Determination of the nick site at oriT of IncI1 plasmid R64: global similarity of oriT structures of IncI1 and IncP plasmids.

The nick site at the origin of transfer, oriT, of IncI1 plasmid R64 was determined. A site-specific and strand-specific cleavage of the phosphodiester bond was introduced during relaxation of the oriT plasmid DNA. Cleavage occurred between 2'-deoxyguanosine and thymidine residues, within the 44-bp oriT core sequence. The nick site was located 8 bp from the 17-bp repeat. A protein appeared to be associated with the cleaved DNA strand at the oriT site following relaxation. This protein was observed to bind to the 5' end of the cleaved strand, since the 5'-phosphate of the cleaved strand was resistant to the phosphate exchange reaction by polynucleotide kinase. In contrast, the 3' end of the cleaved strand appeared free, since it was susceptible to primer extension by DNA polymerase I. The global similarity of the oriT structures of IncI1 and IncP plasmids is discussed.     

Relaxation,linearization and fragmentation of supercoiled circular DNA by tungsten microprojectiles

The aim of the study was to characterize DNA lesions caused by microprojectile bombardment and by the postbombardment presence of tungsten particles in transformed cells. For the sake of simplicity, plasmid DNA was used as a target for bombardment with naked tungsten particles. Unexpectedly extensive DNA degradation was observed under standard bombardment conditions. However, no further DNA fragmentation occurred under postbombardment conditions, simulated by incubation of plasmid DNA with a suspension of tungsten particles. Instead, relaxation and linearization of supercoiled circular plasmids (pAHC25 and others) took place. It is concluded that the observed linearization (a single site double–strand break in DNA circle) results from the ability of tungsten to catalyse the hydrolysis of phosphodiester bonds in torsionally strained sites of native DNA selectively.     

Poly(ADP-ribose) polymerases PARP1 and PARP2 modulate topoisomerase II beta (TOP2B) function during chromatin condensation in mouse spermiogenesis

To achieve the specialized nuclear structure in sperm necessary for fertilization, dramatic chromatin reorganization steps in developing spermatids are required where histones are largely replaced first by transition proteins and then by protamines. This entails the transient formation of DNA strand breaks to allow for, first, DNA relaxation and then chromatin compaction. However, the nature and origin of these breaks are not well understood. We previously reported that these DNA strand breaks trigger the activation of poly(ADP-ribose) (PAR) polymerases PARP1 and PARP2 and that interference with PARP activation causes poor chromatin integrity with abnormal retention of histones in mature sperm and impaired embryonic survival. Here we show that the activity of topoisomerase II beta (TOP2B), an enzyme involved in DNA strand break formation in elongating spermatids, is strongly inhibited by the activity of PARP1 and PARP2 in vitro, and this is in turn counteracted by the PAR-degrading activity of PAR glycohydrolase. Moreover, genetic and pharmacological PARP inhibition both lead to increased TOP2B activity in murine spermatids in vivo as measured by covalent binding of TOP2B to the DNA. In summary, the available data suggest a functional relationship between the DNA strand break-generating activity of TOP2B and the DNA strand break-dependent activation of PARP enzymes that in turn inhibit TOP2B. Because PARP activity also facilitates histone H1 linker removal and local chromatin decondensation, cycles of PAR formation and degradation may be necessary to coordinate TOP2B-dependent DNA relaxation with histone-to-protamine exchange necessary for spermatid chromatin remodeling.     

Effects of cooling, cryopreservation and heating on sperm proteins, nuclear DNA, and fertilization capability in mouse

In the present study, we used confocal microscopy and electrophoresis to study the effects of heating to 5 or 100 degrees C or cooling to 4 degrees C or -- 196 degrees C on the stability of sperm proteins and DNA. We used intracytoplasmic sperm injection (ICSI) to determine the fertilizing capability of treated spermatoza. It was shown that sperm cryopreservation at - 196 degrees C or cooling at 4 degrees C altered neither protein and DNA profiles nor the sperm fertilization capability, while the protein and DNA profiles of sperm heated at 100 degrees C were irreversibly degraded and inactivated. The proteins of sperm were severely damaged while the nuclear DNA still maintained its integrity when heated to 58 degrees C. Observation by laser confocal microscopy showed that after being heated to 58 degrees C and 100 degrees C, the nuclear of mouse sperm lost their ability to activate oocytes and they could not transform to male pronuclei though the membrane of some sperm could degrade and induce the formation of sperm asters in ICSI oocytes. The results indicate that the use of 58 degrees C heating only causes the degradation of sperm proteins, while the 100 degrees C heating elicits the irreversible degradation of both sperm proteins and nuclear DNA, and the damage of sperm proteins is primarily responsible for the observed decrease in sperm fertilizing capability.     

