A universal description for the experimental behavior of salt-(in)dependent oligocation-induced DNA condensation

We report a systematic study of the condensation of plasmid DNA by oligocations with variation of the charge, Z, from +3 to +31. The oligocations include a series of synthetic linear ε-oligo(l-lysines), (denoted εKn, n = 3–10, 31; n is the number of lysines with the ligand charge Z = n+1) and branched α-substituted homologues of εK10: εYK10, εLK10 (Z = +11); εRK10, εYRK10 and εLYRK10 (Z = +21). Data were obtained by light scattering, UV absorption monitored precipitation assay and isothermal titration calorimetry in a wide range concentrations of DNA and monovalent salt (KCl, C_KCl). The dependence of EC₅₀ (ligand concentration at the midpoint of DNA condensation) on C_KCl shows the existence of a salt-independent regime at low C_KCl and a salt-dependent regime with a steep rise of EC₅₀ with increase of C_KCl. Increase of the ligand charge shifts the transition from the salt-independent to salt-dependent regime to higher C_KCl. A novel and simple relationship describing the EC₅₀ dependence on DNA concentration, charge of the ligand and the salt-dependent dissociation constant of the ligand–DNA complex is derived. For the ε-oligolysines εK6–εK10, the experimental dependencies of EC₅₀ on C_KCl and Z are well-described by an equation with a common set of parameters. Implications from our findings for understanding DNA condensation in chromatin are discussed.     

A complex experimental assessment for objective description of hierarchical psychophysiological behavior as human regulatory phenotype

For the objective and valid identification of different human regulatory phenotypes it should be useful to analyze the behavior of different regulatory subsystems (Anochin 1976) in one multivariate design. Therefore in a DARA supported project a fully computerized and reliable laboratory assessment was developed and tested. We used a set of electrophysiological parameters that should indicate the activity of different functional regulation systems on different "behavioral levels". Skin conductance, skin temperature and voice pitch were used as indicators of sympathico-parasympathical activity. Breathing, heart rate variability and bloodpressure should indicate cardiovascular activity and electromyogram and mimic variablity were thought as indicators of locomotional external behavioral activity. To identify physiological reactions which are influenced by emotional stress we used voice stress measures. Even in the field of aviation and space medicine there exist data about the correlation of voice pitch with emotional excitation (Hecker et. al. 1968, Williams et.al. 1969, Friedrich, Vaic 1978, Vaic et.al. 1981,1982, Griffin, Williams 1987). In our former study (MOSAIC-study, Johannes 1990) the voice pitch and its variation range correlated with perceived emotional excitation but were independent of real bloodpressure variations. Two different types of pitch reaction to this experimental design were correlated to psychological personality scales and assigned subjects to "sensitizers" and "suppressors".     

The condensation of DNA by chromium (III) ions

Cr(III), one of the most potent inorganic carcinogens, induces condensation of DNA into a very compact product at 37 degrees, as shown by electron microscopy. The condensation begins with the appearing of some supercoil structures and complete condensation occurs at relatively low Cr(III) concentrations; for 3 and 30 mM ionic strength they are 4.5 and 45 microM, respectively. Under these conditions, Cr(III) inhibits the interaction between ethidium and DNA as shown by absorption and fluorescence spectra.     

A phosphate bound universal linker for DNA synthesis.

A uridine-based linker immobilized onto polystyrene beads at the 5' terminus via a phosphodiester group and then used as a universal DNA synthesis support gives post synthesis DNA cleavage in 8 hrs or less without alkali metal salts. DNA produced with the new support was analyzed by HPLC, MALDI mass spectroscopy and PAGE. Each analysis showed DNA of equivalent quality to that produced with standard CPG supports, without contaminating materials resulting from linker or support backbone decomposition.     

Condensation of DNA by multivalent cations: experimental studies of condensation kinetics

