Changes in DNA topology during spermatogenesis

DNA topology in histone- and protamine-depleted nuclei (nucleoids) from somatic cells, sperm, and spermatogenic cells was studied to determine if the superhelical configuration of DNA looped domains is altered during spermatogenesis. The expansion and contraction of nucleoid DNA was measured with a fluorescence microscope following exposure of nucleoids to different concentrations of ethidium bromide (EB). Nucleoids from Xenopus laevis erythrocytes, primary spermatocytes, and round spermatids, and from Rana catesbeiana sperm all exhibited a biphasic change (condensed-relaxed-condensed) in size as a function of exposure to increasing concentrations (0.5–100 g/ml) of EB, indicating that they contain negatively supercoiled DNA. In contrast, DNA in sperm nucleoids from Xenopus laevis and Bufo fowleri was relaxed and expanded at low (0.5–6 g/ml) EB concentrations, but became gradually condensed as the EB concentration was increased (6–100 g/ml). Nucleoids prepared from all cell types retained the general shape of the nucleus regardless of the superhelical configuration of the nucleoid DNA. Sperm nucleoid DNA condensed by 100 g/ml EB was relaxed by exposure to UV light, DNase I, proteinase K, or 4 M urea, but not by RNase A or 10 mM dithiothreitol. These results demonstrate that the DNA in sperm nucleoids is constrained in domains of supercoiling by nonbasic nuclear proteins. Negatively supercoiled DNA is present in nucleoids from cells with a full complement of histones, including Rana sperm, but not in nucleoids from Xenopus and Bufo sperm in which histones are replaced by intermediate-type protamines. Histone replacement in these species, therefore, is accompanied by unfolding of nucleosomal DNA and active removal of the negative supercoils. Results presented also suggest an important role for the nonbasic nuclear proteins of sperm in the morphogenesis of the nucleus and the arrangement of DNA.     

Variations in chloroplast nucleoid number during the cell cycle in the algaScenedesmus quadricauda grown under different light conditions

Summary Synchronous cultures of the green algaScenedesmus quadricauda were grown at different mean irradiances (ranging from 15 Wm^–2 to 130Wm^–2). At each irradiance, the algae were exposed to illumination regimes which differed in light duration and dark intervals (222 to 240 hours). The cells from these cultures were sampled during their cycles, stained with DAPI and the number of nuclei and chloroplast nucleoids estimated.The nucleoids divided semisynchronously in steps which represented doublings in their number. For each doubling a constant amount of light energy (defined as the product of irradiance and light duration) had to be converted by the cells to become committed to this division. The times to the start of the nucleoid divisions were therefore inversely proportional to the irradiances applied and the final number of nucleoids was proportional to the light duration.Temporal relationships between nuclear and nucleoid divisions were also light dependent. Shortage of light energy caused delay in nucleoid division. The cell division rate was higher than the rate of nucleoid division and consequently, the cells tended to decrease their nucleoid number with decreasing irradiance. With increasing irradiance the start of nucleoid division was gradually shifted toward the beginning of the cell cycle. The rate of nucleoid division exceeded the rate of nuclear and cellular division, thus with increasing irradiance cells with increasing numbers of nucleoids were formed.Abbreviations DAPI 46-diamidino-2-phenylindole - pt-DNA chloroplast DNA     

Isolation of omikron-endosymbionts from mass cultures of Euplotes aediculatus and characterization of their DNA

Omikron-particles were isolated from Euplotes aediculatus cell homogenates. Purified preparations were used to extract the DNA of omikron. The melting profile suggests a GC content of 47.5% for omikron DNA. This value is 2.8% lower if it is calculated from buoyant density which was found to be 1.704 g/cm³. Similar experiments suggest a GC content of 33% for Euplotes DNA. Omikron has a DNA content of ˜3.5 × 10⁹ D per particle, and of ˜0.5 × 10⁹ D per nucleoid, since the DNA is organized in 3–9 distinct nucleoids. The kinetic complexity of omikron DNA as derived from renaturation kinetics is 0.64 × 10⁹ D and therefore by a factor of four smaller than the genome of Escherichia coli. The genome size is similar to that found for lambda-particles of Paramecium aurelia and for certain mycoplasmas.     

