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PubMed1988

Effect of adriamycin and hyperthermia on the sedimentation of nucleoids from L1210 cells.

We report on the individual and combined effects of adriamycin (ADR) and hyperthermia (HYP) on the sedimentation behavior of L1210 mouse leukemia cell nucleoids in neutral sucrose gradients. Nucleoid sedimentation profiles obtained from cells incubated with ADR (1-10 microM; 30 min; 37 degrees C) exhibited an increased sedimentation rate associated with an increased protein content of these subnuclear units. Exposure of cells to HYP (1-3 h; 42 degrees C) produced similar results. Simultaneous exposure of L1210 cells to conditions of HYP and ADR which resulted in minimal changes in nucleoid sedimentation when used singly, produced an enhanced effect. A similar enhancement was observed with other intercalating antineoplastic agents believed to exert their effect, at least partially, via free radicals (daunorubicin, amsacrine, bisantrene, mitoxantrone). However, enhancement with HYP was not observed with (a) the classic intercalating agent, ethidium bromide; (b) non-intercalating DNA-breaking agents (bleomycin, lithocholic acid, etoposide); (c) inhibitors of poly (ADP-ribose) polymerase (m-methoxybenzamide, benzamide); or (d) non-intercalating antineoplastic agents capable of causing free radical formation (bleomycin). The results suggest that DNA intercalating agents capable of initiating free radical processes may show an enhanced toxicity with simultaneous HYP treatment, and that the nucleoid assay may be a means of screening agents with these biological properties for potential clinical usefulness in combination with HYP.

Animals↗

PubMed2003

A function for the mitochondrial chaperonin Hsp60 in the structure and transmission of mitochondrial DNA nucleoids in Saccharomyces cerevisiae.

The yeast mitochondrial chaperonin Hsp60 has previously been implicated in mitochondrial DNA (mtDNA) transactions: it is found in mtDNA nucleoids associated with single-stranded DNA; it binds preferentially to the template strand of active mtDNA ori sequences in vitro; and wild-type (rho+) mtDNA is unstable in hsp60 temperature-sensitive (ts) mutants grown at the permissive temperature. Here we show that the mtDNA instability is caused by a defect in mtDNA transmission to daughter cells. Using high resolution, fluorescence deconvolution microscopy, we observe a striking alteration in the morphology of mtDNA nucleoids in rho+ cells of an hsp60-ts mutant that suggests a defect in nucleoid division. We show that rho- petite mtDNA consisting of active ori repeats is uniquely unstable in the hsp60-ts mutant. This instability of ori rho- mtDNA requires transcription from the canonical promoter within the ori element. Our data suggest that the nucleoid dynamics underlying mtDNA transmission are regulated by the interaction between Hsp60 and mtDNA ori sequences.

Cell Division↗

PubMed1981

Human tumor strains defective in the repair of alkylated DNA fail to regenerate rapidly-sedimenting nucleoids after N-methyl-N'-nitro-N-nitrosoguanidine treatment.

Upon treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), human cell strains characterized as either proficient or defective both in repair of alkylation-damaged DNA and in supporting the growth of MNNG-treated adenovirus (Mer+ and Mer- phenotypes (1,2)), all underwent a rapid relaxation of nucleoid DNA, as judged by sedimentation in 15--30% neutral sucrose gradients. DNA in the repair-proficient Mer+ strains (normal fibroblast and tumor) was restored to the rapidly-sedimenting (control) form within 2--4 h after the removal of MNNG. In contrast, nucleoid DNA of the repair-deficient Mer- tumor strains remained slowly-sedimenting even after 48 h of incubation. The delayed recovery of Mer- nucleoid DNA was specific for MNNG damage, since after u.v. irradiation, to which Mer+ and Mer- strains are equally resistant (2), all cell lines tested underwent DNA relaxation within the first hour after irradiation (3 J/m2) and regenerated rapidly-sedimenting nucleoids within 4--6 h of repair incubation.

Cell Line↗

PubMed1996

Analysis of mitochondrial DNA nucleoids in wild-type and a mutant strain of Saccharomyces cerevisiae that lacks the mitochondrial HMG box protein Abf2p.

