Non-nucleosomal configuration of chromatin in nucleolar organizer regions of metaphase chromosomes in situ

The structure of metaphase chromatin in a human tumor cell line, TG cells, was investigated using thin sections selectively stained for DNA with the Feulgen-like osmium-ammine reaction. The bulk of metaphase chromatin was characterized by the nucleosomal configuration. Some specimens were pretreated by silver staining for selective visualization of acidic proteins of the nucleolar organizer regions. In these specimens, the osmium-amine DNA tracer revealed that the chromatin present at the sites of silver granule localization had a completely extended configuration, and never gave rise to nucleosomal structures.     

Effects of camptothecin, an inhibitor of DNA topoisomerase I on ribosomal gene structure and function in TG cells.

The effects of camptothecin treatment and topoisomerase I inhibition on ribosomal gene structure and function were investigated in TG cells, a human tumour cell line. 90- and 180-min treatments with 25 microM camptothecin resulted in an increased DNA fragmentation and decreased activity of topoisomerase I in cell extracts. After 180-min treatment, the incorporation of labelled uridine into total cell RNA was reduced to 39% and the ribosomal RNA synthesis to 10%, as compared to values of control cells. At the ultrastructural level, the nucleolar components appeared to be segregated; after selective DNA staining, with osmium-amine complex, a part of the nucleolar chromatin of treated cells showed the presence of thin, extended DNA filaments, superimposable to those present in control cells.     

Synthesis and application of novel bifunctional spin labels.

The synthesis of new bifunctional spin-labeled cross-linking reagents is described. Covalent attachment to papain was achieved via a thiol-specific thiosulfonate residue and, for the second anchor point, via a nonspecific photoreactive azido function. The thiosulfonate formed a reversible disulfide linkage, which could be cleaved again reductively by dithiothreitol. The spin label, a pyrroline-1-oxyl radical, was highly immobilized after attachment to papain by both functional groups and showed little if any relative motion with respect to the protein.     

Endogenous polyamines are intimately associated with highly condensed chromatin in vivo. A fluorescence cytochemical and immunocytochemical study of spermine and spermidine during the cell cycle and in reactivated nuclei

Polyamines are low molecular weight aliphatic polycations essential for cell proliferation and differentiation. By immunocytochemistry, as well as by two independent fluorescence cytochemical methods, we show that polyamines are associated with highly condensed chromatin in nucleated erythrocytes and in metaphase and anaphase chromosomes. In other cells, polyamines mainly occur in cytoplasm. The association between polyamines and DNA in condensed chromatin is so close that DNase treatment is necessary for making polyamines available for reaction with antibodies. Studies of chick/HeLa cell heterokaryons reveal that polyamines disappear from the chick erythrocyte nuclei concomitantly with DNA decondensation and initiation of RNA synthesis. Our data strongly suggest that polyamines are important for chromatin condensation in vivo.     

Relationship between the Ag-NOR proteins and ribosomal chromatin in situ during drug-induced RNA synthesis inhibition

In human TG tumor cells, the role of silver-NOR proteins was investigated by examining their relationship with the chromatin structure during inhibition of RNA synthesis by actionomycin-D treatment. This induced segregation of the nucleoli into four distinct zones and weakened the silver reaction. The fibrillar components were found to constitute the site of silver-stained proteins segregation. Feulgen-like osmium-ammine staining revealed that the DNA disappeared from the segregated nucleoli except for a network of nonnucleosomal filaments. When Ag-NOR protein detection was combined with chromatin visualization, we found constant overlapping of the silver reaction sites with the nonnucleosomal DNA filaments. Our results indicate that certain Ag-NOR proteins are not directly linked to active rRNA synthesis, but might rather affect the structure of ribosomal genes.     

Differential effects of triphenyltin and 8-azido-ATP on the ATP synthesis, ATP-Pi exchange, and ATP hydrolysis in liposomes containing ATP synthase and bacteriorhodopsin

The ATP hydrolysis activity of purified ATP synthase reconstituted in liposomes was inhibited by triphenyltin in a manner different from that of other thiol-specific reagents. In liposomes containing ATP synthase and bacteriorhodopsin, ATP hydrolysis and ATP-Pi exchange were inhibited by triphenyltin to a greater extent than the ATP synthesis, in contrast to what was found with an F1-specific inhibitor, 8-azido-ATP. The possibility is discussed that ATP hydrolysis and ATP synthesis are differently coupled to proton conduction through F0.     

