Electron Microscope Study of DNA-Containing Plasms : II. Vegetative and Mature Phage DNA as Compared with Normal Bacterial Nucleoids in Different Physiological States

The nucleoids of Escherichia coli, independently of the physiological state of the bacteria, are shown to be preserved as a fine-stranded fibrillar nucleoplasm by an OsO₄ fixation under defined conditions: acetate-veronal buffer pH 6, presence of Ca⁺⁺ and amino acids, stabilization with uranyl-acetate before dehydration. The same fixation procedure applied to the DNA of vegetative phage reveals a pool of homogeneous fibrillar structure very similar to the nucleoplasm. The "versene test," which produces a coarse coagulation of these plasms, emphasizes the similar behaviour of the pool and the nucleoids. The heads of mature phage are preserved in their true polyhedral shape by the standard fixation procedure, although they may be badly distorted when fixed under different conditions. Lanthanum nitrate and uranyl-acetate are shown to increase markedly the contrast of both phage and cytoplasm. The consequences of the fibrillar structure of the genetic material are discussed in relation to the probable division process.     

Properties of condensed bacteriophage T4 DNA isolated from Escherichia coli infected with bacteriophage T4.

Methods developed for isolating bacterial nucleoids were applied to bacteria infected with phage T4. The replicating pool of T4 DNA was isolated as a particle composed of condensed T4 DNA and certain RNA and protein components of the cell. The particles have a narrow sedimentation profile (weight-average s=2,500S) and have, on average, a T4 DNA content similar to that of the infected cell. Their dimensions observed via electron and fluorescence microscopy are similar to the dimensions of the intracellular DNA pool. The DNA packaging density is less than that of the isolated bacterial nucleoid but appears to be roughly similar to its state in vivo. Host-cell proteins and T4-specific proteins bound to the DNA were characterized by electrophoresis on polyacrylamide gels. The major host proteins are the RNA polymerase subunits and two envelope proteins (molecular weights, 36,000 and 31,000). Other major proteins of the host cell were absent or barely detectable. Single-strand breaks can be introduced into the DNA with gamma radiation or DNase without affecting its sedimentation rate. This and other studies of the effects of intercalated ethidium molecules have suggested that the average superhelical density of the condensed DNA is small. However, these studies also indicated that there may be a few domains in the DNA that become positively supercoiled in the presence of high concentrations of ethidium bromide. In contrast to the Escherichia coli nucleoid, the T4 DNA structure remains condensed after the RNA and protein components have been removed (although there may be slight relaxation in the state of condensation under these conditions).     

Ultrastructure of the ring-shaped nucleoli in the hepatocytes of chick embryos]

The fine structure of ring-shaped nucleoli in hepatocytes of chick embryos at different terms of development was studied. Structural differences were shown between annular nucleoli and nucleoli with typical nucleolonemal organization. Ring-shaped nucleoli, as a rule, are devoid of nucleolonemal structure and their fibrillar component is reduced. The material which fills the central cavity always appears similar to the nucleoplasm in its structure. On the basis of serial sections, we propose that central cavity is isolated from the nucleoplasm. From the hypotonical treatment and EDTA staining application it is concluded that the central cavity of ring-shaped nucleoli contains DNP associated with the intranucleolar chromatin. The number of these nucleoli increased after the injection of a liver homogenate cytoplasmic fraction extracted from adult hen. The physiological significance of such nucleoli is discussed.     

Scattering of the silver-stained proteins of nucleolar organizer regions in Ishikawa cells by actinomycin D.

Scattering of the silver-stained proteins of nucleolar organizer regions (Ag-NOR proteins) was produced by actinomycin D in Ishikawa cells. Scattering of Ag-NOR proteins was found only in cells treated with actinomycin D and various other agents had no effect. Scattering was dose-dependent up to 10(-2) micrograms/ml of actinomycin D, but it was not found at higher concentrations that caused marked inhibition of total DNA and RNA synthesis. Actinomycin D (10(-2) micrograms/ml) caused the following changes: (i) nucleolar segregation and (ii) emergence of dense fibrillar bodies in the nucleoplasm. Ag-NOR proteins were observed on the fibrillar centers and surrounding fibrillar components in control nucleoli, on the fibrillar and amorphous zones in segregated nucleoli, and on the dense fibrillar bodies emerging in the nucleoplasm. The scattering of Ag-NOR proteins was due to the argyrophilic nature of the dense fibrillar bodies. Actinomycin D (10(-1) micrograms/ml) also caused similar morphological alterations in the nucleolus and nucleoplasm, but Ag-NOR proteins were observed only on nucleolar remnants.     