DNA in viruses and cells exists in highly condensed, tightly packaged states. We have undertaken an in vitro study of the kinetics of DNA condensation by the trivalent cation hexaammine cobalt (III) with the aim of formulating a quantitative, mechanistic model of the condensation process. Experimental approaches included total intensity and dynamic light scattering, electron microscopy, and differential sedimentation. We determined the average degree of condensation, the distribution of condensate sizes, and the fraction of uncondensed DNA as a function of reaction time for a range of [DNA] and [Co(NH(3))(3+)(6)]. We find the following: (1) DNA condensation occurs only above a critical [Co(NH(3))(3+)(6)] for a given DNA and salt concentration. At the onset of condensation, [Co(NH(3))(3+)(6)]/[DNA-phosphate] is close to the average value of 0.54, which reflects the 89-90% charge neutralization criterion for condensation. (2) The equilibrium weight average hydrodynamic radius of the condensates first decreases, then increases with increasing [Co(NH(3))(3+)(6)] as they undergo a transition from intramolecular (monomolecular) to intermolecular (multimolecular) condensation. However, is insensitive to [DNA]. (3) The uncondensed DNA fraction decays approximately exponentially with time. The equilibrium uncondensed DNA fraction and relaxation time decrease with increasing [Co(NH(3))(3+)(6)] but are insensitive to [DNA]. (4) The condensation rate in its early stages is insensitive to [DNA] but proportional to [Co(NH(3))(3+)(6)](xs) = [Co(NH(3))(3+)(6)] - [Co(NH(3))(3+)(6)](crit). (5) Data for low [DNA] and low [Co(NH(3))(3+)(6)] at early stages of condensation are most reliable for kinetic modeling since under these conditions there is minimal clumping and network formation among separate condensates. A mechanism with initial monomolecular nucleation and subsequent bimolecular association and unimolecular dissociation steps with rate constants that depend on the number of DNA molecules in the condensate, accounts reasonably well for these observations.     

Doxorubicin hinders DNA condensation promoted by the protein bovine serum albumin (BSA)

In this work, we have studied the interaction between the anticancer drug doxorubicin (doxo) and condensed DNA, using optical tweezers. To perform this task, we use the protein bovine serum albumin (BSA) in the working buffer to mimic two key conditions present in the real intracellular environment: the condensed state of the DNA and the abundant presence of charged macromolecules in the surrounding medium. In particular, we have found that, when doxo is previously intercalated in disperse DNA, the drug hinders the DNA condensation process upon the addition of BSA in the buffer. On the other hand, when bare DNA is firstly condensed by BSA, doxo is capable to intercalate and to unfold the DNA condensates at relatively high concentrations. In addition, a specific interaction between BSA and doxo was verified, which significantly changes the chemical equilibrium of the DNA–doxo interaction. Finally, the presence of BSA in the buffer stabilizes the double‐helix structure of the DNA–doxo complexes, preventing partial DNA denaturation induced by the stretching forces.     

A simplified universal genomic DNA extraction protocol suitable for PCR

Conventional genomic DNA extraction protocols need expensive and hazardous reagents for decontamination of phenolic compounds from the extracts and are only suited for certain types of tissue. We developed a simple, time-saving and cost-efficient method for genomic DNA extraction from various types of organisms, using relatively innocuous reagents. The protocol employs a single purification step to remove contaminating compounds, using a silica column and a non-hazardous buffer, and a chaotropic-detergent lysing solution that hydrolyzes RNA and allows the selective precipitation of DNA from cell lysates. We used this system to extract genomic DNA from different tissues of various organisms, including algae (Dunaliella salina), human peripheral blood, mouse liver, Escherichia coli, and Chinese hamster ovary cells. Mean DNA yields were 20-30 μg/cm(3) from fresh tissues (comparable to yields given by commercial extraction kits), and the 260/280 nm absorbance ratio was 1.8-2.0, demonstrating a good degree of purity. The extracted DNA was successfully used in PCR, restriction enzyme digestion and for recombinant selection studies.     

Exchange of counterions in DNA condensation

We measured the fluorescence intensity of DNA-bound fluorescent dyes YO-PRO-1 (oxazole yellow) and YOYO-1 (dimer of oxazole yellow) at various spermidine concentrations to determine how counterions on DNA are exchanged in the process of DNA condensation. A decrease of fluorescence intensity was observed with an increase of spermidine. Considering the chemical equilibrium under the competition between the dye and spermidine for counterion condensation on DNA, the theoretical curve well describes the decrease of the fluorescence intensity. These results indicate that dyes are exchanged for spermidine at the binding site on DNA; that is, the exchange of counterions occurs. The parameters associated with the decrease of the fluorescence intensity show that the relative affinity of the dye and spermidine for DNA depends on the state of DNA. Moreover, YOYO-1 prevents the DNA condensation, but the effect of YO-PRO-1 on the condensation is very slight, though both dyes intercalate for DNA; the high affinity of YOYO-1 compared to YO-PRO-1 enables prevention of the condensation.     

