Chromatin condensation and sensitivity of DNA in situ to denaturation during cell cycle and apoptosis--a confocal microscopy study

The goal of this study was to construct high resolution 3D confocal images of regions of condensed and extended chromatin in cell nuclei and individual chromosomes. It has been shown previously that sensitivity of DNA in situ to denaturation correlates with chromatin condensation and varies during cell cycle and apoptosis. Thus, detection of DNA which was partially denatured in situ provided a means to image areas of condensed chromatin. DNA denaturation was detected using a metachromatic dye acridine orange (AO) which differentially stains single stranded (ss) and double-stranded (ds) DNA sections. Early studies of denaturability of cellular DNA utilized flow cytometry and standard fluorescence microscopy. These techniques could not reveal small local differences in DNA denaturability within cell nucleus or in individual chromosomes. For instance, it was not possible to detect the initial points of chromosome condensation in G2-phase of the division cycle or in apoptosis. In order to achieve this goal we have recently extended these studies by applying confocal microscopy. We investigated DNA denaturability in normal human fibroblasts and HL-60 leukemic cells, at different stages of cell cycle and apoptosis. Following removal of RNA and partial denaturation of DNA with acid cells were stained with AO. Green (530 nm) and red (640 nm) fluorescence (exc. 457 nm) of non-denatured and denatured DNA was imaged by confocal microscopy. Blind deconvolution was used to further improve the quality of 3D images. Photobleaching of AO fluorescence was minimized and a correction for chromatic aberration and register shift was implemented. Nuclei of interphase cells exhibited predominantly green fluorescence representing AO binding to ds DNA. Punctuate areas of red fluorescence representing AO binding to denatured DNA and most likely associated with local regions of condensed chromatin were also present in all interphase nuclei. The proportion of denatured DNA increased in cells entering mitosis. In prophase individual condensing chromosomes exhibited varied proportions of green and red fluorescence indicating different content of denatured chromatin. In some chromosomes bands of denatured and denaturation-resistant chromatin were clearly resolved. In metaphase and anaphase chromosomes exhibited red fluorescence along all length of their arms indicating the highest and uniform susceptibility to denaturation. In telophase chromosomes contained predominantly denaturation-resistant DNA again and denaturated regions were significantly less abundant. At cytokinesis some decondensing chromosomes were still resolved. At this stage almost all regions of denatured DNA were located close to nuclear envelope. These regions may correspond to pockets of heterochromatin reforming at nuclear periphery. In early apoptosis condensation of chromatin appeared to commence in several distinct regions within nucleus. Some apoptotic bodies contained condensed chromatin surrounding central regions of extended chromatin. At late stages of apoptosis the whole volume of apoptotic bodies was occupied by condensed chromatin.     

Different chromatin fractions of tomato (Solanum lycopersicum L.) and related species

Conventional chromosome staining has suggested that more than 75% of the tomato chromosomes are constituted by heterochromatin. In order to determine whether more deeply stained proximal regions are classic heterochromatin, the distributions of C-bands and chromomycin A₃ (CMA) bands, and the prophase condensation patterns, were analysed in tomato. In this and most other species of the tomato clade, the 5S and 45S rDNA sites were also localised. In tomato, CMA banding was similar to C-banding. After conventional staining, all species displayed large condensed heteropycnotic regions that did not correspond to C-bands or CMA bands. Analyses of the CMA banded karyotypes revealed a low heterochromatin content. Around 12–17% of the chromatin of tomato was CMA⁺ and 1/4 to 1/5 of this heterochromatin corresponded to 45S rDNA. In other species, the CMA⁺ heterochromatin showed extensive variation (8–35%), but was never near the values found in the literature for tomato. These data suggest the existence of three principal fractions of chromatin in tomato and related species: the late condensed euchromatin corresponding to the terminal regions of the chromosomes, the precocious condensed euchromatin that occupies the major part of the chromosomes and the constitutive heterochromatin that represents those regions revealed by C-bands.     