DNA-protein complexes (nucleoids) are believed to be the segregating unit of mitochondrial DNA (mtDNA) in Saccharomyces cerevisiae. A mitochondrial HMG box protein, Abf2p, is needed for maintenance of mtDNA in cells grown on rich dextrose medium, but is dispensible in glycerol grown cells. As visualized by 4',6'-diamino-2-phenylindole staining, mtDNA nucleoids in mutant cells lacking Abf2p ( delta abf2) are diffuse compared with those in wild-type cells. We have isolated mtDNA nucleoids and characterized two mtDNA-protein complexes, termed NCLDp-2 and NCLDs-2, containing distinct but overlapping sets of polypeptides. This protocol yields similar nucleoid complexes from the delta abf2 mutant, although several proteins appear lacking from NCLDs-2. Segments of mtDNA detected with probes to COXII, VAR1 and ori5 sequences are equally sensitive to DNase I digestion in NCLDs-2 and NCLDp-2 from wild-type cells and from the delta abf2 mutant. However, COXII and VAR1 sequences are 4-to 5-fold more sensitive to DNase I digestion of mtDNA in toluene-permeabilized mitochondria from the delta abf2 mutant than from wild-type cells, but no difference in DNase I sensitivity was detected with the ori5 probe. These results provide a first indication that Abf2p influences differential organization of mtDNA sequences.

DNA, Fungal↗

PubMed2004

DNA gyrase is involved in chloroplast nucleoid partitioning.

DNA gyrase, which catalyzes topological transformation of DNA, plays an essential role in replication and transcription in prokaryotes. Virus-induced gene silencing of NbGyrA or NbGyrB, which putatively encode DNA gyrase subunits A and B, respectively, resulted in leaf yellowing phenotypes in Nicotiana benthamiana. NbGyrA and NbGyrB complemented the gyrA and gyrB temperature-sensitive mutations of Escherichia coli, respectively, which indicates that the plant and bacterial subunits are functionally similar. NbGyrA and NbGyrB were targeted to both chloroplasts and mitochondria, and depletion of these subunits affected both organelles by reducing chloroplast numbers and inducing morphological and physiological abnormalities in both organelles. Flow cytometry analysis revealed that the average DNA content in the affected chloroplasts and mitochondria was significantly higher than in the control organelles. Furthermore, 4',6-diamidino-2-phenylindole staining revealed that the abnormal chloroplasts contained one or a few large nucleoids instead of multiple small nucleoids dispersed throughout the stroma. Pulse-field gel electrophoresis analyses of chloroplasts demonstrated that the sizes and/or structure of the DNA molecules in the abnormal chloroplast nucleoids are highly aberrant. Based on these results, we propose that DNA gyrase plays a critical role in chloroplast nucleoid partitioning by regulating DNA topology.

Base Sequence↗

PubMed1981

A new approach to the study of the E. coli nucleoid.

E. coli were examined by the freeze-fracture thaw-fix technique, embedded in thin fibrin gels. After glutaraldehyde fixation the bacterial nucleoid was found spread out over the surrounding fibrin. Addition of calcium and uranyl acetate to the fixative preserved the nucleoid in compact form. The spread nucleoid was then examined against a smooth mica background after freeze-thaw and osmotic lysis. These spreads were critical-point dried, rotary shadowed with platinum-carbon and viewed as stereo-pair micrographs. Structures seen are tentatively interpreted as clusters of polyribosomes, extended DNA, and supercoiled DNA complexed with proteins or polyamines. After osmotic lysis, glutaraldehyde alone preserves the nucleoid in compact form. Only where strands are broken, in freeze-fracture or freeze-thaw lysis, must uranyl acetate be added to the fixative to preserve a compact structure.

Bacterial Proteins↗

PubMed1992

Involvement of FtsZ in coupling of nucleoid separation with septation.

