Electromagnetic field effect on separation of nucleotide sequences and unwinding of a double helix during DNA replication.

Our previous pre-clinic experimental results have showed that the bacterium infection can be suppressed and the epithelialization can be enhanced by the externally applied rectangular pulsed electrical current stimulation (RPECS). The results are clinically significant for patients, especially for those difficult patients whose skin wounds need long periods to heal. However, the results also raise questions: How does the RPECS accelerate the epithelium cell proliferation? What is the relationship among the bacterium infection, the epithelialization and the RPECS? To answer these questions, we have previously modeled mitosis and cytokinesis mechanisms for animal cells and amitosis for bacteria at a cellular level and in a view of physics. In this paper, we model the separation of nucleotide sequences and the unwinding of a double helix during DNA replication at a molecular level and also in wild types of cells. Firstly, we define a new concept of nucleotide (NT) electromagnetic field (EMF) box (sequence) which carries genetic information: The continuous NT EMF boxes compose a nucleotide strand. Then, we hypothesize the symmetry, repulsion and attraction of NT EMF boxes: If a pair of NT EMF boxes are (quasi) mirror or complementary symmetric about a plane (curve) or point, they repulse or attract from each other because there is a repulsive or attractive EMF force between them. Our models suggest, the repulsive EMF force from children DNA strands simultaneously separates the children DNA strands, splits the hydrogen bonds of parental base pairs, and unwinds the parental double helix while DNA polymerases are synchronously synthesizing the new children DNA strands. To understand the mechanism of epithelialization enhanced with the externally applied RPECS at a molecular level, we hypothesize that the normal separation of nucleotide sequences and unwinding of a double helix during DNA replication could be suppressed in the bacteria but not in the epithelium cells because: (a) the spontaneous EMF in the epithelium could be 1000 times stronger than that in bacteria; (b) the epithelium cells have one more non-conducting envelope (nuclear membrane) to protect the normal separation and unwinding; (c) based on our previous experimental data, the RPECS amount received by the bacteria are three times as much as the amount the epithelium cells receive. Therefore, the epithelium cellular proliferation may be directly, as well as indirectly (e.g., somatic reflex) accelerated by the RPECS.

Nucleosides and nucleotides. 208. Alternate-strand triple-helix formation by the 3'-3'-linked oligodeoxynucleotides with the anthraquinonyl group at the junction point.

The synthesis of 3'-3'-linked oligodeoxynucleotides (ODNs) with the anthraquinonyl group at the junction point is described. The ODNs were synthesized on a DNA synthesizer using a controlled pore glass (CPG) carrying pentaerythritol that has an intercalator at one of the four hydroxymethyl groups. Stability of the triplexes with the target duplexes was studied by thermal denaturation. The 3'-3'-linked ODNs with the anthraquinonyl group enhanced the thermal stability of the triplexes when compared with those without the intercalator and the unmodified nonamer 10. It was found that the ODNs 12 and 13 carrying the anthraquinonyl groups can form thermally stable triplexes by skipping two or three extra base pairs between two binding domains of the target duplexes. The ability of the 3'-3'-linked ODNs to inhibit cleavage of the target DNA 22 by the restriction enzyme Hind III was tested. It was found that the 3'-3'-linked ODN 16 with the anthraquinonyl group at the junction point inhibited the cleavage by the enzyme more effectively than the nonamer 14 and the 3'-3'-linked ODN 15 without the intercalator.

Influence of semen processing technique on human sperm DNA integrity.

OBJECTIVES: To compare the effects of density-gradient centrifugation and swim-up technique on sperm DNA integrity. METHODS: Semen samples (n = 22) were obtained from consecutive nonazoospermic men presenting for infertility evaluation. Individual samples were divided into three aliquots (whole semen, density-gradient centrifugation, and swim-up) for subsequent analysis of sperm motility and DNA integrity. Sperm DNA integrity was evaluated by flow cytometry analysis of acridine orange-treated spermatozoa and expressed as the percentage of spermatozoa demonstrating denatured DNA. RESULTS: Mean sperm motility (+/-SEM) improved significantly after processing with two-layer density-gradient and swim-up compared with whole semen (65.6% +/- 4.0% and 73.0% +/- 3.0% versus 52.0% +/- 3.6%, respectively, P <0.005), with no significant difference in motility between Percoll-treated and swim-up-treated spermatozoa. In contrast, the percentage of spermatozoa with denatured DNA was reduced significantly in swim-up-treated but not in Percoll-treated spermatozoa compared with whole semen (4.8% +/- 1. 2% and 13.6% +/- 3.6% versus 10.1% +/- 2.3%, respectively, P <0. 0001). CONCLUSIONS: Although density-gradient centrifugation is comparable to swim-up technique in recovering spermatozoa with enhanced motility, spermatozoa recovered after swim-up possess higher DNA integrity. These data urge us to reexamine our current sperm processing techniques in order to minimize sperm DNA damage.