Epifluorescent microscopic studies on the mechanism of preferential destruction of chloroplast nucleoids of male origin in young zygotes ofChlamydomonas reinhardtii

Summary The studies on the kinetics of nucleoid destruction reported here showed that destruction of chloroplast nucleoids (ct nucleoids) of male origin began to occur at about 30 minutes after mixing of male (mt^–) and female (mt⁺) gametes. The timing of initiation of the destruction differed among zygotes but usually occurred during 50–120 minutes after mixing. About 10 minutes was required for complete digestion of the ct nucleoids. UV irradiation on young zygotes or addition of an RNA-synthesis inhibitor, actinomycin D, to the incubation medium during the first 0–30 minutes after mixing almost completely inhibited the incorporation of³H uridine into the cell nuclei and the preferential destruction without inhibiting cell nuclear fusion. These results suggest that soon after mating,de novo RNA synthesis is concerned for the preferential destruction of ct nucleoids. To determine in which of the two cell nuclei in the zygotes the RNA is synthesized, each gamete (mt^–, mt⁺) was irradiated with UV and mated with unirradiated gametes of opposite mating type. This treatment of the male gametes had no effect on the incorporation of³H uridine into cell nuclei and the preferential destruction of ct nucleoids but UV irradiation of female gametes almost completely inhibited the incorporation of³H uridine into cell nuclei and the preferential destruction of ct nucleoids. Similar phenomena occurred in other crosses. The UV effect was photoreactivated in about 50% by white light, suggesting that the UV target is DNA. Thus, RNA synthesized in the cell nucleus of female origin soon after mating may be responsible for the preferential destruction of ct nucleoids of male origin     

The repair of double-strand breaks in the nuclear DNA of Saccharomyces cerevisiae and its genetic control

Summary With the use of neutral sucrose sedimentation techniques, the size of unirradiated nuclear DNA and the repair of double-strand breaks induced in it by ionizing radiation have been determined in both wild-type and homozygous rad52 diploids of the yeast Saccharomyces cerevisiae. The number average molecular weight of unirradiated DNA in these experiments is 3.0×10⁸±0.3 Daltons. Double-strand breaks are induced with a frequency of 0.58×10^-10 per Daltonkrad in the range of 25 to 100 krad. Since repair at low doses is observed in wild-type but not homozygous rad52 strains, the corresponding rad52 gene product is concluded to have a role in the repair process. Cycloheximide was also observed to inhibit repair to a limited extent indicating a requirement for protein synthesis. Based on the sensitivity of various mutants and the induction frequency of double-strand breaks, it is concluded that there are 1 to 2 double-strand breaks per lethal event in diploid cells incapable of repairing these breaks.     

Variable amounts of DNA related to the size of chloroplasts III. Biochemical determinations of DNA amounts per organelle

Plastid genomes (plastomes) are part of the integrated compartmentalised genetic system of photoautotrophic eukaryotes. They are highly redundant and generally dispersed in several regions (nucleoids) within organelles. DNA quantities and number of DNA-containing regions per plastid vary and are developmentally regulated in a way not yet understood. Reliable quantitative data describing these patterns are scarce. We present a protocol to isolate fractions of pure plastids with varying average sizes from leaflets (≤1 mm) and leaves of different developmental stages continuously up to maturity (25 cm) from Beta vulgaris L. (sugar beet) to determine DNA amounts per organelle. The approach is based on plastid purification from homogenates of moderately fixed tissue by differential and isopycnic gradient centrifugations and on application of two different DNA specific colorimetric reactions after removing potentially interfering compounds. The sensitive fluorochrome DAPI (4′,6-diamidino-2-phenylindole) was used to estimate numbers and emission intensity of nucleoids per plastid. The amounts determined ranged from 0.15 to 4.9 × 10⁻² pg DNA for plastids of 1→8 μm average diameter, corresponding from approximately a dozen to 330 genome equivalents per organelle and on average four to seven copies per nucleoid. The ratio of plastid/nuclear DNA changed continuously during leaf development from as little as 0.4% to about 20% in fully developed leaves. On the other hand, mesophyll cells of mature leaves differing in ploidy (di-, tri- and tetraploid) appeared to maintain a relatively constant nuclear genome/plastome ratio, equivalent to about 1,700 copies per C-value.     