Isolation and characterization of chromatin replication bands and macronuclei from Euplotes eurystomus

A method is described for isolating replication bands (RBs) from Euplotes eurystomus in quantities sufficient for biochemical analysis. The method involves the disruption of whole cells in a low ionic strength buffer that maintains RB integrity while dispersing macronuclear chromatin. The RBs are then stabilized with MgCl2 and spermidine phosphate and isolated by gradient centrifugation. Staining with silver nitrate and thiol-specific coumarin maleimide has been demonstrated in the RBs of Euplotes and other hypotrichs; both of these properties were maintained in isolated RBs. A method is also described in this study for isolating highly purified macronuclei. Examination of isolated macronuclei and RBs with electron microscopy (EM) indicates that the morphology of both structures remain essentially intact during purification. We also observe with EM an increase in the number of replicating molecules in RBs compared to macronuclei. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrates a consistent but minor enrichment of a 55 kilodalton protein in RBs when compared to macronuclear proteins.     

Photochemical crosslinking of protein and DNA in chromatin. II. Synthesis and application of psoralen-cystamine-arylazido photocrosslinking reagents

The synthesis and testing of a new type of nucleic acid-protein photocrosslinking reagent is described. The reagents are composed of a psoralen ligand for nucleic acid photoattachment, which is linked to an azidobenzoyl group, for protein photoattachment. The linker contains a disulfide bridge which can be opened by reduction with mercaptans. The reagents were tested in a chromatin system, where it was found that they induced cleavable crosslinks between the histones and the DNA upon irradiation with long-wavelength ultraviolet light (lambda greater than 300 nm).     

Effect of thiol-specific reagents on the activity of PaeI and PaeII restrictases from Pseudomonas aeruginosa

5,5'-dithiobis-2-nitrobenzoic acid, N-ethylmaleimide, and parachloromercuribenzoate have been demonstrated to inhibit the activity of restrictases PaeI and PaeII from Ps. aeruginosa bacterial cells. Restrictase PaeII was more sensitive to the action of thiol-specific reagents, as compared to PaeI. The minimal concentration of reagents for SH-groups that completely inhibited the activity of restrictases PaeI and PaeII was determined. The protective effect against the inhibitory action of 5,5'-dithiobis-2-nitrobenzoic acid on the activity of PaeII was observed after preincubation of these enzymes with phage lambda DNA and Mg2+ cations. It is suggested that restrictase PaeI and PaeII molecules contain SH-groups, essential for the enzymatic activity. They are believed responsible for restrictase binding with DNA substrate.     

Microautoradiographic study of cell proliferation in compensatory renal growth.

DNA synthesis in the renal parenchyma was studied by electron microscopic autoradiography 48 and 72 hours after unilateral nephrectomy in mice and weanling rats. The proportion of labelled nuclei belonging to the epithelium, endothelium, interstitial areas, or circulating cells was evaluated. Most of cells showing DNA synthesis were epithelial but many belonged to stroma. All the nephron segments were found to participate in compensatory hyperplasia, with a greater contribution, however, of the proximal convoluted tubules. DNA synthesis by the capillary endothelial cells occurred later than the peak epithelial mitotic activity. The site of DNA synthesis in nuclei was the euchromatin, the silver grains being uniformly distributed throughout the nuclear areas with condensed chromatin, and more seldom in the nuclear envelope areas or that of the perinucleolar satellites.     

FINE STRUCTURAL ALTERATIONS OF INTERPHASE NUCLEI OF LYMPHOCYTES STIMULATED TO GROWTH ACTIVITY IN VITRO

This report describes fine structural changes of interphase nuclei of human peripheral blood lymphocytes stimulated to growth by short-term culture with phytohemagglutinin. Chromatin is found highly labile, its changes accompanying the sequential increases of RNA and DNA synthesis which are known to occur in lymphocyte cultures. In "resting" lymphocytes, abundant condensed chromatin appears as a network of large and small aggregates. Early in the response to phytohemagglutinin, small aggregates disappear during increase of diffuse chromatin regions. Small aggregates soon reappear, probably resulting from disaggregation of large masses of condensed chromatin. Loosened and highly dispersed forms then appear prior to the formation of prophase chromosomes. The loosened state is found by radioautography to be most active in DNA synthesis. Small nucleoli of resting lymphocytes have concentric agranular, fibrillar, and granular zones with small amounts of intranucleolar chromatin. Enlarging interphase nucleoli change chiefly (1) by increase in amount of intranucleolar chromatin and alteration of its state of aggregation and (2) by increase in granular components in close association with fibrillar components.     