Effect of Triton X-100 on the physical properties of liposomes

The interaction of the nonionic detergent Triton X-100 with phospholipid bilayers of liposomes made of egg yolk phosphatidylcholine was studied through the behavior of several physical properties. The dielectric permittivity spectra between 30 kHz and 13 MHz, the viscosity, the density, and the d.c. conductivity (1 kHz) of aqueous liposomes suspensions at various mole ratios were measured at 22 degrees C. For detergent-to-phospholipid ratios lower than 3, a dielectric relaxation process of characteristic frequency of about 50 kHz was recorded. This process does not appear for the liposomes in water, and becomes smaller and smaller for detergent-to-phospholipid ratios higher than 3. The viscosity of these suspensions showed a biphasic behavior, being remarkably increased by the detergent for concentration ratios lower than 3. The measured d.c. conductivity of these samples showed no relation with this process, being slightly increased when the detergent content is increased. As a conclusion of these results a well defined concentration range appears where the phospholipid organization changes forming highly asymmetrical structures.     

Enhanced resistance to nuclease degradation of nucleic acids complexed to asialoglycoprotein-polylysine carriers.

We have previously shown targeting of DNA to hepatocytes using an asialoorosomucoid-polylysine (AsOR-PL) carrier system. The AsOR-PL conjugate condenses DNA and facilitates entry via specific receptor-ligand interactions. In these studies, our objective was to determine if AsOR-PL conjugates protect bound DNA from nuclease attack. Double-stranded plasmid or single-stranded oligonucleotide DNA, alone or bound to conjugate, was incubated under conditions mimicking those encountered during in vitro and in vivo transfections. The results showed that complexed DNA was effectively protected from degradation by serum nucleases. Degradation of single-stranded oligonucleotides was inhibited 3- to 6-fold in serum during 5 hours of incubation. For complexed plasmids, greater than 90% remained full-length during 1.5 and 3 hour incubations in serum or culture medium containing 10% serum, respectively. Uncomplexed plasmid was completely degraded after 15 minutes in serum or 60 minutes in medium. In cell lysates, the conjugate was not effective in inhibiting endonuclease activity; plasmids were readily converted from supercoiled to open circular and linear forms. However, the resultant nicked forms were substantially protected from further degradation during one hour of incubation compared to plasmid alone. Under all conditions complexed DNA did not readily dissociate from the conjugate. Overall, for both single and double-stranded DNA, AsOR-PL conjugates conferred substantial protection from nuclease degradation.     

Radiation protection of DNA by ferulic acid under in vitro and in vivo conditions

The effect of ferulic acid was studied on γ-radiation-induced relaxation of plasmid pBR322 DNA and induction of DNA strand breaks in peripheral blood leukocytes and bone marrow cells of mice exposed to whole body γ-radiation. Presence of 0.5 mM ferulic acid significantly inhibited the disappearance of supercoiled (ccc) plasmid pBR322 with a dose modifying factor (DMF) of 2.0. Intraperitoneal administration of different amounts (50, 75 and 100 mg/kg body weight) of ferulic acid 1 h prior to 4 Gy γ-radiation exposure showed dose-dependent decrease in the yield of DNA strands breaks in murine peripheral blood leukocytes and bone marrow cells as evidenced from comet assay. The dose-dependent protection was more pronounced in bone marrow cells than in the blood leukocytes. It was observed that there was a time-dependent disappearance of radiation induced strand breaks in blood leukocytes (as evidenced from comet parameters) following whole body radiation exposure commensuration with DNA repair. Administration of 50 mg/kg body weight of ferulic acid after whole body irradiation of mice resulted disappearance of DNA strand breaks at a faster rate compared to irradiated controls, suggesting enhanced DNA repair in ferulic acid treated animals. (Mol Cell Biochem xxx: 209–217, 2005)     

Cell line specific control of polyethylenimine-mediated transient transfection optimized with "Design of experiments" methodology