The cell-cycle parameters of an Escherichia coli strain expressing essential division gene ftsZ at one-fifth of its normal level, because of antisense regulation by DicF RNA, have been analysed. Inhibition of FtsZ expression affects neither the generation time nor the replication initiation mass, the C period, or the constriction period, but it does dramatically retard the initiation of constriction relative to replication termination. Separation of the nucleoids is equally postponed, indicating that division is not coupled to termination of replication, but to partitioning. The severe inhibition of nucleoid separation by DicF RNA, and its suppression by overproduction of FtsZ, suggest a role for FtsZ in the control of separation, and consequently in the coupling of separation and division. We suggest that the normal pattern of nucleoid separation previously found in cells deficient in ftsZ function was a consequence of the loss of a negative effect exerted by FtsZ on separation. In agreement with this view, we find that nucleoid separation is temporarily inhibited after arrest of FtsZ synthesis, but is later resumed as FtsZ is further diluted into the elongating filaments.

Bacterial Proteins↗

PubMed2024

The Lrs14 family of DNA-binding proteins as nucleoid-associated proteins in the Crenarchaeal order Sulfolobales.

Organization of archaeal chromatin combines bacterial, eukaryotic, and unique characteristics. Many archaeal lineages harbor a wide diversity of small and highly expressed nucleoid-associated proteins, which are involved in DNA structuring. In Sulfolobales, representing model organisms within the Crenarchaeota, Sul7d, Cren7, Sul10a, and Sul12a are well-characterized nucleoid-associated proteins. Here, we combine evidence that the Lrs14 family of DNA binders is part of the repertoire of nucleoid-associated proteins in Sulfolobales. Lrs14-encoding genes are widespread within genomes of different members of the Sulfolobales, typically encoded as four to nine homologs per genome. The Lrs14 proteins harbor a winged helix-turn-helix DNA-binding domain and are typified by a coiled-coil dimerization. They are characterized by distinct sequence- and structure-based features, including redox-sensitive motifs and residues targeted for posttranslational modification, allowing a further classification of the family into five conserved clusters. Lrs14-like proteins have unique DNA-organizing properties. By binding to the DNA nonsequence specifically and in a highly cooperative manner, with a slight preference for AT-rich promoter regions, they introduce DNA kinks and are able to affect transcription of adjacent transcription units either positively or negatively. Genes encoding Lrs14-type proteins display considerable differential expression themselves in response to various stress conditions, with certain homologs being specific to a particular stressor. Taken together, we postulate that members of the Lrs14 family can be considered nucleoid-associated proteins in Sulfolobales, combining a DNA-structuring role with a global gene expression role in response to stress conditions.

DNA-Binding Proteins↗

PubMed1975

Nuclear and cell division in Bacillus subtilis: dormant nucleoids in stationary-phase cells and their activation.

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.

Bacillus subtilis↗

PubMed1981

Assimilation of single-stranded donor deoxyribonucleic acid fragments by nucleoids of competent cultures of Bacillus subtilis.

Lysates containing folded chromosomes of competent Bacillus subtilis were prepared. The chromosomes were supercoiled, as indicated by the biphasic response of their sedimentation rates to increasing concentrations of ethidium bromide. Limited incubation of the lysates with increasing concentrations of ribonucleases resulted in a gradual decrease in the sedimentation velocity of the deoxyribonucleic acid (DNA) until finally a constant S value was reached. Incubation with sonicated, 4,5',8-trimethylpsoralen-monoadducted, denatured, homologous donor DNA molecules at 37 degrees C and concomitant irradiation with long-wave ultraviolet light of the nucleoid-containing lysates resulted in the formation of complexes of the donor DNA molecules and the recipient chromosomes. This complex formation was stimulated when nucleoids were previously (i) unfolded by ribonuclease incubation, (ii) (partially) relaxed by X irradiation, or (iii) subjected to both treatments. Monoadducts were not essential. On the other hand, the complex-forming capacity of recipient chromosomes previously cross-linked by 4,5',8-trimethylpsoralen diadducts was greatly reduced, suggesting that strand separation of the recipient molecule was involved in the formation of the complex. None of these effects has been observed when heterologous (Escherichia coli) donor DNA has been used. When the same kind of experiments were carried out at 70 degrees C, donor-recipient DNA complexes were also formed and required strand separation and homology similar to donor-recipient complex formation at 37 degrees C. However, in contrast to what was found at 37 degrees C, unfolding plus relaxation of the nucleoids, as well as the absence of monoadducts in the donor DNA fragments, resulted in a decrease in complex formation. On the basis of these results, we assume that superhelicity can promote the in vitro assimilation of single-stranded donor DNA fragments by nucleoids of competents B. subtilis cells at 70 degrees C, but that at 37 degrees C a different mechanism is involved.