Susceptibility of sperm chromatin to acid denaturation in situ: a study in endogenous FSH-deprived adult male bonnet monkeys (Macaca radiata).

Acid denaturation of calf thymus DNA in vitro followed by acridine orange (AO) binding induced a 112% increase in the emission of red, a 58% decrease in green, and a consequential decrease in the ratio of green:red fluorescences from 1.7 to 0.9. This metachromatic property of AO on binding to DNA following acid denaturation was utilized to study the susceptibility of normal and ovine follicle-stimulating hormone (oFSH) actively immunized bonnet monkey spermatozoa voided throughout the year. For analyses, the scattergram generated by the emission of red and green fluorescences by 10,000 AO-bound sperm from each semen sample was divided into 4 quadrant zones representing percentage cells fluorescing high green-low red (Q1), high green-high red (Q2), low green-low red (Q3) and low green-high red. (Q4). Normal monkey sperm obtained during the months of July-December exhibited 76, 13, and 11% cells in Q2, Q3, and Q4 quadrants, respectively. However, during January-June, when the females of the species are markedly subfertile, noncycling, and amenorrhoeic, the spermatozoa ejaculated by the male monkeys exhibited 38, 39, and 23% sperm in Q2, Q3, and Q4, respectively, the differences being highly significant (p < .01-.001). FSH deprivation induced significant shifts in fluorescence emissions, from respective controls, with 39, 33, and 28% cells in Q2, Q3, and Q4, respectively, during July-December, and 15, 48, and 37% sperm in Q2, Q3, and Q4 quadrants, respectively, during January-June. It is postulated that the altered kinetics of germ cell transformations and the deficient spermiogenesis observed earlier following FSH deprivation in these monkeys may have induced the enhanced susceptibility to acid denaturation in sperm.

High salt and solvent induced Z-conformation in native calf thymus DNA.

The Z-DNA forming potentiality of native calf thymus DNA using spectrophotometric measurements and diethyl pyrocarbonate reactivity have been investigated. The Z value was evaluated from absorbance ratio for B- and Z-DNA and compared with the values obtained for native DNA. The results suggest that native DNA undergoes B- to Z-/Z-analogous transition to an extent of 20 per cent under constraint conditions. Possible Z-DNA formation was also characterized by hyper-reactivity of native DNA towards diethyl pyrocarbonate in aqueous solvent. The thermal melting profile of native and modified DNA and nuclease S1 digestibility data, in addition, reflects B- to Z-/Z-analogous transition in native DNA.

Unwound regions in yeast centromere IV DNA.

The DNA of the centromere of chromosome IV (CEN4) of Saccharomyces cerevisiae is found to be sensitive to single-strand specific nuclease P1 when inserted into a negatively supercoiled plasmid. Fine mapping identifies two P1-sensitive segments: one segment maps to essential centromere element CDEI and bordering CDEII bases, and the other segment is located in element CDEIII. The AT-rich element CDEII, which is expected to be early melting, is for the most part resistant to nuclease P1. Cleavage is inhibited by NaCl, MgCl2 and polyamines. The cleavage rate is only weakly dependent on P1 concentration in the range of 0.5 to 20 munits/microliters. The two P1-sensitive segments are also modified by the DNA-confirmation-specific reagent KMnO4. Negative superhelicity is required for all modifications. Two-dimensional topoisomer analysis indicates the unwinding of 80(+/- 10) bases within the negatively supercoiled CEN4-containing plasmid. The data best fit a model in which the DNA of the CEN4 region undergoes a transition into a paranemic intermediate in which each strand is folded into an RNA-like foldback structure.

Monitoring of the cooperative unfolding of the sunY group I intron of bacteriophage T4. The active form of the sunY ribozyme is stabilized by multiple interactions with 3' terminal intron components.

