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PubMed2006

Linear connection of condensing chromosomes in nuclei of synchronized CHO cells.

Reversibly permeabilized cells allowed the analysis of intermediates of large-scale chomatin condensation in a cell cycle-dependent manner. This paper summarizes major intermediates of chromatin condensation and visualizes connectivity between different forms of chromosomes. At the early S phase the veil-like fibrillary chromatin is supercoiled to form chromatin bodies representing the earliest visible chromosomes. Supercoiling results in a chromatin fiber, which turns to rope (thick fiber) forming loops and chromatin ribbon. The elongated shape of prechromosomes indicates that they are arranged head to tail. Bent forms (loop, c-, and v-shaped structures) of interphase chromosomes open at the end of S phase. Linear arrangement of chromosomes was observed to the end of the condensation process, suggesting that connectivity of chromosomes is maintained throughout the cell cycle.

Animals↗

PubMed2000

Mitotic chromosome condensation requires Brn1p, the yeast homologue of Barren.

In vitro studies suggest that the Barren protein may function as an activator of DNA topoisomerase II and/or as a component of the Xenopus condensin complex. To better understand the role of Barren in vivo, we generated conditional alleles of the structural gene for Barren (BRN1) in Saccharomyces cerevisiae. We show that Barren is an essential protein required for chromosome condensation in vivo and that it is likely to function as an intrinsic component of the yeast condensation machinery. Consistent with this view, we show that Barren performs an essential function during a period of the cell cycle when chromosome condensation is established and maintained. In contrast, Barren does not serve as an essential activator of DNA topoisomerase II in vivo. Finally, brn1 mutants display additional phenotypes such as stretched chromosomes, aberrant anaphase spindles, and the accumulation of cells with >2C DNA content, suggesting that Barren function influences multiple aspects of chromosome transmission and dynamics.

Cell Cycle Proteins↗

PubMed2000

Chromosome condensation factor Brn1p is required for chromatid separation in mitosis.

This work describes BRN1, the budding yeast homologue of Drosophila Barren and Xenopus condensin subunit XCAP-H. The Drosophila protein is required for proper chromosome segregation in mitosis, and Xenopus protein functions in mitotic chromosome condensation. Mutant brn1 cells show a defect in mitotic chromosome condensation and sister chromatid separation and segregation in anaphase. Chromatid cohesion before anaphase is properly maintained in the mutants. Some brn1 mutant cells apparently arrest in S-phase, pointing to a possible function for Brn1p at this stage of the cell cycle. Brn1p is a nuclear protein with a nonuniform distribution pattern, and its level is up-regulated at mitosis. Temperature-sensitive mutations of BRN1 can be suppressed by overexpression of a novel gene YCG1, which is homologous to another Xenopus condensin subunit, XCAP-G. Overexpression of SMC2, a gene necessary for chromosome condensation, and a homologue of the XCAP-E condensin, does not suppress brn1, pointing to functional specialization of components of the condensin complex.

Cell Cycle Proteins↗

PubMed2003

Drosophila MCM10 interacts with members of the prereplication complex and is required for proper chromosome condensation.

Mcm10 is required for the initiation of DNA replication in Saccharomyces cerevisiae. We have cloned MCM10 from Drosophila melanogaster and show that it complements a ScMCM10 null mutant. Moreover, Mcm10 interacts with key members of the prereplication complex: Mcm2, Dup (Cdt1), and Orc2. Interactions were also detected between Mcm10 and itself, Cdc45, and Hp1. RNAi depletion of Orc2 and Mcm10 in KC cells results in loss of DNA content. Furthermore, depletion of Mcm10, Cdc45, Mcm2, Mcm5, and Orc2, respectively, results in aberrant chromosome condensation. The condensation defects observed resemble previously published reports for Orc2, Orc5, and Mcm4 mutants. Our results strengthen and extend the argument that the processes of chromatin condensation and DNA replication are linked.

Animals↗

PubMed1989

32P-postlabelling analysis of DNA adducts in the skin of mice treated with petrol and diesel engine lubricating oils and exhaust condensates.