Phosphorylation of D-glucose in Escherichia coli mutants defective in glucosephosphotransferase, mannosephosphotransferase, and glucokinase.

The morphology of nucleoids and mesosomes of Bacillus subtilis in stationary-and lag-phase cultures was studied by making three-dimensional cell reconstructions in plastic of electron micrographs of serial sections. In cells from stationary cultures, the dormant nucleoids are frequently, but not always, spherical and the mesosomes are small and compact. It is suggested that the spherical nucleoids represent the resting stage in which replication and segregation have been completed. In cells from lag-phase cultures, the compact mesosomes develop into an elaborate system of tubes and wider sacs which become wrapped around the elongating nucleoids and invade the nucleoplasm in preparation for division.     

Nuclear and cell division in Bacillus subtilis: cell development from spore germination.

The changes in the morphology of the nucleoids and the mesosomes in Bacillus subtilis cells during synchronous outgrowth after spore germination were followed in large-scale three-dimensional cell reconstructions. Shortly after outgrowth of the cell begins in Spizizen medium with glucose, the mesosome becomes an elongated structure in close contact with a rounded nucleoid. When nuclear replication reaches full activity, the mesosome develops into a single, complicated versatile system, with tubules that traverse the cytoplasm and have elaborations in and near the nucleoplasm. Later the system may retract to form large rounded mesosomes; the tubules and strings of vesicles within these mesosomes probably have been collected from the cytoplasm. Shortly after the first cell division, both sister cells have two nucleoids, but with longer generation times induced by growth in media containing acetate instead of glucose; these sister cells have only one nucleoid each. In acetate-grown cells rounded nucleoids that have no contact with a mesosome may represent nucleoids in a temporary stage of rest. On the other hand, the nucleoids of cells growing in glucose-containing medium are always penetrated by mesosomal material, superficially or deeply. Since the mesosome appears capable of traversing the nuclear fibrils, and even reaching the last strands connecting the dividing nucleoids, it is suggested that this organelle may play a vital role in the Bacillus division cycle.     

Nucleoid structure and distribution in thermophilic Archaea.

Nucleoid structure and distribution in thermophilic organisms from the Archaea domain were studied. Combined phase-contrast and fluorescence microscopy of DAPI (4',6-diamidino-2-phenylindole)-stained Sulfolobus acidocaldarius and Sulfolobus solfataricus cells revealed that the nucleoids were highly structured. Different nucleoid distribution within the cells, representing different partition stages, was observed. The conformation of the nucleoids differed between exponentially growing and stationary-phase cells. Also, the stationary-phase cells contained two chromosomes, and the nucleoids occupied a larger part of the interior of the cells than in the exponentially growing cells. The part of the cell cycle during which fully separated nucleoids could be detected was short. Since the postreplication period is long in these organisms, there was a considerable time interval between termination of chromosome replication and completion of nucleoid separation, similar to the G2 phase in eukaryotic cells. The length of the visible cell constriction period was found to be in the same range as that of eubacteria. Finally, cell-cell connections were observed under certain conditions. Possible eubacterial, eukaryotic, and unique features of nucleoid processing and cell division in thermophilic archaea are discussed.     

Spectrofluorometric measurement of the binding of ethidium to superhelical DNA from cell nuclei.

Structures retaining many of the morphological features of nuclei may be released by lysing HeLa cells in solutions containing non-ionic detergents and high concentrations of salt. These nucleoids contain few chromatin proteins. We have shown that the DNA of nucleoids is quasicircular and supercoiled by measure spectrofluorometrically the amount of the intercalating dye, ethidium, bound to unirradiated and gamma-irradiated nucleoids. Ethidium binds to nucleoids in the manner characteristic of the binding to superhelical DNA: at low concentrations more ethidium binds to unirradiated nucleoids than to their gamma-irradiated counterparts with broken DNA, and at higher concentrations less ethidium binds to the unirradiated nucleoids. The quasi-circles in nucleoids are 22 times less sensitive to gamma-irradiation than are circles of pure PM2 DNA: they must contain about 2.2 X 10(5) base pairs. The constraints that maintain the quasi-circularity of nucleoid DNA are very resistant to extremes of temperature and alkali; some remain under conditions in which the duplex is denatured. The constraints are destabilised by ethidium suggesting that they are stabilised by free energy of supercoiling. Proteolytic enzymes, but not ribonucleases, remove the constraints. Possible structures for the constraining mechanism are discussed.     