Temperature dependent release of beta-beta'' subunits of DNA dependent RNA polymerase from the folded chromosome of a dnaAts mutant of Escherichia coli.

Summary DNA-dependent RNA polymerase has been found to be preferentially released at 43° C from the folded nucleoids of an E. coli dnaA ^ts mutant when compared with the same nucleoids at 30° C or with nucleoids of a dnaA ⁺ strain at either 30° or 43° C. The polypeptides released are identical in molecular weight with those of the and constituent polypeptides of the core enzyme of a known E. coli RNA polymerase. In addition, these polypeptides are precipitated by specific anti-RNA polymerase rabbit IgG. The implications of the interactions of RNA polymerase with the dnaA gene product are discussed.     

Division of chloroplast nucleoids and replication of chloroplast DNA during the cell cycle ofDunaliella salina grown under blue and red light

Summary Synchronous cultures of the algaDunaliella salina were grown in blue or red light. The relationships between replication of chloroplast DNA, cell size, cell age and the number of chloroplast nucleoids were studied. The replication of chloroplast DNA and the division of chloroplast nucleoids occurred in two separate periods of the chloroplast cycle. DNA replication was concomitant with that in the nucleocytoplasmic compartment but nucleoid division occurred several hours earlier than nuclear division. Red-light-grown cells were bigger and grew more rapidly than those grown in blue light. In newly formed daughter cells, the chloroplast nucleoids were small and spherical and they were localized around the pyrenoid. During the cell cycle they spread to other parts of the chloroplast. The number of DNA molecules per nucleoid doubled during DNA replication in the first third of the cell cycle but decreased several hours later when the nucleoids divided. Their number was fairly constant independent of the different light quality. Cells grown in red light replicated their chl-DNA and divided their nucleoids before those grown in blue light and their daughter cells possessed about 25 nucleoids as opposed to 15.Abbreviations DAPI 4,6-diamidino-2-phenylindole - chl-DNA chloroplast DNA - PAR photosynthetically active radiation     

The ultrastructure of the nuclear envelope of amphibian oocytes

Summary In order to investigate the chemical composition of the nuclear pore complexes isolated nuclei from matureXenopus laevis oocytes were manually fractioned into nucleoplasmic aggregates and the nuclear envelopes. The whole isolation procedure takes no more than 60–90 sec, and the pore complexes of the isolated envelopes are well preserved as demonstrated by electron microscopy. Minor nucleoplasmic and cytoplasmic contaminations associated with the isolated nuclear envelopes were determined with electron microscopic morphometry and were found to be quantitatively negligible as far as their mass and nucleic acid content is concerned. The RNA content of the fractions was determined by direct phosphorus analysis after differential alkaline hydrolysis. Approximately 9% of the total nuclear RNA of the matureXenopus egg was found to be attached to the nuclear envelope. The nonmembranous elements of one pore complex contain 0.41×10^–16 g RNA. This value agrees well with the content estimated from morphometric data. The RNA package density in the pore complexes (270×10^–15 g/³) is compared with the nucleolar, nucleoplasmic and cytoplasmic RNA concentration and is discussed in context with the importance of the pore complexes for the nucleo-cytoplasmic transport of RNA-containing macromolecules.Additionally, the results of the chemical analyses as well as of the³H-actinomycin D autoradiography and of the nucleoprotein staining method of Bernhard (1969) speak against the occurence of considerable amounts of DNA in the nuclear pore complex structures.The author thanks Miss Ulrika Lempert, Miss Marianne Winter, and Miss Sigrid Krien for skilful technical help as well as Dr. W. W. Franke for many helpful discussions. The work has been supported by a Deutsche Forschungsgemeinschaft grant given to Dr. W. W. Franke (SFB Molgrudent, 46).     

Possible involvement of DNA-repair events in the transdifferentiation of mesophyll cells ofZinnia elegans into tracheary elements

In cultures of isolated mesophyll cells ofZinnia elegans, transdifferentiation into tracheary elements is induced by a combination of auxin and cytokinin and is blocked by inhibitors of DNA synthesis and poly (ADP-ribose) synthesis. During transdifferentiation, a very low level of synthesis of nuclear DNA was found in some cultured cells by microautoradiography after pulse-labeling with [³H]thymidine. Density profiles of nuclear DNA that had been double-labeledin vivo with bromodeoxyuridine (BrdU) and [³H]thymidine indicated that this DNA synthesis was repair-type synthesis. The sedimentation velocity of nucleoids increased during the culture of isolated mesophyll cells and the increase was dependent on phytohormones. This phenomenon may reflect the rejoining of DNA strand breaks after repair-type DNA synthesis during transdifferentiation. Treatment of cells with inhibitors of DNA synthesis or of poly(ADP-ribose) synthesis prevented the increase in the sedimentation velocity of nucleoids. The data suggest the involvement of DNA-repair events in the transdifferentiation of mesophyll cells into tracheary elements.     