Bacillus subtilis↗

PubMed1981

Repair of thermal damage to the Escherichia coli nucleoid.

The folded chromosome or nucleoid of Escherichia coli was analyzed by low-speed sedimentation in neutral sucrose gradients after heat treatment (30 min at 50 degrees C) and subsequent incubation of cells at 37 degrees C for various times. Heat treatment resulted in in vivo association of the nucleoids with cellular protein and in an increase in sedimentation coefficient. During incubation at 37 degrees C, a fraction of the nucleoids, from heated cells, because dissociated from cellular protein and regained their characteristic sedimentation coefficients. The percentage of nucleoids which returned to their control sedimentation position in the sucrose gradients corresponded to the percentage of cells able to repair thermal damage as assayed by enumeration on agar plates.

Bacterial Proteins↗

PubMed1981

Nucleoid structure in freeze fractures of Streptococcus faecalis: effects of filtration and chilling.

With the techniques used in this study, the nucleoid of Streptococcus faecalis could not be seen in freeze-etch preparations unless glutaraldehyde had been added to cultures of cells before they were frozen. With time, the nucleoid became visible as a network of fibers, apparently as a result of the aggregation of individual chromosomal elements in the presence of glutaraldehyde. When glutaraldehyde was added to undisturbed cultures, the fibers that became visible were observed in small patches that were seemingly scattered throughout the cytoplasm. However, if cells were chilled or placed on filters before glutaraldehyde was added, the fibers which then developed were seen in large central areas. The appearance of centralized nucleoids in freeze fractures of cells that had been chilled or filtered could be correlated with a decrease in the central density of the cytoplasm, as seen by light microscopy, in cells embedded in gelatin or bovine serum albumin. These observations are discussed in relation to a model for the normal structure of the nucleoid which suggests that the treatments routinely used to study the morphology-physiology of cells (chilling, filtration, and fixation) result in a reorganization of the cytoplasm, leading to an increase in the centralization of nuclear material.

Cell Nucleus↗

PubMed1985

Confocal scanning light microscopy of the Escherichia coli nucleoid: comparison with phase-contrast and electron microscope images.

The nucleoid of living and OsO4- or glutaraldehyde-fixed cells of Escherichia coli strains was studied with a phase-contrast microscope, a confocal scanning light microscope, and an electron microscope. The trustworthiness of the images obtained with the confocal scanning light microscope was investigated by comparison with phase-contrast micrographs and reconstructions based on serially sectioned material of DNA-containing and DNA-less cells. This comparison showed higher resolution of the confocal scanning light microscope as compared with the phase-contrast microscope, and agreement with results obtained with the electron microscope. The effects of fixation on the structure of the nucleoid were studied in E. coli B/r H266. Confocal scanning light micrographs and electron microscopic reconstructions showed that the shape of the nucleoid remained similar after OsO4 or glutaraldehyde fixation; however, the OsO4 nucleoid appeared to be somewhat smaller and more centralized within the cell.

Cell Nucleus↗

PubMed1989

Cell length, nucleoid separation, and cell division of rod-shaped and spherical cells of Escherichia coli.

By comparing the dimensions and DNA contents of normal rod-shaped Escherichia coli with those of mutants that grow as spheres or ellipsoids, we have determined that two parameters remain unchanged: the DNA/mass ratio and the average cell length (diameter, for spherical cells). In consequence, the average volumes and DNA contents of the spherical mutant cells are about four to six times greater than those of rod-shaped cells growing at a similar rate. In addition, it was found that cells of both rod and sphere forms had approximately the same number of nucleoids (as seen when the DNA was condensed after inhibition of protein synthesis). The nucleoids of the spherical cells therefore consist of four to six completed chromosomes each (polytene nucleoids). We suggest that the attainment of a minimum cell length is necessary for nucleoid separation after chromosome replication and that such a separation is itself a prerequisite for septum formation.

Cell Division↗

PubMed1991

Condensation of the forespore nucleoid early in sporulation of Bacillus species.