Samples of unused or used petrol and diesel engine lubricating oils were applied to the shaved dorsal skin of 4- to 6-week-old male Parkes mice, either as a single treatment (50 microliters/mouse) or as four consecutive daily treatments (50 microliters/application). DNA isolated from the skin 24 h after the final treatment was digested to 3'-mononucleotides and analysed by 32P-postlabelling for the presence of aromatic adducts. Enhancement of sensitivity using butanol extraction or nuclease P1 digestion of the DNA hydrolysates led to the detection of up to eight adduct spots on polyethyleneimine-cellulose thin-layer chromatograms with samples of DNA from skin treated with used engine oils, at levels of 40-150 amol total adducts/micrograms DNA. Multiple treatments with the used oils gave rise to similar patterns of adducts in lung DNA. A single treatment of mouse skin with petrol engine exhaust condensate (50 microliters), or diesel engine exhaust condensate (50 microliters), containing 20 and 46 micrograms benzo[a]pyrene (BaP)/g respectively, gave rise to approximately 75 amol total adducts/micrograms DNA in skin. A significant proportion, 31 and 48% respectively, of the adducts formed by the petrol and diesel engine exhaust condensates co-chromatographed with the major BaP-DNA adduct, but with the used engine oils, only petrol engine oil, and not diesel engine oil, produced significant amounts of an adduct (22% of total) that corresponded to the BaP-DNA adduct.

Adenosine Triphosphate↗

PubMed1993

A hypothesis for chemical carcinogen induced chromatin condensation.

A hypothesis for chemical carcinogen induced condensation of nuclear and nucleolar chromatin is proposed. Chromatin condensation is believed to be a result of cascade effects initiated by the inhibition of messenger RNA synthesis after exposure to chemical carcinogen in vivo. Inhibition of messenger RNA synthesis leads to the loss of protein production, which in turn causes the dephosphorylation of histone H1 and triggers the condensation of chromatin in vivo.

Amanitins↗

PubMed2001

Calibration of an optical condensate measurement technique using indirect static headspace gas chromatography.

Researchers in the Department of Mechanical Engineering seek to obtain a means to measure less than 3 mg of H2O that adheres to a metal surface as condensate. The objective is to calibrate optical reflectance measurements of an aluminum surface as a function of the condensate thickness present. Collaboration with the Hazardous Substance Research Center at Michigan State University results in the development of an indirect static headspace gas chromatographic technique capable of measuring H2O in low-milligram quantities. The technique utilizes manual headspace sampling, a megabore capillary column, and a flame ionization detector. A correlation of r2 = 0.999 is obtained for the calibration of the indirect measurement technique. The calibration of the analytical instrument demonstrates adequate precision (< +/- 50 microg at a 95% confidence interval) for such a heterogeneous sample. The calibration samples consist of a strip of aluminum metal, H2O, and pulverized calcium carbide to convert the H2O to acetylene. A scaled calibration technique is used to simplify handling trace water volumes. The surface reflectance measurements are found to correlate well (r2 = 0.935) with measurements of the condensate mass. This result facilitates the development of an optical mass-transfer measurement technique. This study focuses on the analytical method and its relationship with engineering research.

Calibration↗

PubMed2005

Light and an exogenous transcription factor qualitatively and quantitatively affect the biosynthetic pathway of condensed tannins in Lotus corniculatus leaves.