The effect of light on the number of chloroplast nucleoids in daughter cells of the algaScenedesmus quadricauda

Summary Synchronous cultures of the green algaScenedesmus quadricauda (Turp.) Bréb. grown at mean irradiances 25Wm^–2, 75Wm^–2, and 130Wm^–2 PhAR were exposed to different illumination regimes (ratio of light to dark interval varied from 2:22 to 24:0 hours). The populations of daughter cells released under these conditions differed markedly in their progress in the cell cycle. The cells from these populations were stained with DAPI and the shape, localization and number of chloroplast nucleoids were examined. The nucleoids were of spherical shape, divided asynchronously having dumbbell shape during fission. In the chloroplast, nucleoids were located symmetrically about the transverse axis of the cells. The mean number of nucleoids varied from two in the least developed daughter cells to 16 in the daughter cells of the highest developmental stage. The progress of these cells and thus also the number of nucleoids were proportional to the portion of the light energy amount which these daughter cells shared from the total light energy amount obtained by their mother cells.Abbreviations DAPI 4, 6-diamidino-2-diphenylindole - PhAR photosynthetically active radiation (400–700 nm)     

RNA Polymerase B levels during the cell cycle ofPhysarum polycephalum

Summary Tritiated -amanitin has been used as a specific and sensitive probe to estimate the number of RNA polymerase B molecules in isolated nuclei, chromatin and nucleoids, obtained from macroplasmodia ofPhysarum polycephalum. During mitosis (metaphase±10 min) there is at least 10-fold less RNA polymerase B than at all phases of the cell cycle, even if DNA replication has been blocked in vivo. It is concluded that many of the RNA polymerase B molecules leave the chromatin during decondensation of the chromosomes in telophase of the synchronous nuclear division ofPhysarum.     

Mutagenic and Genotoxic Effects of <Emphasis Type="Italic">cis</Emphasis>-(Dichloro)tetraammineruthenium(III) Chloride on Human Peripheral Blood Lymphocytes

Chemotherapeutic agents play an important role in cancer treatment mostly due their systemic action on human organism allowing access to liquid tumors and even metastases. Among these drugs, ruthenium compounds have been showing promising results to treat tumors and represent an important development of new antitumor therapy. This study presents the evaluation of cis-(dichloro)tetraammineruthenium(III) chloride, cis-[RuCl₂(NH₃)₄]Cl, genotoxic effects using human peripheral blood lymphocytes cultured in vitro. Mitotic index (MI), chromosome aberrations (CA), and DNA damage using the comet assay were analyzed. MI in human peripheral blood lymphocyte cultures treated with 1, 10, 100, and 1,000 μg mL⁻¹ cis-[RuCl₂(NH₃)₄]Cl were 5.9%, 4.6%, 3.9%, and 0%, respectively. Doxorubicin chloridate was used as the positive control. CA derived from 1, 10, and 100 μg mL⁻¹ concentrations were defined as spontaneous when compared with the negative control, and at the concentration of 1,000 μg mL⁻¹, the cell cycle was inhibited (IM = 0%). Results obtained for the comet assay using cis-[RuCl₂(NH₃)₄]Cl suggest that this compound has no genotoxic activity against cultured human peripheral blood lymphocytes.     

