Curved DNA molecules migrate anomalously slowly in free solution

The electrophoretic mobility of a curved DNA restriction fragment taken from the VP1 gene in the SV40 minichromosome has been measured in polyacrylamide gels and free solution, using capillary electrophoresis. The 199 bp restriction fragment has an apparent bend angle of 46 ± 2° located at SV40 sequence position 1922 ± 2 bp [Lu Y.J., Weers B.D. and Stellwagen N. C. (2005) Biophys. J., 88, 1191–1206]. The ‘curvature module’ surrounding the apparent bend center contains five unevenly spaced A- and T-tracts, which are responsible for the observed curvature. The parent 199 bp fragment and sequence mutants containing at least one A-tract in the curvature module migrate anomalously slowly in free solution, as well as in polyacrylamide gels. Hence, the anomalously slow mobilities observed for curved DNA molecules in polyacrylamide gels are due in part to their anomalously slow mobilities in free solution. Analysis of the gel and free solution mobility decrements indicates that each A- or T-tract contributes independently, but not equally, to the curvature of the 199 bp fragment and its A-tract mutants. The relative contribution of each A- or T-tract to the observed curvature depends on its spacing with respect to the first A-tract in the curvature module

H NMR Studies of Eukaryotic Cytochrome c

H NMR resonance assignments in the spectra of horse, tuna, Neurmpora crassa and Candida krusei cyto-chromes c are described. Assignments have been made using NMR double-resonance techniques in conjunction with electron-exchange experiments, spectral comparison of related proteins, and consideration of the X-ray structure of tuna cytochrome c. Resonances arising from 11 residues of horse cytochrome c have been assigned

Two-State Migration of DNA in a structured Microchannel

DNA migration in topologically structured microchannels with periodic cavities is investigated experimentally and with Brownian dynamics simulations of a simple bead-spring model. The results are in very good agreement with one another. In particular, the experimentally observed migration order of Lambda- and T2-DNA molecules is reproduced by the simulations. The simulation data indicate that the mobility may depend on the chain length in a nonmonotonic way at high electric fields. This is found to be the signature of a nonequilibrium phase transition between two different migration states, a slow one and a fast one, which can also be observed experimentally under appropriate conditions.Comment: Revised edition corresponding to the comments by the referees, submitted to Physical Review

Modeling DNA beacons at the mesoscopic scale

We report model calculations on DNA single strands which describe the equilibrium dynamics and kinetics of hairpin formation and melting. Modeling is at the level of single bases. Strand rigidity is described in terms of simple polymer models; alternative calculations performed using the freely rotating chain and the discrete Kratky-Porod models are reported. Stem formation is modeled according to the Peyrard-Bishop-Dauxois Hamiltonian. The kinetics of opening and closing is described in terms of a diffusion-controlled motion in an effective free energy landscape. Melting profiles, dependence of melting temperature on loop length, and kinetic time scales are in semiquantitative agreement with experimental data obtained from fluorescent DNA beacons forming poly(T) loops. Variation in strand rigidity is not sufficient to account for the large activation enthalpy of closing and the strong loop length dependence observed in hairpins forming poly(A) loops. Implications for modeling single strands of DNA or RNA are discussed.Comment: 15 pages, 17 figures, submitted to Eur. J. Phys.

Liminality of NHS research ethics committees: navigating participant protection and research promotion across regulatory spaces

NHS research ethics committees (RECs) serve as the gatekeepers of health research involving human participants. They have the power to decide, through a regulatory ‘event licensing’ system, whether or not any given proposed research study is ethical and therefore appropriate to undertake. RECs have several regulatory functions. Their primary function has been to protect the interests of research participants and minimise risk of harm to them. Yet RECs, and other actors connected to them, also provide stewardship for the promotion of ethical and socially valuable research. While this latter function traditionally has been seen as secondary, the ‘function hierarchy’ is increasingly blurred in regulation. Regulatory bodies charged with managing RECs now emphasise that the functions of RECs are to both protect the interests of research participants, and also promote ethical research that is of potential benefit to participants, science, and society. Though the UK has held in some of its previous regulations (broadly defined) that RECs equally function to facilitate (ethical) health research, I argue that the ‘research promotionist’ ideology has moved ‘up the ladder’ in the regulation of RECs and in the regulation of health research, all the way to implementation in law, specifically in the Care Act 2014, and in the regulatory bodies charged with overseeing health research, namely the Health Research Authority. This thesis therefore asks: what impact does this ostensibly twinned regulatory objective then have on the substantive and procedural workings of RECs? I invoke a novel ‘anthropology of regulation’ as an original methodological contribution, which enables me to study empirically the nature of regulation and the experiences of actors within a regulatory space (or spaces), and the ways in which they themselves are affected by regulation. Anthropology of regulation structures my overall empirical inquiry to query how RECs, with a classic primary mandate to protect research participants, now interact with regulatory bodies charged with promoting health research and reducing perceived regulatory barriers. I further query what this changing environment might do to the bond of research and ethics as seen through REC processes of ethical deliberation and decision-making, by invoking the original concept of ‘regulatory stewardship’. I argue that regulatory stewardship is a critical, but hitherto invisible, component of health research regulation, and requires fuller recognition and better integration into the effective functioning of regulatory oversight of research involving human participants

Modeling ssDNA electrophoretic migration with band broadening in an entangled or cross-linked network

We use a coarse-grained model proposed by Graham and Larson based on the temporary network model by Schieber et al.. [1] to simulate the electrophoretic motion of ssDNA and corresponding band broadening due to dispersion. With dimensionless numbers reflecting the experimental physical properties, we are able to simulate ssDNA behavior under weak to moderate electric field strengths for chains with 8–50 entanglements per chain (∼1000–8500 14base pairs), and model smoothly the transition from reptation to oriented reptation. These results are fitted with an interpolation equation, which allows the user to calculate dimensionless mobilities easily from input parameters characterizing the gel matrix, DNA molecules, and field strengths. Dimensionless peak widths are predicted from mobility fluctuations using the central limit theorem and the assumption that the mobility fluctuations are Gaussian. Using results from previous studies of ssDNA physical properties (effective charge Ξq and Kuhn step length b K ) and sieving matrix properties (pore size or tube diameter a ), we give scaling factors to convert the dimensionless values to “real” experimental values, including the mobility, migration distance, and time. We find that the interpolation equation fits well the experimental data of ssDNA mobilities and peak widths, supporting the validity of the coarse-grained model. The model does not account for constraint release and hernia formation, and assumes that the sieving network is a homogeneous microstructure with no temperature gradients and no peak width due to injection. These assumptions can be relaxed in future work for more accurate prediction.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/56125/1/2783_ftp.pd