We describe a design of experiments (DoE) response surface modeling strategy to optimize the concentration of basal variables underpinning polyethylenimine (PEI) mediated transfection of different CHO-K1 derived parental cell populations in a chemically defined medium, specifically the relative concentration of linear 25 kD PEI, host CHO cells and plasmid DNA. Using recombinant secreted alkaline phosphatase (SEAP) reporter activity as the modeled response, a discrete simple maximum was predicted for each CHO host cell population. Differences between the modeled optima derived from host cell specific differences in PEI cytotoxicity, such that the PEI:cell interaction effectively limited PEI-DNA polyplex load at a relatively constant PEI:DNA ratio. However, across the three CHO host cell populations, SEAP reporter production was not proportional to plasmid DNA input at the host cell specific predicted basal variable optima. A 10-fold variation in SEAP reporter output per mass of plasmid DNA delivered was observed. To determine the cellular basis of this difference in transient productivity, host CHO cells were transfected with fluorescently labeled polyplexes followed by flow cytometric analysis. Each CHO host cell population exhibited a distinct functional phenotype, varying in the extent of PEI-DNA polyplex binding to the cell surface and degree of polyplex internalization. SEAP production was directly proportional to the level of polyplex internalization and heparan sulfate proteoglycan level. Taken together, these data show that choice of host CHO cell line is a critical parameter, which should rationally precede cell line specific transient production platform design using DoE methodology.     

Electric permittivity and dielectric dispersion of low-molecular weight DNA at low ionic strength.

The dielectric properties of sonicated calf-thymus DNA (MW approximately 3 X 10(5) g mol-1) have been investigated in a frequency range between a few kHz and 100 MHz. Two samples, sonicated in a different way were used after proper characterization including light-scattering, viscometry and contour length distribution by electron microscopy. Dielectric measurements were performed at several concentrations between 10(-4) and 3 X 10(-3) monomol 1-1 and 22 degrees C. Under all circumstances two separated dispersion regions were observed, the corresponding specific increments of which decreased with increasing concentration. The same was observed with the mean relaxation time of the high frequency dispersion. Both the frequency and concentration dependence was largely analogous to what is observed with other polyelectrolytes. Values of the dielectric parameters extrapolated to infinite dilution could also be interpreted in the same manner as for more simple, charged macromolecules and no specific effects had to be taken into account.     

小花棘豆(Oxytripis glabra DC)毒素基因的cDNA克隆及序列测定

在完成小花棘豆毒素 95 %氨基酸序列的基础上 ,根椐已知的氨基酸序列 ,设计合成了特异简并引物 .以小花棘豆总RNA为模板 ,逆转录合成cDNA第一链 ,用置换法合成双链cDNA .用特异引物对此双链cDNA进行PCR扩增 ,将扩增后的目的基因与用SmaⅠ酶切的质粒pUC 18连接 ,转化大肠杆菌JM10 7.筛选阳性克隆进行序列分析 ,获得了OXY基因的全部序列 .经测序后测得基因序列与原氨基酸序列对照完全一致 .GenBnak数据检索说明 ,OXY基因编码序列确定是一个从未报道的序列 .此研究结果对该毒素的应用研究奠定了基础 .     

Sperm chromatin damage associated with male smoking

Cigarette smoke is a rich source of mutagens and carcinogens; thus, we have investigated the effects of male smoking on the DNA of human sperm. This was performed using the sperm chromatin structure assay (SCSA), which measures the sensitivity of sperm DNA to acid induced denaturation, and the terminal deoxynucleotidyl transferase assay (TdTA), which measures DNA strand breaks by addition of the biotinylated nucleotide dUTP to 3'-OH ends of DNA, sites of DNA breakage, using the enzyme terminal deoxynucleotidyl transferase. Sperm from subjects who smoked were significantly more sensitive to acid induced denaturation than non-smokers (P<0.02) and possessed higher levels of DNA strand breaks (P<0.05). We hypothesise that smoking damages the chromatin structure and produces endogenous DNA strand breaks in human sperm. These changes may result in sperm DNA mutations, that predispose offspring to greater risk of malformations, cancer and genetic diseases.     

