2,193 research outputs found
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
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