Evidence from studies on segregated nucleoli that nucleolar silver staining proteins C23 and B23 are in the fibrillar component

Several procedures for the silver staining of nucleoli have been evaluated at the electron microscopic level to determine optimal conditions for ultrastructural preservation and staining specificity. The present study shows that a brief fixation with 1% buffered formaldehyde followed by methanol: acetic acid (3 : 1) fixation yielded optimal preservation and silver staining of nucleoli. Using this procedure for electron microscopic studies of interphase nucleoli, it was found that the punctate silver grains observed by light microscopy were composed of fine silver granules, of approx. 100 Å diameter, organized in discrete clusters. In similar studies on adriamycin-induced segregated nucleoli, it was observed that the silver staining reaction was mainly limited to the fibrillar portion of the nucleolus. Accordingly, nucleolar proteins C23 and B23, found earlier to be the major silver binding proteins of the nucleolus, are mainly concentrated in the fibrillar nucleolar component.     

Immunocytochemistry of Lipids: Chemical Fixatives have Dramatic Effects on the Preservation of Tissue Lipids

We report here the effects of chemical fixatives on lipids studied under conditions simulating the immunogold labelling of phosphatidylserine. Using anti-phosphatidylserine antibodies, it is shown that the labelling intensity of a phosphatidyl-serine/phosphatidylcholine coating depends largely on the conditions of fixation. In fact, the usual aldehydic fixatives washed out most of the phostphatidylserine, thus preventing the binding of anti-phosphatidylserine antibodies. This was confirmed on biological samples such as rat liver and brain by measuring the loss of radiolabelled lipids during the fixation procedure. Furthermore, the complete procedure of tissue preparation for electron microscopical observation was investigated. The loss of (radiolabelled) lipids was studied in tissue samples during fixation and resin embedding. The results showed that the classical procedure (glutaraldehyde fixation followed by epoxy resin embedding) results in the loss of 73–91% of the tissue lipids whereas in unfixed, freeze-substituted samples, more than 76% of the tissue lipids are preserved.     

Bacteriophage T4 head morphogenesis. VII. Terminal stages of head maturation.

Several aspects of the terminal stages of T4 head maturation were investigated using ts and am mutants blocked at single steps of the assembly pathway. We had previously found that cells infected with mutants of gene 13, e.g., tsN38 and amE609, accumulated both stable (10 to 20%)- and fragile (80%)-filled head precursors (Hamilton and Luftig, 1972). Here we showed the following for such gene 13-defective, mutant-infected cells. (i) Using thin-section analysis the pool of phage precursor structures observed under nonpermissive conditions was one-third of that observed when the cells were cultured under permissive conditions. (ii) In order for complete conversion of the precursors into viable phage to occur, there were apparent requirements of metabolic energy, protein, and DNA synthesis. (iii) The intracellular DNA pool under nonpermissive conditions exhibited a 50% distribution between 63S (mature size) and 200 S (concatenate size) DNA, with the latter DNA serving as a precursor pool. Further, this DNA pool when spread onto a protein monolayer exhibited a dispersed array of DNA, strands around a core, which was less dense than that found for the greater than 1,000S DNA concatenate isolated from gene 49-defective infected cells. (iv) When precuations were taken to stabilize the head precursors, such as lysis of the cells into glutaraldehyde, there was a 30% increase in the yield of 1,200S filled heads. Correlating these results and previous results concerning gene 49-defective unfilled heads, we propose that there are several forms of gene 13 fragile head precursors which serve as intermediates between gene 49 unfilled heads and gene 13 stable filled heads. We cannot, however, rule out the possibility that all gene 13-defective heads represent a single class of unstable particles, which decay slowly. In either case, we have shown that gene 13-defective particles are unstable to some degree inside the cell and are highly unstable outside the cell; yet all particles can still be efficiently converted to phage in vivo.     

Isolation of the Escherichia coli nucleoid

Numerous protocols for the isolation of bacterial nucleoids have been described based on treatment of cells with sucrose-lysozyme-EDTA and subsequent lysis with detergents in the presence of counterions (e.g., NaCl, spermidine). Depending on the lysis conditions both envelope-free and envelope-bound nucleoids could be obtained, often in the same lysate. To investigate the mechanism(s) involved in compacting bacterial DNA in the living cell, we wished to isolate intact nucleoids in the absence of detergents and high concentrations of counterions. Here, we compare the general lysis method using detergents with a procedure involving osmotic shock of Escherichia coli spheroplasts that resulted in nucleoids free of envelope fragments. After staining the DNA with DAPI (4',6-diamidino-2-phenylindole) and cell lysis by either isolation procedure, free-floating nucleoids could be readily visualized in fluorescence microscope preparations. The detergent-salt and the osmotic-shock nucleoids appeared as relatively compact structures under the applied ionic conditions of 1 M and 10 mM, respectively. RNase treatment caused no dramatic changes in the size of either nucleoid.     

