Efficiency of nonhomologous DNA end joining varies among somatic tissues, despite similarity in mechanism

Failure to repair DNA double-strand breaks (DSBs) can lead to cell death or cancer. Although nonhomologous end joining (NHEJ) has been studied extensively in mammals, little is known about it in primary tissues. Using oligomeric DNA mimicking endogenous DSBs, NHEJ in cell-free extracts of rat tissues were studied. Results show that efficiency of NHEJ is highest in lungs compared to other somatic tissues. DSBs with compatible and blunt ends joined without modifications, while noncompatible ends joined with minimal alterations in lungs and testes. Thymus exhibited elevated joining, followed by brain and spleen, which could be correlated with NHEJ gene expression. However, NHEJ efficiency was poor in terminally differentiated organs like heart, kidney and liver. Strikingly, NHEJ junctions from these tissues also showed extensive deletions and insertions. Hence, for the first time, we show that despite mode of joining being generally comparable, efficiency of NHEJ varies among primary tissues of mammals.

DNA Binding, Coprotease, and Strand Exchange Activities of Mycobacterial RecA Proteins: Implications for Functional Diversity among RecA Nucleoprotein Filaments

One of the fundamental questions concerning homologous recombination is how RecA or its homologues recognize several DNA sequences with high affinity and catalyze all the diverse biological activities. In this study, we show that the extent of single-stranded DNA binding and strand exchange (SE) promoted by mycobacterial RecA proteins with DNA substrates having various degrees of GC content was comparable with that observed for Escherichia coli RecA. However, the rate and extent of SE promoted by these recombinases showed a strong negative correlation with increasing amounts of sequence divergence embedded at random across the length of the donor strand. Conversely, a positive correlation was seen between SE efficiency and the degree of sequence divergence in the recipient duplex DNA. The extent of heteroduplex formation was not significantly affected when both the pairing partners contained various degrees of sequence divergence, although there was a moderate decrease in the case of mycobacterial RecA proteins with substrates containing larger amounts of sequence divergence. Whereas a high GC content had no discernible effect on E. coli RecA coprotease activity, a negative correlation was apparent between mycobacterial RecA proteins and GC content. We further show clear differences in the extent of SE promoted by E. coli and mycobacterial RecA proteins in the presence of a wide range of ATP:ADP ratios. Taken together, our findings disclose the existence of functional diversity among E. coli and mycobacterial RecA nucleoprotein filaments, and the milieu of sequence divergence (i.e., in the donor or recipient) exerts differential effects on heteroduplex formation, which has implications for the emergence of new genetic variants.

Theoretical and Computational Analysis of Static and Dynamic Anomalies in Water-DMSO Binary Mixture at Low DMSO Concentrations

Experiments have repeatedly observed both thermodynamic and dynamic anomalies in aqueous binary mixtures, surprisingly at low solute concentration. Examples of such binary mixtures include water-DMSO, water-ethanol, water-tertiary butyl alcohol (TBA), and water-dioxane, to name a few. The anomalies have often been attributed to the onset of a structural transition, whose nature, however, has been left rather unclear. Here we study the origin of such anomalies using large scale computer simulations and theoretical analysis in water-DMSO binary mixture. At very low DMSO concentration (below 10%), small aggregates of DMSO are solvated by water through the formation of DMSO-(H2O)(2) moieties. As the concentration is increased beyond 10-12% of DMSO, spanning clusters comprising the same moieties appear in the system. Those clusters are formed and stabilized not only through H-bonding but also through the association of CH3 groups of DMSO. We attribute the experimentally observed anomalies to a continuum percolation-like transition at DMSO concentration X-DMSO approximate to 12-15%. The largest cluster size of CH3-CH3 aggregation clearly indicates the formation of such percolating clusters. As a result, a significant slowing down is observed in the decay of associated rotational auto time correlation functions (of the S = O bond vector of DMSO and O-H bond vector of water). Markedly unusual behavior in the mean square fluctuation of total dipole moment again suggests a structural transition around the same concentration range. Furthermore, we map our findings to an interacting lattice model which substantiates the continuum percolation model as the reason for low concentration anomalies in binary mixtures where the solutes involved have both hydrophilic and hydrophobic moieties.

A quasi-steady performance prediction model for dynamic characteristics of a volute pump

A performance prediction model generally applicable for volute-type centrifugal pumps has been extended to predict the dynamic characteristics of a pump during its normal starting and stopping periods. Experiments have been conducted on a volute pump with different valve openings to study the dynamic behaviour of the pump during normal start-up and stopping, when a small length of discharge pipeline is connected to the discharge flange of the pump. Such experiments have also been conducted when the test pump was part of a hydraulic system, an experimental rig, where it is pumping against three similar pumps, known as supply pumps, connected in series, with the supply pumps kept idle or running. Instantaneous rotational speed, flowrate, and delivery and suction pressures of the pump were recorded and it was observed in all the tested cases that the change of pump behaviour during the transient period was quasi-steady, which validates the quasi-steady approach presented in this paper. The nature of variation of parameters during the transients has been discussed. The model-predicted dynamic head-capacity curves agree well with the experimental data for almost all the tested cases.