A robust universal method for extraction of genomic DNA from bacterial species

The intactness of DNA is the keystone of genome-based clinical investigations, where rapid molecular detection of life-threatening bacteria is largely dependent on the isolation of high-quality DNA. Various protocols have been so far developed for genomic DNA isolation from bacteria, most of which have been claimed to be reproducible with relatively good yields of high-quality DNA. Nonetheless, they are not fully applicable to various types of bacteria, their processing cost is relatively high, and some toxic reagents are used. The routine protocols for DNA extraction appear to be sensitive to species diversity, and may fail to produce high-quality DNA from different species. Such protocols remain time-consuming and tedious, thus to resolve some of these impediments, we report development of a very simple, rapid, and high-throughput protocol for extracting of high-quality DNA from different bacterial species. Based upon our protocol, interfering phenolic compounds were removed from extraction using polyvinylpyrrolidone (PVP) and RNA contamination was precipitated using LiCl. The UV spectrophotometry and gel electrophoresis analysis resulted in high A ₂₆₀/A ₂₈₀ ratio (>1.8) with high intactness of DNA. Subsequent evaluations were performed using some quality-dependent techniques (e.g., RAPD marker and restriction digestions). The isolated DNA from 9 different bacterial species confirmed the accuracy of this protocol which requires no enzymatic processing and accordingly its low-cost making it an appropriate method for large-scale DNA isolation from various bacterial species.     

A method for the experimental study of DNA conformational transitions in fibers

The method proposed for the study of DNA conformational transitions is based on the proportionality, experimentally observed, between the length of a DNA fiber and the axial rise per nucleotide characterizing the molecular helix. Precise curves for the A-B and B-C transitions as a function of the relative humidity are obtained by using X-ray fiber data and measurements of fiber dimensions. It is thus shown that the A-B transition is a cooperative process between two different states, whereas the B-C transition can be considered as a progressive change of conformation. The present method is applied on two natural DNAs differing in base composition so that the effect of the nucleotide content on the conformational changes can be estimated.     

Fluorescence correlation spectroscopy (FCS) as a tool to study DNA condensation with hexadecyltrimethylammonium bromide (HTAB)

The presented data show that the FCS technique can be used to detect the DNA condensation process induced with the cationic compound hexadecyltrimethylammonium bromide (HTAB). We have shown that HTAB induces plasmid condensation upon interaction with it. Condensation can be considered to be complete when the diffusion constant reaches its maximum. The HTAB induced increase in diffusion time does not correlate well with the changes observed when count rate and particle number are considered. This observation contradicts data published for another cationic agent, spermine. This apparent discrepancy proves that the mechanisms of interaction between these compounds and DNA are different. Consequently, the different characters of the plots of count rate, diffusion time, and particle number versus condensing agent concentration can be a source of additional information about the nature of cationic compound-DNA interaction.     

A simple reaction kinetic model of rapid (G protein dependent) and slow (beta-Arrestin dependent) transmission

In this paper the qualitative dynamic behavior of reaction kinetic models of G protein signaling is examined. A simplified basic G protein signaling structure is defined, which is extended to be able to take the effect of slow transmission, RGS mediated feedback regulation and ERK-phosphatase mediated feedback regulation into account. The resulting model gives rise to an acceptable qualitative approximation of the G protein dependent and independent ERK activation dynamics that is in good agreement with the experimentally observed behavior.     

Poly(A) dependent translation in rabbit reticulocyte lysate

Poly(A) tail has been known to enhance mRNA translation in eukaryotic cells. However, the effect of poly(A) tail in vitro is rather small. Rabbit reticulocyte lysate (RRL) is widely used for studying translation in vitro. Translation in RRL is typically performed in nuclease-treated lysate in which most of the endogenous mRNA have been removed. In this condition, the difference in the translational efficiency between poly(A)⁺ and poly(A)⁻ mRNAs is about two-fold. We studied the effect of poly(A) tail on luciferase mRNA translation in nuclease uncreated reticulocyte lysate, in which endogenous globin mRNAs were actively translated. In the case of capped mRNAs. stimulation of translation by poly(A) addition was about 1.5- to 1.6-fold and the effect of the poly(A) length was small. However, in the case of uncapped mRNAs, the addition of poly(A) tail increased luciferase expression over 10-fold. The effect of the poly(A) tail was dependent on its length. The difference in the translational efficiency was not due to the change of mRNA stability. These data indicate that RRL has the potential to translate mRNA in a poly(A) dependent manner.