Specific Mg binding to AT-rich regions of chromatin in the evolution of eukaryotes

At SIMS XIV, we reported SIMS evidence of specific Mg²⁺ binding to the AT-rich regions of human metaphase chromosomes represented by G-bands. Subsequent Mg²⁺-depletion experiments supported a direct role for Mg²⁺ in promoting and maintaining the higher order chromatin structure originating G-bands, possibly due to both Mg²⁺-DNA and Mg²⁺-protein interactions. An in-depth study, reported elsewhere, implicated also Ca²⁺ in the maintenance of chromatin ultrastructure in the scaffold of mammalian chromosomes, in association with topoisomerase II.We examine here the association of Mg²⁺ with AT-rich regions of chromatin in the chromosomes of the Indian muntjac deer (IMD), leading to conclusions similar to the above. To answer the question whether the presumed divalent cation role in the chromosomes of advanced eukaryotes had an evolutionary history to be traced back to earlier evolutionary stages, we have SIMS-mapped Ca²⁺ and Mg²⁺ in BrdU-labeled polytene chromosomes from the salivary gland of the Dipteran Drosophila melanogaster. Striking Ca²⁺ and Mg²⁺ SIMS banding patterns correlating with those of the Br label (a thymidine analogue) implicate unequivocally a close association of both these cations with the AT-rich regions of DNA for these primitive eukaryotes.     

Diffusion-enhanced resonance energy transfer shows that linker-DNA accessibility decreases during salt-induced chromatin condensation

Accessibility of linker-DNA chromatin during salt-induced condensation of chicken erythrocytes chromatin was studied by diffusion-enhanced resonance energy transfer. A terbium complex was covalently bound to linker-DNA and fluorescein molecules bound to latex particles with diameters ranging from 14 to 2470 nm were used as acceptor. The accessibility of linker-DNA to molecules with a diameter superior to 14 nm diminished during condensation, but for an acceptor diameter of 14 nm or less, no accessibility variation was observed. It can be concluded that (1) linker-DNA is located inside the fiber when chromatin is in the condensed state, (2) chromatin condensation can prevent the approach to DNA due to steric hindrance, (3) salt-induced chromatin condensation is a gradual process, and (4) condensed chromatin models containing a central cavity are more likely.Abbreviations DTPA diethylene tetramine pentacetic acid - FDL-DERET fast diffusion limit of diffusion-enhanced resonance energy transfer - Pso-Tb psoralen-terbium complex - PAS paraamino salicylic acid - TREF time-resolved emission of fluorescence     

Organization of chromatin in the interphase mammalian cell

The use of imaging techniques has become an essential tool in cell biology. In particular, advances in fluorescence microscopy and conventional transmission electron microscopy have had a major impact on our understanding of chromatin structure and function. In this review we attempt to chart the conceptual evolution of models describing the organization and function of chromatin in higher eukaryotic cells, in parallel with the advances in light and electron microscopy over the past 50 years. In the last decade alone, the application of energy filtered transmission electron microscopy (EFTEM), also referred to as electron spectroscopic imaging (ESI), has provided many new insights into the organization of chromatin in the interphase nucleus. Based on ESI imaging of chromatin in situ, we propose a 'lattice' model for the organization of chromatin in interphase cells. In this model, the chromatin fibers of 10 and 30nm diameter observed by ESI, produce a meshwork that accommodates an extensive and distributed interchromosomal (IC) space devoid of chromatin. The functional implications of this model for nuclear activity are discussed.     

DNA damage induced by laser-hematoporphyrin derivative

Irradiation caused DNA single-strand breaks in S180 cells in the presence oflaser-hematoporphyrin derivative(HPD).The number of single strand breaks was 3.69 ×10~(10)break dolton~(-1).The analysis of base composition of DNA showed that the effect of laser-HPDirradiation on guanine was the highest,being 5-12 times as high as those of the three others(adenine,cytosine and thymine).It was different from the nature of DNA damage caused by γ-ray irradiation.     

Discovery of bisulfite-mediated cytosine conversion to uracil, the key reaction for DNA methylation analysis--a personal account

Methylation at position 5 of cytosine in DNA is being intensively studied in many areas of biological sciences, as the methylation is intimately associated with the control of gene functions. The principal analytical method for determining the sites of 5-methylcytosine in genome at the sequence level involves bisulfite modification of DNA. The utility of this chemical treatment is based on the property of bisulfite to selectively deaminate cytosine residues. The bisulfite-mediated cytosine deamination was discovered in 1970 by us in the University of Tokyo. At the same time, Shapiro and his coworkers in New York University found the same reaction independently. We also reported that 5-methylcytosine was deaminated by bisulfite only very slowly. These findings were later utilized by a group of Australian scientists to devise a means to analyze 5-methylcytosine in DNA; thus, a method called 'bisulfite genomic sequencing' was invented by these researchers in 1992. This review describes the author's reflection of the discovery of bisulfite reactions with pyrimidine bases. The author's recent work that has resulted in an improvement of the procedure of analysis by use of a newly devised high concentration bisulfite solution is also described.     

