Number Of DNA Base Pairs Research Articles

Advances in Recovering Plasmids from Wastewater: A State of the Science

Plasmids are small rings of double stranded DNA that are found in all three domains of life: the Bacteria, the Archaea, and the Eukarya. Plasmids encode for proteins that provide their host organisms multiple abilities, such as the ability to transfer genetic information, degrade xenobiotic compounds, resist antibiotics, and outcompete other cells. Plasmids are similar in size to viruses, mostly in the submicron size range; however, plasmid size is normally described by the number of DNA base pairs as opposed to the physical size.Although plasmids are found in all three domains of life, plasmids are most commonly associated with bacteria. Because wastewater harbors bacteria and WRRFs harness bacteria for biological treatment processes, wastewater contains a variety of plasmids and plasmid hosts. Sources of plasmids in wastewater include bacteria excreted from humans and bacteria present in the environment. Researchers have found high levels of bacteria with antibiotic resistant plasmids in the fecal matter of antibiotic-treated children and adults. Bacterial hosts transport plasmids throughout wastewater treatment processes, and as bacteria die and rupture the plasmids are released into the wastewater. Although research suggests that naked plasmids degrade in wastewater, WRRF effluents and biosolids have been shown to contain plasmids and may contribute to the spread of antibiotic resistance.Plasmids and their associated genetic information represent a potential recoverable resource from wastewater. These small rings of intracellular DNA not only encode the genes that facilitate their own transfer and propagation but also harbor functional genes related to a variety of processes. The biotechnology industry has harnessed the information on plasmids to improve multiple industries, such as agriculture, chemical production, paper, textiles, health care, environment, and biotechnology. Plasmids that function to produce antibiotics, degrade xenobiotic compounds, and serve as biocatalysts have been isolated from wastewater. At the same time, the purposes and functions of significant proportions of plasmid DNA recovered from wastewater samples remain undetermined and represent an unknown value to society. Nevertheless, research suggests that wastewater contains genetic information that is of value to society.Plasmids are unlike traditional recoverable commodities, because they are not sold by weight. The information encoded on plasmids and the intellectual property protections provide the value. The actual plasmid has little to no value. Therefore, traditional methods for valuing resource recovery are not transferable to plasmids, as bulk plasmids with no information on the encoded functions or intellectual property protection have little to no value. Hence, a methodology for valuing plasmids must involve sequencing and/or culture-based determination of the gene functions available thereon. Once determined, plasmid products suitable for market could take one or more of several forms, consisting of the plasmid itself, its intellectual property rights, the products of its functional genes, or any combination of the above.Plasmid based products, such as enzymes, are used in multiple industries. Using market estimates from multiple biotechnology sectors, the market for plasmids and plasmid based products and intellectual property could be expected to grow between 5% and 10% combined annual growth rate, and the biotechnology sector has a current value of greater than $270 billion. Yet plasmid recovery, if evaluated using an ‘expected value’ economic approach, likely represents a net loss for WRRFs at this time due to the substantial risks and capital costs involved compared to the potential returns. As a result, plasmid recovery and the recovery of genetic materials from municipal wastewater is a long-term objective, and a pathway for developing the product recovery potential of plasmids is provided over a 20-year time horizon.This title belongs to WE&RF Research Report SeriesISBN: 9781780408545 (eBook)

On the scattering of DNA replication completion times

Stochasticity of Eukaryotes’ DNA replication should not lead to large fluctuations of replication times, which could result in mitotic catastrophes. Fundamental problem that cells face is how to be ensured that entire genome is replicated on time. We develop analytic approach of calculating DNA replication times, that being simplified and approximate, leads, nevertheless, to results practically coincident with those that were obtained by some sophisticated methods. In the framework of that model we consider replication times' scattering and discuss the influence of repair stopping on kinetics of DNA replication. Our main explicit formulae for DNA replication time tr ∝ \(\sqrt {1nN} \) (N is the total number of DNA base pairs) is of general character and explains basic features of DNA replication kinetics.