The effects of increasing light and of a heterologous bHLH transcription factor on the accumulation of condensed tannins (CT) were investigated in leaves of Lotus corniculatus, a model legume species which accumulates these secondary metabolites in leaves as well as reproductive tissues. Light and expression of the transgene increased the level of CT in a synergistic way. To monitor how the changes in accumulation of condensed tannins were achieved, the level of expression of four key genes in the flavonoid pathway was estimated by real-time RT-PCR analysis. Early genes of the pathway (PAL and CHS) were affected less in their expression and so appeared to be less involved in influencing the final level of CT than later genes in the pathway (DFR and ANS). Steady-state levels of DFR and ANS transcripts showed a strong positive correlation with CT and these genes might be considered the first rate-limiting steps in CT biosynthesis in Lotus leaves. However, additional factors mediated by light are limiting CT accumulation once these genes are up-regulated by the transgene. Therefore, the increment of the steady-state mRNA level for DFR and ANS might not be sufficient to up-regulate condensed tannins in leaves. The real-time RT-PCR approach adopted showed that members within the CHS and DFR gene families are differentially regulated by the exogenous bHLH gene and light. This finding is discussed in relation to the approaches for controlling CT biosynthesis and for studying the expression profile of multi-gene families.

Amino Acid Sequence↗

PubMed1998

The observation of the local ordering characteristics of spermidine-condensed DNA: atomic force microscopy and polarizing microscopy studies.

Condensation of DNA by multivalent cations can provide useful insights into the physical factors governing the folding and packaging of DNA in vivo. In this work, local ordered structures of spermidine-DNA complexes prepared from different DNA concentrations have been examined by using atomic force microscopy (AFM) and polarizing microscopy (PM). Two types (I and II) of DNA condensates, significantly different in sizes, were observed. It was found that for extremely dilute solutions (DNA concentrations around 1 ng/microl or below), the DNA molecules would collapse into toroidal structures with a volume equivalent to a single lambda-DNA (type I). In relatively dilute solutions (DNA concentrations between 1 and 10 ng/microll), a significantly larger structure of multimolecular toroids (circular and elliptical, type II) were formed, which were constructed by many fine particles. Measurements show that the average diameter of these fine particles was similar to the outer diameter of the monomolecular toroids observed in extremely dilute solutions, and the thickness of the multimolecular toroids had a distribution of multi-layers with height increments of 11 nm, indicating that the multimolecular toroidal structures have lamellar characteristics. Moreover, by enriching the DNA-spermidine complexes in very diluted solution, branch-like structures constructed by subunits were observed by using AFM. The analysis of the pellets in polarizing microscopy reveals a liquid-crystal-like pattern. These observations suggest that DNA-spermidine condensation could have multiple stages, which are very sensitive to the DNA and spermidine concentrations.

Bacteriophage lambda↗

PubMed1999

Layer-by-layer deposition of oppositely charged polyelectrolytes on the surface of condensed DNA particles.

DNA can be condensed with an excess of poly-cations in aqueous solutions forming stable particles of submicron size with positive surface charge. This charge surplus can be used to deposit alternating layers of polyanions and polycations on the surface surrounding the core of condensed DNA. Using poly-L-lysine (PLL) and succinylated PLL (SPLL) as polycation and polyanion, respectively, we demonstrated layer-by-layer architecture of the particles. Polyanions with a shorter carboxyl/backbone distance tend to disassemble binary DNA/PLL complexes by displacing DNA while polyanions with a longer carboxyl/backbone distance effectively formed a tertiary complex. The zeta potential of such complexes became negative, indicating effective surface recharging. The charge stoichiometry of the DNA/PLL/SPLL complex was found to be close to 1:1:1, resembling poly-electrolyte complexes layered on macrosurfaces. Recharged particles containing condensed plasmid DNA may find applications as non-viral gene delivery vectors.

Anions↗

PubMed2002

Unique condensation patterns of triplex DNA: physical aspects and physiological implications.

Triple-stranded DNA structures can be formed in living cells, either by native DNA sequences or following the application of antigene strategies, in which triplex-forming oligonucleotides are targeted to the nucleus. Recent studies imply that triplex motifs may play a role in DNA transcription, recombination and condensation processes in vivo. Here we show that very short triple-stranded DNA motifs, but not double-stranded segments of a comparable length, self-assemble into highly condensed and ordered structures. The condensation process, studied by circular dichroism and polarized-light microscopy, occurs under conditions that mimic cellular environments in terms of ionic strength, ionic composition and crowding. We argue that the unique tendency of triplex DNA structures to self-assemble, a priori unexpected in light of the very short length and the large charge density of these motifs, reflects the presence of strong attractive interactions that result from enhanced ion correlations. The results provide, as such, a direct experimental link between charge density, attractive interactions between like-charge polymers and DNA packaging. Moreover, the observations strongly support the notion that triple-stranded DNA motifs may be involved in the regulation of chromosome organization in living cells.