Potentiation of a nucleolytic activity in Bacillus subtilis

In several strains of Bacillus subtilis extensive breakdown of chromosomal DNA may be potentiated by osmotic lysis of protoplasts. At its most severe, in strains originating from Farmer & Rothman's thymine auxotroph, the rate of DNA breakdown was greater than 50% per hour at 40 degrees C. The rate of DNA breakdown in most other strains tested was approximately 5% per hour except for SP beta- strains, in which the rate of DNA breakdown was only 0.3%. DNA degradation was attributed to relaxation of control of a nuclease specified by the prophage of SP beta or a related phage. The most potent nuclease in lysates was an ATP-activated protein of Mr 280 000. Derivatives of Farmer and Rothman's strain containing integrated plasmids had the highest rate of DNA degradation. Although the chromosome was completely destroyed, covalently closed circular plasmids were generated from the integrated sequence. These showed massive deletions of the B. subtilis part of the integrated plasmid but the vector sequence remained intact. The nucleolytic activity therefore appears to recognize specific sequences in B. subtilis DNA. We suggest that activation of SP beta genes during development of competence may be a cause of deletion of cloned genes in the early stages of establishment of cloned sequences.     

Dielectric relaxation measurements of 12 kbp plasmid DNA

The dielectric properties of 12 kbp plasmid DNA have been measured as a function of temperature in the range 5 degrees C to 40 degrees C. Time domain reflectometry was used to obtain dielectric data over the frequency range from 200 kHz to 3 GHz. Values of the frequency dependent polarisability per DNA macromolecule have been determined from the measurements. Possible mechanisms that could account for the dielectric dispersion are also discussed, in particular the counterion fluctuation model of Manning-Mandel-Oosawa.     

Dielectric analysis for estimation of oil content in the mycelia of Mortierella alpina.

The dielectric behavior of the filamentous fungi Mortierella alpina SAM2104 and 1S-4, which produce polyunsaturated fatty acid enriched oil in the mycelia, was investigated. During the cultivation carried out in a 10-kL fermentor for 12-15 days, the relative permittivity and conductivity of the broth were measured in the frequency range of 100 kHz to 30 MHz. The dielectric parameters, i.e., the amplitude of dielectric relaxation (Deltaepsilon) and the characteristic frequency (f(c)), were obtained by fitting the Cole-Cole equation to the observed dielectric relaxation, and the conductivity of the medium (kappa(a)) was also measured. The value of Deltaepsilon gradually increased from the second day through the end of cultivation, suggesting that volume fraction of the cell increased with oil accumulation. The conductivity of the cytoplasm (kappa(i)) was calculated from the experimental values of f(c) and kappa(a), using a theoretical equation based on an ellipsoidal cell model. As a result, good correlation between the calculated kappa(i) and the oil content was obtained. These findings indicate that dielectric analysis enables us to estimate the oil content in the mycelia of oleaginous fungi and also provides a useful tool for monitoring cell growth and for controlling the cultivation process.     

Influence of temperature and ionic strength on the low-frequency dielectric dispersion of DNA solutions

The dielectric properties of DNA solutions at low frequencies (5 Hz to 2 kHz) have been measured by means of a four-terminal bridge method utilized to minimize electrode polarization errors. At 24°C native salt-free DNA has a very large specific dielectric increment, Δε/c = 9.8 × 10⁶ l/mol and a very low frequency relaxation centered at 18 Hz. Both the dielectric increment and the relaxation time are greatly decreased by partial heat denaturation at temperatures above 60°C or by addition of salt, the effects being much larger for divalent anions. These results are shown to be in qualitative agreement with theoretical treatments of counterion fluctuation polarization by McTague and Gibbs for the equilibrium case and by Mandel for relaxation. The ratio of the relaxation time for the low-frequency process to that previously observed at much higher frequencies suggests that these relaxations result from counterion fluctuations along the longitudinal and transverse axes of the molecule, respectively.     

Dielectric behavior of DNA--dye complex. II

The dielectric relaxation of native DNA and the effect of aminoacridine dyes, such as acridine orange (AO), proflavine (PF), and ethidium bromide (EB) have been investigated at different molar DNA phosphate (P) to dye (D) ratios in the frequency range 100 Hz–100 kHz. The static dielectric constant was observed to decrease with increasing binding of aminoacridines. This was interpreted as arising from the neutralization of the surface changes of the DNA molecules as a result of dye binding. At any P/D ratio the extent of charge neutralization was greatest for AO and least for the EB–DNA complex. The relaxation time (τ) for dye-bound DNA was greater compared to that for native DNA. This increase in τ was ascribed to the increase in the length of the dye-bound DNA. The maximum value of τ occurred at P/D = 20, 10, and 2 for AO-, PF-, and EB-treated DNA, respectively. The variation of τ at various levels of binding gave a qualitative idea about the conformational changes of DNA due to its binding with the dyes.     