Mechanism of chromatin remodeling

Results from biochemical and structural studies of the RSC chromatin-remodeling complex prompt a proposal for the remodeling mechanism: RSC binding to the nucleosome releases the DNA from the histone surface and initiates DNA translocation (through one or a small number of DNA base pairs); ATP binding completes translocation, and ATP hydrolysis resets the system. Binding energy thus plays a central role in the remodeling process. RSC may disrupt histone-DNA contacts by affecting histone octamer conformation and through extensive interaction with the DNA. Bulging of the DNA from the octamer surface is possible, and twisting is unavoidable, but neither is the basis of remodeling.

Preferential binding specificity of silver cation to a single nucleobase over base pairs evaluated by abasic site-containing DNA

The binding specificity of silver cations to abasic (AP) site-containing DNA was electrochemically investigated by comparison with the fully matched DNA without the AP site. AP site-containing DNA is designed in a way that only the nucleotide opposite the AP site is variable to allow for coexistence of an unpaired nucleotide and a number of DNA base pairs. The surface of a gold electrode was modified by AP site-containing DNA duplex on which Ag + binding specificity was evaluated. Electrochemical investigations on the AP-DNA-modified electrodes reveal that Ag + preferentially associates to the unpaired nucleotides instead of the coexisted base pairs and shows sequence-dependant binding, especially stronger for purines than for pyrimidines. Additionally, the hydrogen bond pattern moieties of the unpaired nucleotides should be involved in Ag + binding evidenced by a decrease of the redox signal when introducing a ligand with its hydrogen bond moiety complementary to the nucleotide deoxycytidine. This is the first attempt to make a comparison in one DNA molecule for metal ion binding to coexisted unpaired nucleotide and DNA base pairs. The present method demonstrates an easy way for investigating binding specificity of heavy metal ions to AP site in the presence of coexisted DNA base pairs.

Quantitative analysis of DNA–porphyrin interactions

The binding of manganese(III)-tetra(4-N-methylpyridyl)porphyrin (MnTMpyP) with synthetic poly(dA-dT)2, poly(dI-dC)2, and poly(dG-dC)2 DNAs as well as calf thymus (CT) DNA has been quantitatively studied in detail using induced CD (circular dichroism) spectroscopy in the Soret absorption band. The CD spectra, which changed greatly depending on the porphyrin to DNA base-pair molar ratio (r), were normalized with respect to DNA concentration and deconvoluted. Three independent component binding modes (named mode 1, 2, and 3 in the order of increasing r values) were identified, which successfully simulated the observed CD spectra with negligibly small residuals for a wide range of r values. In the case of poly(dA-dT)2, poly (dI-dC)2, and CT DNA, all the three modes appeared, whereas in the case of poly(dG-dC)2 DNA, only modes 1 and 3 appeared in the r range studied. The r dependence of each binding mode, i.e., its relative affinity toward DNA, has been revealed by this analysis. Mode 1, which appeared as a single binding mode at very low r values (r < or = ca. 0.05), was inhibited by the addition of methyl green, a drug that preferentially binds to the major groove of poly (dA-dT)2 DNA. Berenil, a known minor groove binder to poly(dA-dT)2 or poly(dI-dC)2 DNA, inhibited modes 2 and 3. From these inhibition experiments as well as comparison of the component spectra for DNAs of different sequence, a binding site on DNA was proposed for each component binding mode. The number of DNA base pairs covered by a single molecule of porphyrin was estimated.

DNA-assisted binding of microspheres on glass substrates and their laser-induced release

DNA hybridization has been increasingly adopted in materials sciences due to its complementary nature of single stranded DNAs. This unique property could be potentially used in the realization of 2 dimensional (2D) arrays of colloidal microspheres as a precursor to further build more complicated superstructures. In order to precisely understand this DNA-assisted assembly of colloidal particles, we quantitatively assessed the surface density of grafted and hybridizing accessible DNA oligomers on both substrate and colloidal particles. The DNA grafting densities were determined by UV–Vis of dye-functionalized complementary DNA oligomers, in conjunction with theoretical models. The variations of the concentration of hybridized DNA as a function of parameters such as the number of DNA base pairs (bp), the length of spacer and the size of sphere were also investigated to determine the immobilization strength of colloidal microspheres on the substrate. Dehybridization of the particle was conducted by utilizing a focused laser beam. These results were also compared with the particle hybridization energies and modeled according to the sum of DNA bindings as a function of the number of hybridized bases.