Chromosomes↗

PubMed2004

Isolation and characterization of a novel DNA methyltransferase complex linking DNMT3B with components of the mitotic chromosome condensation machinery.

Proper patterns of genome-wide DNA methylation, mediated by DNA methyltransferases DNMT1, -3A and -3B, are essential for embryonic development and genomic stability in mammalian cells. The de novo DNA methyltransferase DNMT3B is of particular interest because it is frequently overexpressed in tumor cells and is mutated in immunodeficiency, centromere instability and facial anomalies (ICF) syndrome. In order to gain a better understanding of DNMT3B, in terms of the targeting of its methylation activity and its role in genome stability, we biochemically purified endogenous DNMT3B from HeLa cells. DNMT3B co-purifies and interacts, both in vivo and in vitro, with several components of the condensin complex (hCAP-C, hCAP-E and hCAP-G) and KIF4A. Condensin mediates genome-wide chromosome condensation at the onset of mitosis and is critical for proper segregation of sister chromatids. KIF4A is proposed to be a motor protein carrying DNA as cargo. DNMT3B also interacts with histone deacetylase 1 (HDAC1), the co-repressor SIN3A and the ATP-dependent chromatin remodeling enzyme hSNF2H. Further more, DNMT3B co-localizes with condensin and KIF4A on condensed chromosomes throughout mitosis. These studies therefore reveal the first direct link between the machineries regulating DNA methylation and mitotic chromosome condensation in mammalian cells.

Adenosine Triphosphatases↗

PubMed1980

Legionnaires' disease outbreak at an Atlanta, Georgia, Country Club: evidence for spread from an evaporative condenser.

During July 1978 an outbreak of Legionnaires' disease characterized by high fever, prostration, and pneumonia occurred at an Atlanta, Georgia, country club. All eight cases involved club members whose primary club activity was golfing. The degree of golfing activity during the likely exposure period was a risk factor for acquiring the illness. Legionella pneumophila was isolated from the evaporative condenser within the clubhouse. The fact that the stream of air blowing from the exhaust duct of the evaporative condenser was directed toward a nearby practice green and the 10th and 16th tees supports the hypothesis that this outbreak represents airborne dissemination of L. pneumophila from the evaporative condenser to an outdoor site where susceptible golfers contracted the illness.

Aged↗

PubMed1996

Premature chromosome condensation of the sperm nucleus after intracytoplasmic sperm injection.

A cytogenetic-cytological study was performed on unfertilized human oocytes (first polar body visible) after intracytoplasmic sperm injection (ICSI) with respect to the rate of prematurely condensed sperm chromosomes (G1-PCC). Out of 163 prepared oocytes derived from 41 ICSI cycles, 133 (approximately 82%) could be analysed successfully. a total of 60 oocytes (45.1%) showed metaphase II chromosomes in the haploid range along with an intact sperm head and 27 oocytes (20.3%) were missing the sperm head, but two of them showed an approximately diploid set of chromosomes; 38 oocytes (28.4%) exhibited the maternal metaphase II chromosomes as well as G1-PCC of the sperm nucleus showing a remarkable variation in the degree of condensation. Ten ICSI cycles (each followed by an embryo transfer) were characterized each by 2-3 oocytes demonstrating G1-PCC. It is concluded that the main cause of failed fertilization after ICSI is the failure of oocyte activation. When the sperm nucleus is able to act with the chromosome condensing factors and the oocyte does not become activated, this will lead to the induction of PCC. Absence of the sperm head might be due to injection or ejection of the spermatozoon in the perivitelline space except for two cases in which fertilization might have occurred. Finally, the observation of both a single chromatin region (n = 6) or two chromatin regions (n = 2) indicated oocyte activation which, however, was followed by developmental arrest.