Chloroplast nucleoids in large number and large DNA amount with regard to maternal inheritance inChlamydomonas reinhardtii

Summary We studied the maternal chloroplast inheritance ofChlamydomonas reinhardtii by epifluorescence microscopy after staining with DNA specific fluorochrome DAPI and by genetic methods, using wild type cells and cells containing previously isolated mutation of cond-1 and cond-2. Wild type cells contained about 7 chloroplast (cp) nucleoids, while mutants, cond-1(+) and cond-2(+), contained about 14 and 23 cp nucleoids, respectively, after one week culture on agar plates. The total cpDNA contents were almost proportional to the numbers of cp nucleoids. When cells containing cond-1 or cond-2 mutation were used as a parental source to cross with wild type cells of the other parent, preferential digestion of cp nucleoids from male parent (mt^–) origin occurred in the zygotes, although the frequencies of the digestion were slightly lower than that in the zygotes from the cross between wild type cells. Western blot analysis of the protein ofzyslB gene, which has been found related to preferential digestion of mt^– origin cp-nucleoids DNA, showed that a high amount of this protein was detected with the initiation of preferential digestion of mt^– cp nucleoids and disappeared with the completion of the digestion. Cp genetic markers for antibiotic resistance were maternally inherited in all crosses. These results showed that although the preferential digestion of cp nucleoids consisting of large number and large cpDNA amount requires a slightly longer period to complete, this high ploidy of the cp nucleoids does not disturb maternal inheritance.     

Double-strand DNA hydrolysis by dilanthanide complexes

 Although there has been progress in developing artificial hydrolytic DNA cleaving agents, none of these has been shown to carry out the double-strand hydrolysis of DNA. We demonstrate that La(III) or Ce(IV) combined with the ligand 1,3-diamino-2-hydroxypropane-N,N,N′,N′-tetraacetate (HPTA) in a 2 : 1 ratio can efficiently cleave supercoiled plasmid DNA at 55  °C within a 3-h period. Analysis of end-labeled restriction fragments cleaved by these complexes reveals 3′- and 5′-ends consistent with a hydrolytic mechanism. Unlike for other polydentate carboxylate complexes, plasmid DNA cleavage by La₂(HPTA) or Ce₂(HPTA) affords a significant amount of linear DNA with a considerable fraction of the supercoiled form still remaining. This result implies that La₂(HPTA) and Ce₂(HPTA) can carry out double-strand cleavage of plasmid DNA. La₂(HPTA) and Ce₂(HPTA) represent the first metal complexes demonstrated to be capable of double-strand hydrolytic cleavage of plasmid DNA. Received: 29 March 1999 / Accepted: 9 July 1999     

Identification of two plasmids determining resistance to tetracycline and to erythromycin in group D streptococcus

Summary Two circular DNA plasmids designated Tet and Ero, distinguishable by their molecular weights (64.5 and 17.6×10⁶ respectively) and their guanine plus cytosine (G+C) content (36.5 and 34.5% respectively) were identified in Streptococcus faecalis strain KR, resistant to tetracycline and erythromycin. Loss of resistance to tetracycline, to erythromycin and to both antibiotics simultaneously in independently isolated cured derivatives corresponds to the loss of the Tet, Ero, and of both plasmids respectively. In the singly resistant strains we estimated that 3 and 6 copies of the Tet and Ero plasmids respectively are present per chromosomal genome equivalent.Abbreviations form I supercoiled circular DNA - form II relaxed circular DNA - form III linear double-strand DNA - Tet tetracycline - Ero erythromycin - R resistant - S sensitive     

Ligation of high-melting-temperature ‘clamp’ sequence extends the scanning range of rare point-mutational analysis by constant denaturant capillary electrophoresis (CDCE) to most of the human genome

Mutations cause or influence the prevalence of many diseases. In human tissues, somatic point mutations have been observed at fractions at or below 4/10 000 and 5/100 000 in mitochondrial and nuclear DNA, respectively. In human populations, fractions for the multiple alleles that code for recessive deleterious syndromes are not expected to exceed 5 × 10^–4. Both nuclear and mitochondrial point mutations have been measured in human cells and tissues at fractions approaching 10^–6 using constant denaturant capillary electrophoresis (CDCE) coupled with high-fidelity PCR (hifiPCR). However, this approach is only applicable to those target sequences (~100 bp) juxtaposed with a ‘clamp’, a higher-melting-temperature sequence, in genomic DNA; such naturally clamped targets represent ~9% of the human genome. To open up most of the human genome to rare point-mutational analysis, a high-efficiency DNA ligation procedure was recently developed so that a clamp could be attached to any target of interest. We coupled this ligation procedure with prior CDCE/hifiPCR and achieved a sensitivity of 2 × 10^–5 in human cells for the first time using an externally attached clamp. At this sensitivity, somatic mutations, each representing an anatomically distinct cluster of cells (turnover unit) derived from a mutant stem cell, may be detected in a series of tissue samples, each containing as many as 5 × 10⁴ turnover units. Additionally, rare inherited mutations may be scanned in pooled DNA samples, each derived from as many as 10⁵ persons.     