Study of DNA accessibility in the condensed chromatin structures by resonance energy transfer

The linker DNA accessibility of chicken erythrocyte chromatin was studied by diffusion-enhanced resonance energy transfer (DERET). The 4″-{9?-[((4-carboxy-3-hydroxyphenyl)-acetatamido)-3?,6?,9?-(triacetyl)-3″,6?,9?-triazanonamido]-2″,6″-diazanonyl}-4,5′,8-trimethyl psoralen-terbium complex was photocovalently bound to linker DNA and transferred its energy to fluorescein free in solution or bound on proteins of different sizes. We observed a diminution of linker DNA accessibility in chromatin as the protein size increased. Free fluorescein and proteins (up to a molecular weight of 24,000) labeled with fluorescein isothiocyanate (FITC) showed no variation in linker accessibility as chromatin condensation from 10- to 30-nm fibers was induced by an increase in ionic strength. We can conclude from these observations that linker DNA is located on the outside of the condensed chromatin fiber or, alternatively, that small proteins are free to diffuse toward an inside-located linker DNA, even in the condensed state of chromatin, possibly through the central cavity of the solenoïd model.     

Magnetophoretic concentrating of diamagnetic bio-particles and formation of liquid-crystalline chromatin structures

The packing of chromatin during the self-assembly was modeled with a diamagnetic compression achieved under a gradient magnetic separation (magnetophoresis). Nucleoprotein granules separated from cultivated connective tissue and nuclei of chicken red blood cells formed various chromatin mesophases and chiral supra-molecular structures. The applied magnetic pressure accelerated mesophase formations due to the diamagnetic alignment and the concentrating of nucleosome nanoparticles and aggregates (germs) in gradient magnetic fields. Under various magnetic force parameters (H · grad H) ∼ 10⁵–10¹¹ Oe²/cm the different mesophases emerged. Chicken nucleated red blood cells were found to be a convenient model to study the chromatin condensation under influences including electromagnetic factors.     

Nuclear alterations associated to programmed cell death in larval salivary glands of Apis mellifera (Hymenoptera: Apidae)

The silk glands of bees are a good model for the study of cell death in insects. With the objective to detect the nuclear features during glandular regression stage, larvae at the last instar and pre-pupae were collected and their silk glands were dissected and processed for ultrastructural analysis and histologically for cytochemical and imunocytochemical analysis. The results showed that the cellular nuclei exhibited characteristics of death by atypical apoptosis as well as autophagic cell death. Among the apoptosis characteristic were: nuclear strangulation with bleb formation in some nuclei, DNA fragmentation in most of the nuclei and nucleolar fragmentation. Centripetal chromatin compaction was observed in many nuclei, forming a perichromatin halo differing from typical apoptotic nuclei. With regards to the characteristics of autophagic-programmed cell death, most relevant was the delay in the collapse of many nuclei.     

Temperature and Base Sequence Dependence of 2-Aminopurine Fluorescence Bands in Single- and Double-Stranded Oligodeoxynucleotides

Fluorescence excitation spectra of 2-aminopurine (2AP) incorporated into single-stranded DNA di- and trinucleotides, as well as into single- and double-stranded pentanucleotides, have been measured as a function of temperature from 5 to 65 °C. Spectral shifts have been precisely quantitated through difference spectroscopy and spectral fits. G(2AP)C and C(2AP)G oligonucleotides have relatively blue-shifted excitation spectra (especially the former) compared to the 2AP free base. The position of the excitation peak of 2AP free base is temperature independent, those of (2AP)T, G(2AP)C, C(2AP)G and TT(2AP)TT shift about 0.4 nm to the blue from 5 to 65 °C, though the spectra of the G-C-containing oligomers also change shape. The temperature dependence of the A(2AP)T spectral position is 2.5-times stronger, and just rises to that of the free base at high temperature. On the other hand, the decrease of yield with increasing temperature is smallest for A(2AP)T, even compared to the free base. The dominant effect when A neighbors 2AP appears to be temperature-dependent stacking with accompanying energy transfer, while in G- and C-containing trinucleotides a temperature-independent interaction keeps the 2AP excitation spectrum blue-shifted. The effect of double strand formation appears to be small compared to stacking interactions. These spectra can be useful in identifying base neighbors and structures of 2AP in unknown 2AP-labeled DNA.     