Combing DNA on CTAB-coated surfaces

A fluorescence microscope (FM) coupled with an intensified charge-coupled device (ICCD) camera was used to investigate the combing of DNA on cetyltrimethyl ammonium bromide (CTAB)-coated glass surfaces. DNA molecules can be combed uniform and straight on CTAB-coated surfaces. Different combing characteristics at different pH values were found. At lower pH (ca. 5.5), DNA molecules were stretched 30% longer than the unextended and DNA extremities bound with CTAB-coated surfaces via hydrophobic interaction. At high pH values (e.g., 6.4 and 6.5), DNA molecules were extended about 10% longer and DNA extremities bound with CTAB-coated surfaces via electrostatic attraction. At pH 6.0, DNA molecules could be extended 30% longer on 0.2-mM CTAB-coated surfaces. CTAB cationic surfactant has both a hydrophobic motif and a positively charged group. So, CTAB-coated surfaces can bind DNA extremities via hydrophobic effect or electrostatic attraction at different pH values. It was also found that combing of DNA on CTAB-coated surfaces is reversible. The number of DNA base pairs binding to CTAB-coated surfaces was calculated.

Giant non-universal critical index and fluctuations in DNA phase transition

Thermal unbinding of a double-stranded DNA molecule is a phase transition with a giant (over 100) critical index. The index is non-universal, it changes with concentrations and melting temperatures of base pairs. At slightly lower temperatures it switches to an essential singularity. A finite DNA completely unbinds below critical temperature. The shift from the critical temperature is stochastic. It is inversely proportional to the logarithm of the DNA length, and is observable even in a DNA solution, where the total number of DNA base pairs is, e.g., ∼10 20. Its macroscopically large fluctuations are characteristic of DNA unbinding.

Water regulation of actinomycin-D binding to DNA: the interplay among drug affinity, DNA long-range conformation, and hydration.

Actinomycin-D (actD) binds to natural DNA at two different classes of binding sites, weak and strong. The affinity for these sites is highly dependent on DNA sequence and solution conditions, and the interaction appears to be purely entropic driven. Although the entropic character of this reaction has been attributed to the release of water molecules upon drug to DNA complex formation, the mechanism by which hydration regulates actD binding and discrimination between different classes of binding sites on natural DNA is still unknown. In this work, we investigate the role of hydration on this reaction using the osmotic stress method. We show that the decrease of solution water activity, due to the addition of sucrose, glycerol, ethylene glycol, and betaine, favors drug binding to the strong binding sites on DNA by increasing both the apparent binding affinity delta G, and the number of DNA base pairs apparently occupied by the bound drug nbp/actD. These binding parameters vary linearly with the logarithm of the molar fraction of water in solution log(chi w), which indicates the contribution of water binding to the energetic of the reaction. It is demonstrated that the hydration change measured upon binding increases proportionally to the apparent size of the binding site nbp/actD. This indicates that nbp/actD, measured from the Scatchard plot, is a measure of the size of the DNA molecule changing conformation due to ligand binding. We also find that the contribution of DNA deformation, gauged by nbp/actD, to the total free energy of binding delta G, is given by delta G = delta Glocal + nbp/actD x delta GDNA, where delta Glocal = -8020 +/- 51 cal/mol of actD bound and delta GDNA = -24.1 +/- 1.7 cal/mol of base pair at 25 degrees C. We interpret delta Glocal as the energetic contribution due to the direct interactions of actD with the actual tetranucleotide binding site, and nbp/actD x delta GDNA as that due to the change in conformation, induced by binding, of nbp/actD DNA base pairs flanking the local site. This interpretation is supported by the agreement found between the value of delta GDNA and the torsional free energy change measured independently. We conclude suggesting an allosteric model for ligand binding to DNA, such that the increase in binding affinity is achieved by increasing the relaxation of the unfavorable free energy of binding storage at the local site through a larger number of DNA base pairs. The new aspect on this model is that the "size" of the complex is not fixed but determined by solutions conditions, such as water activity, which modulate the energetic barrier to change helix conformation. These results may suggest that long-range allosteric transitions of duplex DNA are involved in the inhibition of RNA synthesis by actD, and more generally, in the regulation of transcription.