CAITG sequence at the 5' end of Oligo(A)-tracts strongly modulates DNA curvature

An analysis of the base pair doublet geometries in available crystal structures indicates that the often reported intrinsic curvature of DNA containing oligo-(d(A).d(T)) tracts may also depend on the nature of the flanking sequences. The presence of CA/TG doublet in particular at the 5' end of these tracts is expected to enhance their intrinsic bending property. To test this proposition, three oligonucleotides, d(GAAAAACCCCCC), d(CCCCCCAAAAAG), d(GAAAAATTTTTC), and their complementary sequences were synthesized to study the effect of various flanking sequences, at the 5' and 3' ends of the A-tracts, on the curvature of DNA in solution. An analysis of the polyacrylamide gel electrophoretic mobilities of these sequences under different conditions of salts and temperatures (below their melting points) clearly showed that the oligomer with CA/TG sequence in the center was always more retarded than the oligomer with AC/GT sequence, as well as the oligomer with AT/AT sequence. Hydroxyl radical probing of the sequences with AC/GT and CA/TG doublet junctions gives a similar cutting pattern in the A-tracts, which is quite different from that in the C-tracts, indicating that the oligo(A)-tracts have similar structures in the two oligomers. KMnO4 probing shows that the oligomer with a CA/TG doublet junction forms a kink that is responsible for its inherent curvature and unusual electrophoretic mobility. UV melting shows a reduced thermal stability of the duplex with CA/TG doublet junction, and circular dichroism (CD) studies indicate that a premelting transition occurs in the oligomer with CA/TG doublet step before global melting but not in the oligomer with AC/GT doublet step, which may correspond to thermally induced unbending of the oligomer. These observations indicate that the CA/TG doublet junction at the 5' end of the oligo(A)-tract has a crucial role in modulating the overall curvature in DNA.

Enhancement in Bath Mixing and Plume area in a New Degassing Process - A Computational Fluid Dynamic Study

Reaction between the various species in slag and metal phase is usually mass transfer controlled. There have been continuous efforts to increase the reaction efficiency in slag-metal system, especially during decarburization of steel to produce the ultra low carbon steel (ULCS) in secondary steelmaking. It has been found that the surface reaction is a dominant factor in the final stage of decarburization. In the initial stage, the inner site reaction is major factor in the refining process. The mixing of bath affects the later reaction. However, the former reaction (surface reaction) is affected by the plume size area at the top of the metal surface. Therefore, a computational study has been made to understand the fluid dynamics of a new secondary steelmaking process called Revolutionary Degasser Activator (REDA) to study the bath mixing and plume area. REDA process has been considered as it is claimed that this process can reduce the carbon content in steel below 10ppm in a less time than the other existing processes such as RH and Tank degasser. This study shows that both bath mixing and plume area are increased in REDA process facilitating it to give the desired carbon content in less time. Qualitative comments are made on slag-metal reaction system based on this finding.

DNA polymerase alpha-primase complex from the silk glands of the non-mulberry silkworm Philosamia ricini.

The DNA content in the silk glands of the non-mulberry silkworm Philosamia ricini increases continuously during the fourth and fifth instars of larval development indicating high levels of DNA replication in this terminally differentiated tissue. Concomitantly, the DNA polymerase alpha activity also increases in the middle and the posterior silk glands during development, reaching maximal levels in the middle of the fifth larval instar. A comparable level of DNA polymerase delta/epsilon was also observed in this highly replicative tissue. The DNA polymerase alpha-primase complex from the silk glands of P. ricini has been purified to homogeneity by conventional column chromatography as well as by immunoaffinity techniques. The molecular mass of the native enzyme is 560 kDa and the enzyme comprises six non-identical subunits. The identity of the enzyme as DNA polymerase alpha has been established by its sensitivity to inhibitors such as aphidicolin, N-ethylmaleimide, butylphenyl-dGTP, butylanilino-dATP and antibodies to polymerase alpha. The enzyme possesses primase activity capable of initiating DNA synthesis on single-stranded DNA templates. The tight association of polymerase and primase activities at a constant ratio of 6:1 is observed through all the purification steps. The 180 kDa subunit harbours the polymerase activity, while the primase activity is associated with the 45 kDa subunit.