Iron-mediated DNA damage: sensitive detection of DNA strand breakage catalyzed by iron.

Oxidative DNA damage is involved in mutagenesis, carcinogenesis, aging, radiation effects, and the action of several anticancer drugs. Accumulated evidence indicates that iron may play an important role in those processes. We studied the in vitro effect of low concentrations of Fe(II) alone or Fe(III) in the presence of reducing agents on supercoiled plasmid DNA. The assay, based on the relaxation and linearization of supercoiled DNA, is simple yet sensitive and quantitative. Iron mediated the production of single and double strand breaks in supercoiled DNA. Iron chelators, free radical scavengers, and enzymes of the oxygen reduction pathways modulated the DNA damage. Fe(III)-nitrilotriacetate (NTA) plus either H2O2, L-ascorbate, or L-cysteine produced single and double strand breaks as a function of reductant concentration. A combination of 0.1 microM Fe(III)-NTA and 100 microM L-ascorbate induced detectable DNA strand breaks after 30 min at 24 degrees C. Whereas superoxide dismutase was inhibitory only in systems containing H2O2 as reductant, catalase inhibited DNA breakage in all the iron-mediated systems studied. The effect of scavengers and enzymes indicates that H2O2 and .OH are involved in the DNA damaging process. These reactions may account for the toxicity and carcinogenicity associated with iron overload.     

On the electric polarization of DNA

Dielectric dispersion of DNA was studied in the frequency range 100 Hz–100 kHz at four different temperatures (6–30°C). The dielectric increment ε₀–ε_∞ increased with the rise of temperature. The relaxation time, on the other hand, decreased. Both the increase in dielectric increment and the decrease in relaxation time could not be explained on the basis of the counterion polarization theory. Dipole moment was estimated from Kirkwood theory. It was found to decrease systematically with temperature. Even at 0°C there was a dipole moment of 10⁴D.     

Dielectric analysis of Escherichia coli suspensions in the light of the theory of interfacial polarization.

Dielectric measurements of Escherichia coli suspensions were carried out over a frequency range from 10 kHz to 100 MHz, and marked dielectric dispersions having characteristic frequency of approximately 1 MHz were observed. On the basis of the cell model that a spheroid is covered with two confocal shells, a dielectric theory was developed to determine accurately four electrical parameters for E. coli cells such as the conductivity of the cell wall, the dielectric constant of the cell membrane, and the dielectric constant and the conductivity of the protoplasm. The observed data were analyzed by means of the procedure based on the dielectric theory to yield a set of plausible electrical parameters for the cells. By taking account of the size distribution of the cells and a dielectric relaxation of the protoplasm, the observed dispersion curves were successfully reconstituted by the present theory.     

Plasmid DNA breakage by decay of DNA-associated Auger electron emitters: approaches to analysis of experimental data

Plasmid DNA is a popular substrate for the assay of DNA strand breakage by a variety of agents. The use of the plasmid assay relies on the assumption that individual damaging events occur at random, which allows the application of Poisson statistics. This assumption is not valid in the case of damage arising from decay of DNA-associated Auger electron emitters, since a single decay event can generate a few breaks in the same DNA strand, which is indistinguishable from a single break in the assay. The consequent analytical difficulties are overcome by considering relaxation events rather than single-strand breaks, and linearization events rather than double-strand breaks. A further consideration is that apart from damage at the site of DNA-associated decay, which is the principal interest of the analysis, some DNA damage also arises from the radiation field created by all decay events. These two components of damage are referred to as internal and external breakage, respectively, and they can be separated in the analysis since their contribution depends on the experimental conditions. The DNA-binding ligand Hoechst 33258 labeled with 125I was used in our experiments to study breakage in pBR322 plasmid DNA arising from the decay of this Auger electron emitter. The values obtained for the efficiency (per decay) of plasmid relaxation and linearization by the 125I-labeled ligand were 0.090 +/- 0.035 and 0.82 +/- 0.04, respectively. When dimethylsulfoxide was included as a radical scavenger, the efficiency values for relaxation and linearization were 0.15 +/- 0.02 and 0.65 +/- 0.05, respectively.