exposure to nonphysiological conditions. Despite the fact that only small amounts of material can be obtained, the methods have been used to investigate the structure and function of lampbrush chromosomes, nucleocytoplasmic exchange, the structure of the nuclear envelope, the protein composition of various nuclear components, and aspects of RNA synthesis.