外界核心对喷管内水蒸气凝结流动的影响

建立了考虑摩擦阻力的一维水蒸气超音速流凝结数学模型,对喷管内含有不同初始浓度和半径的外界核心的水蒸气超音速流凝结过程进行了数值计算。结果发现:当外界核心浓度在不同范围内时,外界核心半径对凝结有不同的影响规律;当外界核心半径在不同范围内时,外界核心浓度对凝结也有不同的影响规律;为了促进凝结的发生,要选择匹配的外界核心平均半径和浓度。选定喷管出口的液相质量分数为标准,针对计算的几种情况得出:当外界核心半径为1.0×10~(-9)m,外界核心浓度为1.0×10~(14)kg~(-1)时,对应的喷管出口处的液相质量分数最大。     

Cytochemical characterization of Triatoma infestans and Panstrongylus megistus salivary gland cells (Hemiptera, Reduviidae, Triatominae)

The Triatominae subfamily has medical sanitary importance, since these insects are vectors of Trypanosoma cruzi, etiologic agent of Chagas Disease, and Trypanosoma rangeli, which develops in the salivary glands and it is frequently found in mixed infections with T. cruzi. Triatomines of Triatoma and Panstrongylus genera possess a salivary gland complex composed of a pair with three well differentiated units: the anterior (D1), median (D2) and posterior (D3). Saliva is secreted during blood meal and antagonizes hemostatic, inflammatory and immunological systems imposed by the host, which facilitate the hematophagy. In order to identify nuclear structures, we studied interphase nuclei of salivary gland cells of adult insects, males and females, of Triatoma infestans and Panstrongylus megistus. The glands were removed from insects, fixed in acetic acid (45%) and lactic acid (50%), squashed and submitted to different cytochemical methods: lacto-acetic orcein, silver ion impregnation, Feulgen reaction, Toluidine Blue, Variant method of critical electrolyte concentration and C-banding. The preparations were examined with a Zeiss Jenaval photomicroscope and photographed. The results evidenced predominance of binucleated cells in D1 and D2 glands and mononucleated ones in D3. In all salivary glands were observed bulky and polyploid nucleus, a clear association between nucleolar and heterochromatic corpuscles, cytoplasmatic metachromasy and many pre-secretion vesicles in cytoplasm. Such characteristics were associated with intense synthesis activity to produce the saliva. Species were mainly differentiated by a larger heterochromatic corpuscle observed only in T. infestans (called as chromocenter), while P. megistus showed a predominance of euchromatin, with some heterochromatic corpuscles just in males. Females of both species showed a smaller quantity of heterochromatin, which could be related to the high metabolism because of the oviposition.     

Effect of spermine and DNase on DNA release from bacteriophage T5

Bacterial viruses (bacteriophages) consist of nucleic acid protected by a protein envelope called capsid. At the start of infection, the phage genome is translocated into the bacterial cytoplasm. In vitro (and also in vivo), this DNA release can be triggered by binding a specific receptor protein to the phage tail. The force responsible for the release arises from energy stored in the capsid due to strong confinement of the DNA. We show that this force can be modified by adding molecules like spermine that affect DNA conformation. The tetravalent cation spermine can reduce the pressure inside the capsid and induce condensation of the released DNA. We examine the effect of spermine on DNA ejection from phage T5 by using light scattering and gel electrophoresis to measure the amount of DNA remaining in the capsid at the end of ejection. We discuss the results in terms of free energy minimization and we demonstrate that the presence of a DNA condensate outside the phage generates an additional force pulling passively on the DNA remaining inside the capsid.     

Observation of single- and double-stranded DNA using non-contact atomic force microscopy

Non-contact atomic force microscopy (NC-AFM) has been applied to observe single- and double-stranded DNA. For the wet processes used to prepare the sample, a strong adhesion force at the surface is observed even in vacuum conditions. Despite the presence of this adhesion force, single- and double-stranded DNA images can be obtained by NC-AFM. Because of the high sensitivity of the tip-sample interaction, NC-AFM images provide stronger contrast than tapping mode (TM)-AFM images. NC-AFM images reveal detailed structures of single- and double-stranded DNA which are not revealed by TM-AFM. In addition, several NC-AFM images show contrast artifacts, which might provide information on the detailed structure of DNA.     

DNA碱基分子胞嘧啶和胸腺嘧啶的太赫兹光谱研究

利用太赫兹时域光谱技术获得了DNA碱基分子胞嘧啶和胸腺嘧啶在0.1―3.5 THz的特征吸收谱,发现胞嘧啶在2.53 THz的特征吸收细节信息。采用考虑了周期性边界条件的赝势平面波密度泛函方法对胞嘧啶分子晶体进行了结构优化和晶格动力学计算,模拟重现其太赫兹特征吸收光谱,并成功辨识了胞嘧啶在0.1―3.5 THz的所有特征吸收峰。研究结果表明,这些重要的生物分子在太赫兹频段表现出鲜明的光谱特性,胞嘧啶分子3.5 THz以下的吸收特性均来源于由分子间氢键支配的外振动模式。