Methods are presented to acquire data from analytical ultracentrifugation experiments
by computer using the absorption optical scanning system of the Beckman Model-E
ultracentrifuge. A computer program was written which analyzes sedimentation velocity
experiments by the van Holde - Weischet method and by the second moment method. The van
Holde - Weischet method allows a high resolution analysis of sedimentation velocity data by
eliminating the effects of diffusion on the shape of the moving boundary to provide
sedimentation coefficients for a heterogeneous composition of a sample. The second moment
method obtains the sedimentation coefficient by calculating the second moment point, by which
the sedimentation coefficient is defined. Since it is impractical to manually analyze
sedimentation velocity data by this method, these computer programs make an important
analysis method available to the researcher. Using this computer program, it is now possible
to analyze data to a higher resolution and accuracy than manual analysis of stripchart
recordings would permit. Moreover, the time required for the analysis is greatly reduced. Data
from sedimentation equilibrium experiments are analyzed by x² minimization.
Further, a program was written for the acquisition of data to measure diffusion
coefficients from quasi elastic light scattering experiments with a Langley Ford correlator. The
analysis of autocorrelation spectra from light scattering experiments is performed by the
Levenberg - Marquardt method, which allows fitting of data to nonlinear models. The model
used allows the analysis of multicomponent systems by fitting to a sum of exponentials and a
baseline. Traditional analysis of autocorrelation data by hand was limited to least squares fitting
of the data to a linear model of one component without an optimized baseline, often an
unrealistic approximation of the system. Analysis of autocorrelation data by nonlinear curve
fitting increases both the accuracy and amount of data that can be analyzed.
The development of the PPOL-1 208-n series of plasmids and of the miniplasmid pMX
is described. These plasmids were designed to allow studies of in vitro transcription and
chromatin structure after reconstitution with histones. The plasmids themselves were analyzed
by sedimentation and diffusion studies using the computer programs. Sedimentation data is
presented which suggests a new method for rapid estimation of S₀ (the sedimentation
coefficient at zero concentration) for molecules which show a concentration dependency of the
sedimentation coefficient. This is accomplished by linearly extrapolating van Holde Weischet
distributions to zero concentration. Manual analysis of sedimentation velocity experiments to
determine nonideality contributions required several experiments, computer analysis can provide
this information in a single experiment due to the increased resolution of the method.
Diffusion data for this plasmid DNA is used to demonstrate the feasibility of the
multicomponent analysis presented here. Also, sedimentation measurements were carried out
on reconstituted chromatin and on the effects of ethidium bromide on reconstituted chromatin.
The programs were used to demonstrate significant changes in chromatin structure upon
ethidium bromide binding. These changes involved the reduction of S of reconstituted plasmids
upon addition of ethidium bromide as well as a reduction of heterogeneity of the sample. The
data indicates the possibility of a forced exchange of nucleosomes between plasmids, as well
as conformational changes in the chromatin structure.