Fluorescence microscopic examination coupled with digital videoimage analysis of 4',6-diamidino-2-phenylindole-stained sporulating cells of Bacillus megaterium or Bacillus subtilis revealed a striking condensation of the forespore nucleoid. While both mother cell and forespore compartments had equal amounts of DNA, the forespore nucleoid became greater than 2-fold more condensed than the mother cell nucleoid. The condensation of the forespore nucleoid began after only the first hour of sporulation, 2 to 3 h before expression of most forespore-specific genes including those for small, acid-soluble spore proteins, and was abolished in spo0 mutants but not in spoII or spoIII mutants. It is possible that this striking condensation of forespore DNA plays some role in modulating gene expression during sporulation.

Bacillus megaterium↗

PubMed1996

Chloramphenicol causes fusion of separated nucleoids in Escherichia coli K-12 cells and filaments.

Chloramphenicol is frequently used for better visualization of the Escherichia coli nucleoid. Here, we show that chloramphenicol causes not only rounding off of the nucleoid but also fusion of as many as four separated nucleoids. Nucleoid fusion occurred in fast-growing cells and in filaments obtained by dicF antisense RNA induction or in ftsZ84(Ts) and pbpB(Ts) mutants. Thus, treatment with chloramphenicol erroneously suggests that DNA segregation is inhibited.

Anti-Bacterial Agents↗

PubMed1997

Association of the histone-like protein HBsu with the nucleoid of Bacillus subtilis.

To investigate the physiological role of the essential histone-like protein of Bacillus subtilis (HBsu) in the nucleoid structure, a fusion to the green fluorescent protein (GFP) of Aequorea victoria was constructed. This purified fusion protein, HBsuGFP, showed a threefold-reduced affinity to DNA compared to unmodified HBsu; however, in gel mobility shift experiments HBsuGFP DNA-binding was greatly enhanced in the presence of low HBsu concentrations. Additional production of HBsu also had a positive effect on the retarded growth of a B. subtilis strain, PK9C8, which expresses only hbs-gfp (encoding HBsuGFP). HBsu seemed to influence not only growth but also nucleoid structure, as monitored by DNA staining and fluorescence microscopy. Without HBsu production, strain PK9C8 showed a relaxed nucleoid structure associated with HBsuGFP. However, a highly compact nucleoid structure that coincides with the fluorescence of the fusion protein was visualized when HBsu synthesis was induced. This provides the first evidence for in vivo association of HBsu in DNA packaging and its consequence on cell growth.

Bacillus subtilis↗

PubMed1965

FINE STRUCTURE OF THE COAT AND NUCLEOID MATERIAL OF FOWLPOX VIRUS.

Hyde, James M. (University of Mississippi School of Medicine, Jackson), Lanelle G. Gafford, and Charles C. Randall. Fine structure of the coat and nucleoid material of fowlpox virus. J. Bacteriol. 89:1557-1569. 1965.-Several morphological forms characteristic of the poxvirus group were demonstrated for fowlpox virus with neutral phosphotungstic acid (PTA). Viral particles (purified from viral inclusion bodies) stained with uranyl acetate (UA) and shadowed with platinum were shown to have an external knobby surface not evident with PTA. The external coat of freshly purified viral particles seemed intact, but as the preparation aged, it appeared to unwind, resulting in twisted "rope-like" structures. This process was facilitated by use of 1% trypsin, and three dense fibrils were identified with UA within the partially detached viral coat. Studies with alkaline PTA (pH 9) were interpreted as revealing a complex nucleoid, but solutions above this pH damaged the particles. The morphology of the nucleoid was better depicted in ultrathin sections of whole virus which, when stained with UA, revealed dense coiled threads. Treatment of virus with sodium lauryl sulfate exposed an underlying coat consisting of small subunits approximately 40 A in diameter. Of great interest was the demonstration that the detergent removed strands of deoxyribonucleic acid (DNA) from the virus without destroying the contour of the particle. The origin of the strands was definitely the fine uranophilic, coiled threads of the nucleoid, which probably represent the DNA molecule(s). That the extracted material was largely DNA was proved by digestion with deoxyribonuclease and resistance to ribonuclease and trypsin. These studies illustrate how a variety of electron microscopic techniques may be utilized alone or in combination to reveal hitherto undescribed fine structure of viral particles.

Animals↗