Cajal bodies, nucleoli, and speckles in the Xenopus oocyte nucleus have a low-density, sponge-like structure

Nuclear organelles, unlike many cytoplasmic organelles, lack investing membranes and are thus in direct contact with the surrounding nucleoplasm. Because the properties of the nucleoplasm and nuclear organelles influence the exchange of molecules from one compartment to another, it is important to understand their physical structure. We studied the density of the nucleoplasm and the density and permeability of nucleoli, Cajal bodies (CBs), and speckles in the Xenopus oocyte nucleus or germinal vesicle (GV). Refractive indices were measured by interferometry within intact GVs isolated in oil. The refractive indices were used to estimate protein concentrations for nucleoplasm (0.106 g/cm3), CBs (0.136 g/cm3), speckles (0.162 g/cm3), and the dense fibrillar region of nucleoli (0.215 g/cm3). We determined similar protein concentrations for nuclear organelles isolated in aqueous media, where they are no longer surrounded by nucleoplasm. To examine the permeability of nuclear organelles, we injected fluorescent dextrans of various molecular masses (3-2000 kDa) into the cytoplasm or directly into the GV and measured the extent to which they penetrated the organelles. Together, the interferometry and dextran penetration data show that organelles in the Xenopus GV have a low-density, sponge-like structure that provides access to macromolecules from the nucleoplasm.     

Ultrastructural studies of the nucleoids of the pleomorphic forms of Chlamydia psittaci 6BC: a comparison with bacteria.

The nucleoids of the various pleomorphic forms of Chlamydia psittaci have been examined by direct observation of infected cells and by observations on isolated particles. The fixation and staining methods used were the same as those routinely used for the examination of bacteria to facilitate the comparison of chlamydial fine structure with that of bacteria. The nucleoids of reticulate bodies were composed of fine fibrils which extended throughout these particles. The nucleoids of intermediate bodies are characterized by an electron-dense mass with which the fibrous elements are associated in a structurally coherent manner. As condensation of the intermediate bodies proceeds, the electron-dense mass becomes eccentrically located and the fibers form a distinct radiating structure. Large elementary bodies have a few fibers associated with their condensed electron-dense nucleoids but the more condensed mature elementary bodies have a very discrete and homogeneous electron-dense nucleoid which is separated from the cytoplasmic elements of these particles by a very distinct electron-transparent space. These highly condensed elementary body nucleoids are usually ovoid, but may be elongated or irregular, and a small number of these structures react very strongly with ruthenium red. While the nucleoid structure of reticulate bodies resembles that of the bacterial cell, both the condensation process and the nucleoid morphologies which result from it in intermediate and elementary bodies have no parallels among the bacteria. Thus we conclude that major differences in nucleoid organization exist between the chlamydia and the bacteria.     

Conformational constraints in nuclear DNA.

We have investigated DNA superstructure in a wide range of nuclei of higher cells by gently lysing cells to release structures that resemble nuclei but are depleted of nuclear proteins. The sedimentation properties of these structures, which we call nucleoids, have been examined in sucrose gradients containing the intercalating agent, ethidium. The sedimentation rate of nucleoids derived from the growing cells of mammals, birds, amphibians and insects varies in the manner characteristic of circular and superhelical molecules of DNA. These characteristic changes in sedimentation rate are abolished by irradiating the nucleoids with low doses of gamma-rays, a procedure known to introduce single-strand scissions into DNA. We have also investigated by similar means DNA superstructure in nucleoids derived from a variety of different chick cells. Nucleoids derived from adult hen erythrocytes differ from the other nucleoids studied in that their sedimentation rate does not vary in the manner characteristic of supercoiled DNA.     

Envelope-associated nucleoid from Caulobacter crescentus stalked and swarmer cells.

Envelope-associated nucleoids have been isolated from Caulobacter crescentus by using a modification of the procedure of T. Kornberg et al. (Proc. Natl. Acad. Sci. U.S.A. 71:3189-3193, 1974). The development of a Ludox density gradient procedure has permitted preparation of large quantities of synchronous cells. The sedimentation coefficients of the envelope-associated nucleoids of stalked and swarmer cells, prepared under conditions of equivalent cell lysis, were 3,000S and greater than 6,000S respectively. Small differences in the relative amounts of deoxyribonucleic acid, ribonucleic acid, and protein in stalked and swarmer cell envelope-associated nucleoids could not account for the large differences in sedimentation behavior. These characteristic sedimentation coefficients were retained in mixing experiments.