Dynamic analysis of three point bend specimens under impact

A simple one dimensional inertial model is presented for transient response analysis of notched beams under impact, and extracting dynamic initiation toughness values. The model includes the effects of striker mass interactions, and contact deformations of the beam. Displacement time history of the striker mass is applied to the model as forcing function. The model is validated by comparison with the experimental investigation on ductile aluminium 6061 alloy and brittle polymer, PMMA.

Dynamic scheduling in manufacturing systems using brownian approximations

Recently, Brownian networks have emerged as an effective stochastic model to approximate multiclass queueing networks with dynamic scheduling capability, under conditions of balanced heavy loading. This paper is a tutorial introduction to dynamic scheduling in manufacturing systems using Brownian networks. The article starts with motivational examples. It then provides a review of relevant weak convergence concepts, followed by a description of the limiting behaviour of queueing systems under heavy traffic. The Brownian approximation procedure is discussed in detail and generic case studies are provided to illustrate the procedure and demonstrate its effectiveness. This paper places emphasis only on the results and aspires to provide the reader with an up-to-date understanding of dynamic scheduling based on Brownian approximations.

Dynamic structure factors of a dense mixture

We compute the dynamic structure factors of a dense binary liquid mixture. These describe dynamics on molecular length scales, where structural relaxation is important. We find that the presence of a few large particles in a dense fluid of small particles slows down the dynamics considerably. We also observe a deep narrowing of the spectrum for a disordered mixture composed of a nearly equal packing of the two species. In contrast, a few small particles diffuse easily in the background of a dense fluid of large particles. We expect our results to describe neutron scattering from a dense mixture.

Dynamic structure of charge carrier in polyaniline by near-infrared excited resonance Raman spectroscopy

New vibrational Raman features characteristic to the conductive form of polyaniline have been observed with the near-infrared excitation at 1047 nm. Based on an analogy with the resonance Raman spectrum of Michler's ketone in the lowest excited triplet (T-1) state, we consider these features as due to a dynamic structure of a diimino-1,4-phenylene unit in the polyaniline chain exchanging a positive charge very rapidly. This consideration directly leads to a conducting mechanism in which a positive charge migrates from one nitrogen to the other through the conjugated chain of polyaniline.

Excision of uracil from the ends of double stranded DNA by uracil DNA glycosylase and its use in high efficiency cloning of PCR products

We show that uracil DNA glycosylase from E. coli excises uracil residues from the ends of double stranded oligos. This information has allowed us to develop an efficient method of cloning PCR amplified DNA. In this report, we describe use of this method in cloning of E. coli genes for lysyl- and methionyl-tRNA synthetases. Efficiency of cloning by this method was found to be the same as that of subcloning of DNA restriction fragments from one vector to the other vector. Possibilities of using other DNA glycosylases for such applications are discussed.

Expression of microinjected foreign DNA in silkworm,bombyx mori

As a prelude to achieving transgenesis in Bombyx mori, conditions have been established for successful microinjection of cloned foreign genes into the silk worm eggs. A sharpened metallic needle is used to pierce the thick chorion layer of the eggsheil, approaching through a droplet of DNA solution deposited on its surface. The microinjection is carried out within 2-2.5 h after oviposition and the injected eggs show 3-5% hatchability and 80-90% survival. Such larvae continuously expressed the microinjected cloned reporter gene, beta-galactosidase, placed under the control of a constitutively expressed cytoplasmic actin A3 gene promoter from B. mori. The expression is seen in different tissues, viz. the fat body, tracheae and the silk glands, till the late larval instars. The microinjected DNA sequences are retained in the adult G(o) moths.

Inefficient excision of uracil from loop regions of DNA oligomers by E.coli uracil DNA glycosylase

Kinetic parameters for uracil DNA glycosylase (E.coli)-catalysed excision of uracil from DNA oligomers containing dUMP in different structural contexts were determined. Our results show that single-stranded oligonucleotides (unstructured) are used as somewhat better substrates than the double-stranded oligonucleotides. This is mainly because of the favourable V-max value of the enzyme for single-stranded substrates. More interestingly, however, we found that uracil release from loop regions of DNA hairpins is extremely inefficient. The poor efficiency with which uracil is excised from loop regions is a result of both increased K-m and lowered V-max values. This observation may have significant implications in uracil DNA glycosylase-directed repair of DNA segments that can be extruded as hairpins. In addition, these studies are useful in designing oligonucleotides for various applications in DNA research where the use of uracil DNA glycosylase is sought.

Influence of anvil supports and overhangs in dynamic analysis of three point bend testing

Optimum design of dynamic fracture test rigs demands a thorough appreciation of beam vibration under impact. Analyses invariably presume rigid anvils, and neglect overhang effects. The beam response predicted analytically and numerically in this paper highlights the significant role of anvil rigidity and beam overhangs on the impact dynamics of three point bend (3PB) specimens.