We have studied the mechanism by which the 3' terminal domain of the sunY intron of bacteriophage T4 activates the group I ribozyme core of this intron, from which it is separated by some 800 nucleotides. As shown by monitoring either UV absorbance or self-splicing reaction kinetics as a function of temperature, intron transcripts undergo highly cooperative unfolding/inactivation upon heating: the two methods yield similar estimates of the thermodynamic parameters associated with this process. Such cooperativity makes it possible in turn to assess the energetic contribution of specific interactions to the overall structure, by comparing the sensitivity to heat inactivation of molecules carrying various nucleotide substitutions. By combining this approach with chemical modification, we have probed several proven or putative interactions between the core and 3' terminal domain of the intron and conclude that the role of the 3' terminal domain is to stabilize the active form of the ribozyme. Interestingly, the P9.0 interaction, which brings 3' terminal nucleotides next to the core site that binds the guanosine cofactor of the self-splicing reaction, is now shown to be composed in fact of two distinct pairings. An isolated base-pair (P9.0a), involving a residue located only six nucleotides upstream of the 3' splice site, participates in the stabilization of the ribozyme and appears to persist during the second stage of self-splicing (exon ligation). In contrast, formation of the previously demonstrated P9.0b pairing, which involves the two penultimate intron nucleotides, contributes no additional stability and results in no detectable rearrangement of the core structure. Implications for the concept of a static ribozyme are discussed in the light of a slightly revised three-dimensional model of the sunY intron.

Biotinylation of denatured double-stranded DNA after transamination of cytidines: molecular biology applications.

Transamination at 100 degrees C of cytosines in denatured double-strand DNA is a rapid and reliable method to obtain DNA molecules containing N4-aminoethylcytosine (4aeC), which can be quantitatively conjugated to biotinyl-N-hydroxysuccinimide ester (BHS) at 37 degrees C, yielding chemically labelled probes for molecular hybridization. The adopted transamination reaction temperature allows for a ten-fold reduction of the time required for labelling at 42 degrees C, and probes obtained by this procedure are equally effective for general use in molecular biology. Dot-blots with 1-5 pg of target lambda DNA were detected by streptavidin-acid phosphatase complex after hybridization with its homologous sequences. Chemically biotinylated mouse satellite DNA has been used in combination with avidin-horseradish peroxidase to detect metaphase and interphase centromeres via in situ hybridization. Moreover probes labelled with differentially spaced linker arms were prepared by this method.

B--Z conformational transition in d(CGCGCGTTAATT), d(CGCGCGTTAA) and d(CGCGCGTT).

Conformational studies on three DNA-oligomers (d(CGCGCGTTAATT), d(CGCGTTAA) and d(CGCGCGTT) in solution by circular dichroism spectroscopy are reported. In low salt solution, all three DNA oligomers exhibit a characteristic B-conformation. However, under the influence of high salt concentration i.e. 5M NaCl, the octamer d(CGCGCGTT) exhibits 'A' conformation whereas the decamer and dodecamer retain B-conformation. On addition of millimolar amount of NiCl2 to the 5M NaCl, solution of oligodeoxynucleotides a B-Z transition is observed in octamer, decamer and dodecamer. However, NiCl2 titrations show that mid point of transition for dodecamer is at 2.25 mM, for decamer is at 13 mM NiCl2 and for octamer is 17 mM at NiCl2. In 60% alcohol all three oligonucleotides remain in the B-conformation. The melting temperatures of oligonucleotides at various salt concentration are also reported. Thermodynamic parameters calculated by melting profile using a two state model show that dodecamer and decamer are most stable in their 5M NaCl, B-form. However, octamer is more stable in its Z form than that of its 'A' form.

Individuality of DNA denaturation patterns in human sperm as measured by the sperm chromatin structure assay.

Eight monthly semen samples from 45 men not known to be exposed to industrial toxicants were measured by the flow cytometric sperm chromatin structure assay (SCSA). This assay determines susceptibility of sperm DNA to in situ, acid-induced denaturation and is quantitated by the metachromatic shift of acridine orange fluorescence from green (native DNA) to red (denatured DNA). The observed green versus red fluorescence scattergram (cytogram) patterns were generally unique between donors and homogeneous within a donor over time. Within a donor, the cytogram patterns were the same whether intact sperm cells or detached nuclei were measured. For some individuals the cytogram patterns differed for some months and then returned to the original pattern. Intraclass correlations for mean and standard deviation of alpha t [alpha t = red/(red + green) fluorescence] were higher (.67 to .90) than any classically measured semen variables, suggesting that SCSA results within an individual were more consistent than other measures. Furthermore, average within-donor CV of alpha t parameters expressed as a percent of any given individual's means was around 10%, which is significantly lower than those derived from common semen measures. The SCSA is an objective, technically sound, biologically stable, sensitive, and feasible measure of semen quality.