Ultrastructure of meristematic cells of dormant and released buds in Tradescantia paludosa

The lateral bud meristems of Tradescantia paludosa show a characteristic cytohistological zonation during dormancy. The cells comprising this so called ‘zone of inhibition’, which is located at the extreme tip of the bud apex, rarely synthesize nuclear DNA or undergo mitotic division. These nuclei are as large as prophase nuclei, yet contain only telophase (2C) amounts of DNA and significantly lower amounts of histone as compared to the 2C nuclei of the actively dividing cells.Ultrastructural observations of the nuclei in the ‘zone of inhibition’ show that a large proportion of the chromatin is organized as less condensed, diffuse, euchromatin fibrils; however, the chromatin of the actively dividing nuclei of the cells outside the ‘zone of inhibition’ or in the released bud meristems is organized to a greater extent as condensed clumps of heterochromatin. When the dormancy is released, the nuclei in the ‘zone of inhibition’ synthesize DNA and histone and undergo cell division in approx. 4 days. Striking changes in the organization of chromatin fibrils take place during this transition period. The diffuse chromatin fibrils of the nuclei in the ‘zone of inhibition’ progressively become more and more condensed as the cell prepares to undergo the first mitotic division after the release of dormancy. This change which is coupled with the synthesis of histones in the nuclei of the ‘zone of inhibition’ suggests a prominent structural role of these basic proteins in the organization of the chromatin. The large volume of 2C nuclei of the ‘zone of inhibition’ seems, therefore, to result not from a great nuclear mass, but probably from a relatively small degree of condensation of chromatin.     

Transmembrane protein topology mapping by the substituted cysteine accessibility method (SCAM(TM)): application to lipid-specific membrane protein topogenesis

We provide an overview of lipid-dependent polytopic membrane protein topogenesis, with particular emphasis on Escherichia coli strains genetically altered in their lipid composition and strategies for experimentally determining the transmembrane organization of proteins. A variety of reagents and experimental strategies are described including the use of lipid mutants and thiol-specific chemical reagents to study lipid-dependent and host-specific membrane protein topogenesis by substituted cysteine site-directed chemical labeling. Employing strains in which lipid composition can be controlled temporally during membrane protein synthesis and assembly provides a means to observe dynamic changes in protein topology as a function of membrane lipid composition.     

Analysis of highly purified satellite DNA containing chromatin from the mouse.

A purification scheme for satellite DNA containing chromatin from mouse liver has been developed. It is based on the highly condensed state of the satellite chromatin and also takes advantage of its resistance to digestion by certain restriction nucleases. Nuclei are first treated with micrococcal nuclease and the satellite chromatin enriched 3-5 fold by extraction of the digested nuclei under appropriate conditions. Further purification is achieved by digestion of the chromatin with a restriction nuclease that leaves satellite DNA largely intact but degrades non-satellite DNA extensively. In subsequent sucrose gradient centrifugation the rapidly sedimenting chromatin contains more than 70% satellite DNA. This material has the same histone composition as bulk chromatin. No significant differences were detected in an analysis of minor histone variants. Nonhistone proteins are present only in very low amounts in the satellite chromatin fraction, notably the HMG proteins are strongly depleted.     

Synthesis of viral and host DNA in isolated chromatin from herpes simplex virus-infected HeLa cells.

DNA synthesis in chromatin isolated from herpes simplex virus type 1-infected HeLa cells (HSV chromatin) was examined in vitro. The HSV chromatin was found to carry out an initial limited synthesis of DNA in vitro, 50 to 64 pmol of dTMP incorporated in 10(6) nuclei per 10 min, which is comparable to that found in nuclei isolated from HSV-infected cells. DNA synthesis in vitro proceeded for only 30 min, and both HSV DNA and host DNA were synthesized in significant amounts. The HSV and host DNA synthesis in isolated chromatin were inhibited to the same extent by anti-HSV antiserum or by phosphonoacetic acid. The results indicate that the HSV-induced DNA polymerase is most likely involved in the synthesis of host and HSV DNA in isolated chromatin, even though this chromatin contains small amounts of the host gamma-polymerase in addition to the HSV-induced DNA polymerase. The HSV chromatin contains no detectable levels of DNA polymerases alpha and beta, even though infected cells have normal, or increased, levels of these enzymes.