Showing papers on "Nucleolar chromatin published in 1978"

Isolation and purification of nucleoli and nucleolar chromatin from mammalian 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.

Nucleosomal and supranucleosomal organization of transcriptionally inactive rDNA circles in Dytiscus oocytes.

Abstract: Oocytes of the water beetle, Dytiscus marginalis, contain large amounts of rDNA most of which is present in the form of rings containing one or several pre-rRNA genes. Electron microscopy of spread preparations of vitellogenic oocytes has shown that the rDNA is extended in chromatin rings with transcribed pre-rRNA genes and is not packed into nucleosomes (Trendelenburg etal., 1976). When similar preparations are made from previtellogenic ooytes in which a large proportion of the nucleolar chromatin is transcriptionally inactive, a different morphological form of this chromatin is recognized. In contrast to the transcribed chromatin rings the inactive nucleolar chromatin circles show the characteristic beaded configuration, indicative of nucleosomal packing. Nucleosomal packing is also indicated by the comparison of the lengths of these chromatin rings with both isolated rDNA circles and transcribed chromatin rings. In addition, these inactive nucleofilaments often appear to be compacted into globular higher order structures of diameters from 21 to 34nm, each composed of an aggregate of 6–9 nucleosomes. While the estimated reduction of the overall length of rDNA, as seen in our preparations, is, on the average, only 2.2–2.4 fold in the nucleosomal state it is 10–13 fold when supranucleosomal globules are present. These data show that the extrachromosomal rDNA of these oocytes goes through a cycle of condensation and extension, as a function of the specific transcriptional activity, and that the beaded state described here is exclusively found in the non-transcribed state.

Morphology of transcriptional units at different states of activity

Abstract: The morphology of two forms of transcriptionally active chromatin, the nucleoli and the loops of lampbrush chromosomes, has been examined after fixation in situ or after isolation and dispersion of the material in media of low ionic strengths, using a variety of electron microscopic preparation techniques (e.g. spread preparations with positive or negative staining or without any staining at all, with bright and dark field illumination, with autoradiography, after pretreatment of the chromatin with specific detergents such as Sarkosyl NL-30; transmission and scanning transmission electron microscopy of ultrathin sections). Nucleolar chromatin and chromosomes from oocytes of various amphibia and insects as well as from green algae of the family of the Dasycladaceae were studied in particular detail. The morphology of transcriptional units that are densely packed with lateral ribonucleoprotein fibrils, indicative of great transcriptional activity, was compared with that of chromatin of reduced lateral fibril density, including stages of drug-induced inhibition. The micrographs showed that under conditions which preserve the nucleosomal organization in condensed chromatin studied in parallel, nucleosomes are not recognized in transcriptionally active chromatin. This holds for the transcribed regions as well as for apparently untranscribed (i.e. fibril-free) regions interspersed between (‘spacer’) and/or adjacent to transcribed genes and for the fibril-free regions within transcriptional units of reduced fibril density. In addition, comparison of lengths of repeating units of isolated rDNA with those observed in spread nucleolar chromatin indicated that this DNA is not foreshortened and packed into nucleosomal structures. Granular particles which were observed, at irregular frequencies and in variable patterns, in some spacer regions, did not result in a proportional shortening of the spacer axis, and were found to be resistant to detergent treatment effective in removing most of the chromatin associated proteins including histones. Thus, these particles behave like RNA polymerases rather than nucleosomes. It is suggested that structural changes from nucleosomal packing to an extended form of DNA are involved in the transcriptional activation of chromatin.

The structure of nucleolar chromatin in Physarum polycephalum

Abstract: The nucleolar DNA of Physarum polycephalum has been differentially labelled with 3H-thymidine and the structure of the nucleolar chromatin investigated by digestion with micrococcal nuclease. Nucleolar chromatin which had been labelled in G2 phase of the cell cycle and then digested before mitosis had an identical DNA repeat length to main band DNA (165 +/- 5 base pairs) but there was a definite indication that the rate of digestion was faster for nucleolar DNA than for main band DNA. Nucleolar chromatin which had been labelled in G2 and the label chased through mitosis into G2 phase of the next cycle showed an identical DNA repeat length to main band DNA and was also digested at the same rate. We conclude that nucleolar chromatin, at least 25 per cent of which is maximally transcriptionally active in G2, has a nucleosome-like structure.