Adult↗

PubMed1995

Recurrent failure in polar body formation and premature chromosome condensation in oocytes from a human patient: indicators of asynchrony in nuclear and cytoplasmic maturation.

All oocytes from a patient who had undergone four unsuccessful in-vitro fertilization attempts showed neither a polar body nor pronuclei when examined for fertilization. In 19 inseminated oocytes that were spread for karyotypic analysis, one haploid set of metaphase II chromosomes and a remarkable condensed structure were found. Hormonal and morphological criteria implied that the oocytes had been mature at the time of retrieval. Since non-inseminated oocytes contained only one set of metaphase II chromosomes, the condensed structure appeared to represent the sperm chromatin in the state of premature chromosome condensation due to a block in oocyte maturation. Since the first and second polar body, as well as their chromatin, were undetectable in all the patient's oocytes, a rapid maturation to metaphase II before retrieval and prolonged arrest in this state before fertilization, accompanied by degeneration of the first polar body, appear to be responsible for the condition. In accordance with this notion, degenerate spindles (typical of post-ovulatory aged oocytes) and separating chromosomes (probably representing presegregating chromatids) were observed by antitubulin immunofluorescence.

Adult↗

PubMed2006

A Monte-Carlo code for the detailed simulation of electron and light-ion tracks in condensed matter.

In an effort to understand the basic mechanism of the action of charged particles in solid radiation dosimeters, we extend our Monte-Carlo code (MC4) to condensed media (liquids/solids) and present new track-structure calculations for electrons and protons. Modeling the energy dissipation process is based on a model dielectric function, which accounts in a semi-empirical and self-consistent way for condensed-phase effects which are computationally intractable. Importantly, these effects mostly influence track-structure characteristics at the nanometer scale, which is the focus of radiation action models. Since the event-by-event scheme for electron transport is impractical above several kilo-electron volts, a condensed-history random-walk scheme has been implemented to transport the energetic delta rays produced by energetic ions. Based on the above developments, new track-structure calculations are presented for two representative dosimetric materials, namely, liquid water and silicon. Results include radial dose distributions in cylindrical and spherical geometries, as well as, clustering distributions, which, among other things, are important in predicting irreparable damage in biological systems and prompt electric-fields in microelectronics.

Algorithms↗

PubMed1999

Do nuclear condensation or fragmentation and DNA fragmentation reflect the mode of neuronal death?

It is generally believed that nuclear condensation and fragmentation as well as DNA fragmentation reflect the events related to the neuronal apoptosis. Our report demonstrates that severe oxygen-glucose deprivation (OGD) induced condensation and fragmentation of nuclear chromatin of neurones in primary cultures of cerebellar granule cells without intemucleosomal DNA fragmentation. DNA fragmentation detected by TUNEL assay was seen only after mild OGD or after addition of colchicine but not after severe OGD. Thus, at least in primary cerebellar granule cell cultures, the chromatin condensation and fragmentation cannot be considered as a hallmark of apoptosis but rather reflect the neuronal death despite of its form.

Animals↗

PubMed1997

Premature chromosome condensation assay for biodosimetry: studies with fission-neutrons.

Characterization of the premature chromosome condensation assay for radiation quality is needed. To that end, human lymphocytes were exposed in vitro to various doses of 250-kVp x rays (Y(D) = 4 keV microm(-1), Y(D) is the dose-mean lineal energy of the absorbed dose distribution, D(y), where y is defined as the energy deposited in a volume by a single event divided by the mean chord length of the volume) and to fission neutrons (Y(D) = 65 keV microm(-1)). The distribution of prematurely condensed chromosome and fragments following exposure to x rays or to neutrons were non-Poisson after repair at 37 degrees C for 24 h. Dose-response curves were constructed for the yield of excess prematurely condensed chromosome fragments as necessary for biodosimetry applications. The curves were fitted to a weighted linear model by the least-squares regression method. The neutron relative biological effectiveness (RBE) value was estimated to be 2.4 +/- 0.39.

Animals↗