DNA-Binding Proteins and Structural Characteristics of Chloroplast Nucleoids

A comparative analysis of proteins from chloroplast nucleoids was performed in two higher-plant species (Pisum sativumL. andArabidopsis thalianaL.) and a green alga Chlamydomonas reinhardtiiDang. In the nucleoids of the higher plants and the alga, 26–27 proteins were detected with their mol wts ranging from 10 to more than 94 kD. In all the species tested, the distribution of nucleoid proteins by their mol wts was similar, especially between the predominant proteins with mol wts of 10 to 40 kD. Six DNA-binding proteins (12–18 kD) were detected in nucleoids fromCh. reinhardtiichloroplasts after in vivocovalent cross-linking between chloroplast DNA and proteins. Under an electron miscroscope, some regular structures resembling nucleosome-like particles of bacterial cells were revealed.     

Damage at two levels of DNA folding measured by fluorescent halo technique inX-irradiated L5178Y-R and L5178Y-S cells

Summary We examined, by the fluorescent halo assay, alterations in the nucleoid structure (structure formed from cells under mild lysis conditions: in non-ionic detergent TritonX-100, 0.0005% and 1.5 mol/1 NaCl) of L5178Y (LY) cell sublines which had been untreated, treated with reducing/chelating agents (ß-mercaptoethanol or sodium diethyl dithiocarbamate (DDTC(Na))) or X-irradiated. These sublines differ in radiation sensitivity: LY-R is more resistant (D ₀ = 1.1 Gy) and LY-S more sensitive (D ₀ = 0.5 Gy). Halo diameters were measured after cell lysis in the presence of propidium iodide (PI)(0.5 to 50 µg/ml) at pH 6.9 or 9. The maximal DNA unwinding in PI was obtained at 7.5 µg/ml PI, at both pH 6.9 and 9 in both sublines; the maximal halo diameter was larger in LY-S than in LY-R cells. In nucleoids from both sublines DNA could be rewound at higher (10–50 µ/ml) PI concentrations both at pH 6.9 and 9. This ability was impaired by mercaptoethanol or DDTC(Na) (at pH 9) or by X-irradiation, indicating damage and/or alteration in the DNA superhelical structure. The susceptibility to reducing/chelating agents was greater in LY-S than in LY-R nucleoids, pointing to differences in chromatin structure between these sublines. The amount of X-ray-inflicted damage was higher, when measured at pH 9 than at pH 6.9 and was about twice larger in LY-S than in LY-R nucleoids, when the cells were irradiated with the same X-ray dose.From analogies between the behaviour of nucleoids under the above-described conditions and nucleoid type I and II sedimentation, as examined by Lebkowski and Laemmli (1982) we conclude that damage at two levels of DNA folding is measured at pH 6.9 and 9.     

Distribution and dynamics of mitochondrial nucleoids in animal cells in culture

Mitochondrial (mt) nucleoids were visualized in living cells in culture by staining with the fluorochrome picoGreen. The cell types included a line derived from Xenopus heart endothelial cells (XTH-2), 3T3 cells, SV40-transformed 3T3 cells and primary cultures of Xenopus tadpole epidermis cells. In the permanent cell lines 6-60% of the mitochondria were found to be devoid of DNA. The peaks of the frequency distribution of mtDNA content, as revealed by microfluorometry, were not very distinct, indicating the presence of a high amount of aneuploid mt nucleoids. The maximum size of nucleoids (as derived from fluorescence intensity) was 10-12 times that of the minimum peak value in proliferating cell cultures. A linear ratio was found between the volume of the nucleoids and their DNA content, which is interpreted as a uniform package density. In terminally differentiating tadpole epidermis cells mitochondria form large bodies containing giant nucleoids, while in mitotic cells the mt nucleoids are small and of uniform size. Fusion and fission of the nucleoids were observed to occur either for no visible reason or in connection with fusion and fission events of the mitochondria.Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.