The effect of intrinsic curvature on conformational properties of circular DNA

Both thermal fluctuations and the intrinsic curvature of DNA contribute to conformations of the DNA axis. We looked for a way to estimate the relative contributions of these two components of the double-helix curvature for DNA with a typical sequence. We developed a model and Monte Carlo procedure to simulate the Boltzmann distribution of DNA conformations with a specific intrinsic curvature. Two steps were used to construct the equilibrium conformation of the model chain. We first specified the equilibrium DNA conformation at the base pair level of resolution, using a set of the equilibrium dinucleotide angles and DNA sequence. This conformation was then approximated by the conformation of the model chain consisting of a reduced number of longer, straight cylindrical segments. Each segment of the chain corresponded to a certain number of DNA base pairs. We simulated conformational properties of nicked circular DNA for different sets of equilibrium dinucleotide angles, different random DNA sequences, and lengths. Only random sequences of DNA generated with equal probability of appearance for all types of bases at any site of the sequence were used. The results showed that for a broad range of intrinsic curvature parameters, the radius of gyration of DNA circles should be nearly independent of DNA sequence for all DNA lengths studied. We found, however, a DNA properly that should strongly depend on DNA sequence if the double helix has essential intrinsic curvature. This property is the equilibrium distribution of the linking number for DNA circles that are 300-1000 bp in length. We found that a large fraction of the distributions corresponding to random DNA sequences should have two separate maxima. The physical nature of this unexpected effect is discussed. This finding opens new opportunities for joined experimental and theoretical studies of DNA intrinsic curvature.

Physical distribution of recombination in B-genome chromosomes of tetraploid wheat

Several studies have indicated a noncorrespondence between genetic and physical distances in wheat chromosomes. To study the physical distribution of recombination, polymorphism for C-banding patterns was used to monitor recombination in 67 segments in 11 B-genome chromosome arms of Triticum turgidum. Recombination was absent in proximal regions of all chromosome arms; its frequency increased exponentially with distance from the centromere. A significant difference was observed between the distribution of recombination in physically short and physically long arms. In physically short arms, recombination was almost exclusively concentrated in distal segments and only those regions were represented in their genetic maps. In physically long arms, while a majority of the genetic distance was again based upon recombination in distal chromosome segments, some interstitial recombination was observed. Consequently, these regions also contributed to the genetic maps. Such a pattern of recombination, skewed toward terminal segments of chromosomes, is probably a result of telomeric pairing initiation and strong positive chiasma interference. Interference averaged 0.81 in 35 pairs of adjacent segments and 0.57 across the entire recombining portions of chromosome arms. The total genetic map lengths of the arms corresponded closely to those expected on the basis of their metaphase-I chiasma frequencies. As a consequence of this uneven distribution of recombination there can be a 153-fold difference (or more) in the number of DNA base pairs per unit (centiMorgan) of genetic length.

Modulation of the B-A Transition of DNA by Potential Antitumor Antibiotics. Influence of the Base Composition of DNA

The B-A transition of DNA in oriented films of DNA-drug complexes is more or less restricted as a consequence of drug binding as revealed by infrared linear dichroism. A fraction of DNA is irreversibly locked into the B form. This behavior is described by the number of DNA base pairs "frozen" in the B form by one drug molecule. This quantity is dependent on the DNA sequence the drug is attached to. In this paper, drug complexes of oriented films of NaDNA with a GC content of 42% from calf thymus and a GC-rich DNA from Micrococcus lysodeikticus were compared. The restriction of the B-A transition of DNA complexes with two intercalating antibiotics, aclacinomycin A and violamycin BI, is not severely influenced by the base composition of DNA. By contrast, the strong groove binding oligopeptide antibiotics netropsin and distamycin A are much less effective to restrict the B-A transition of GC-rich DNA than of AT-rich DNA. This finding is in agreement with previous results by other methods which support a model based upon a strong preference of AT clusters by these two non-intercalating drugs.