Ultrastructural Studies of H-1 Parvovirus Replication VI. Simultaneous Autoradiographic and Immunochemical Intranuclear Localization of Viral DNA Synthesis and Protein Accumulation

Abstract: The localization of H-1 viral replicative-form double-stranded DNA and progeny single-stranded DNA replication in parasynchronously infected, simian virus 40-transformed newborn human kidney cells was studied with high-resolution electron microscope autoradiography (80-nm silver grains) We analyzed wild-type H-1 and ts1 H-1 (a conditional mutant defective in progeny single-stranded DNA synthesis) The proportion of the total DNA synthesis that was viral was estimated to be >90% by comparing the amount of [3H]thymidine uptake in cultures infected with wild-type H-1 versus ts14 (an H-1 mutant defective in DNA replication) Simultaneous staining with cytochrome c-conjugated anti-H-1 immunoglobulin G was performed to ensure that cells incorporating [3H]thymidine (2- to 60-min pulses) were H-1 infected The sites of H-1 replicative-form (in ts1-infected cells) and progeny (in wild-type-infected cells) DNA synthesis were identical Immunospecifically labeled nuclei at the earliest stages of infection exhibited dense clusters of silver grains over material extruded from nucleolar fibrillar centers These foci became larger with increasing cellular damage, forming a limited number of H-1 DNA synthetic centers in the euchromatin Each island-like focus was surrounded by tufts of heterochromatin containing high concentrations of unassembled H-1 capsid proteins In late phases of infection, the heterochromatin became completely marginated, and the nucleoplasm contained only euchromatin that exhibited randomly distributed sites of H-1 DNA replication This indicates that H-1 DNA synthesis begins at localized euchromatic or nucleolar sites and then spreads outward Immunostained heterochromatin and nucleolar chromatin never incorporated [3H]thymidine Our results suggest that H-1 proteins and cellular cofactors associated with the fibrillar component of the nucleolus and the euchromatin may play a role in the regulation of H-1 DNA synthesis

Visualization of mouse DNA transcriptional complexes in mouse kidney cells infected with SV 40 virus.

Abstract: Electron microscopic studies of transcribed cellular deoxynucleoprotein (DNP) fibers from mouse kidney cells abortively infected with Simian Virus 40 (SV 40) revealed two types of transcriptional complexes. Tandemly repeating units representing ribosomal transcriptional events, although few in number because of the procedure employed, clearly confirmed results of other techniques which revealed the unusually long length of the untranscribed spacer intercepts in mouse nucleolar chromatin. In non-nucleolar arrays the density of the ribonucleoprotein (RNP) fibrils varied, as did the length and configuration of the associated DNP fibers. A statistical correlation between a "smooth" appearance of a transcribed portion of a DNP fiber and a high density of nascent RNP fibrils was observed.

Chapter 14 Nucleolar Proteins

Abstract: Publisher Summary This chapter describes methods for nucleolar protein extraction and specific methods for assay and isolation of nucleolar enzymes. The nucleolus contains a variety of proteins or protein subunits. The techniques to study the chromatin components of the nucleus that are also applicable to the nucleolar fraction of chromatin are discussed. The procedures that may be applied directly to isolated nucleoli prior to fractionation of nucleolar subcomponents include (1) sodium chloride-hydrochloric acid extraction of proteins, (2) chloroethanol extraction, (3) urea extraction procedure, (4) acid extraction, and (5) sodium dodecyl sulfate (SDS) extraction. For functional studies, it is advantageous to fractionate the nucleolus into “native” subcomponents prior to protein isolation. Several procedures are available for isolation of substructures such as nucleolar chromatin, preribosomal ribonucleoprotein (RNP) particles, and fibrillar elements. There are numerous enzymes involved in the assembly and processing of preribosomal particles for which no functional assay is available. The chapter also outlines the methods for studies on nucleolar antigens.

Controls of nucleolar function in cancer cells.

Abstract: The protein synthesizing machinery of the cell in which the ribosomes play a key role is of great importance to its rate of growth (1). The availability of ribosomes is dependent upon the activity of the nucleolus (Fig 1), which is the sole site of synthesis of preribosomal RNA and of assembly of preribosomal particles. The high rate of protein synthesis is one reason why the attention of oncologists has been directed to analysis of nucleolar structure and function (2).