The present work is primarily concerned with the measurement, by several different methods (using X-ray and static light scattering experiments or dynamic light scattering experiments), of the elasticity of bilayers made up of n-dodecyl pentaethylene glycol monoether (C12E5) and hexanol molecules. The membrane bending elasticity modulus, κ, has been measured for both the Lα (lamellar) and the L3 (“sponge”) phase of the C12E5/hexanol/water system. For the lamellar phase, κ was measured using both the excess area and the dynamics of the fluctuations, while for the sponge phase κ was measured using only the excess area. The values of κ measured seem to be of the same order (approximately k B T) for both the lamellar phase and the sponge phase, although the molar ratio of hexanol to C12E5 is 4 times bigger in the sponge phase. The effect of temperature on κ was also studied: the slight increase in κ found for this system with temperature seems to be related to a decreasing polar head area of the C12E5 molecules. For the sponge phase, κ cannot be measured from the dynamics of the fluctuations; however, some results can be extracted from the dynamic light scattering experiments. The most interesting one was that the collective diffusion coefficient D is proportional to the membrane volume fraction, φm, and is a function of the membrane elastic constants, κ and κ, as well as of the thermal energy k B T, leading to an estimate of κ.
Molecular combing is a powerful procedure for aligning a large array of DNA molecules onto a surface. This technique usually leads to an overstretching of about 150% of the molecules' contour length. By changing the magnitude of capillary forces during the combing process, we were able to reduce the relative extension of the DNA molecules. Thus we achieved combing of T7 DNA with an extension close to its molecule contour length. We checked the ability of combed DNA to interact with DNA binding proteins. Using the T7 bacteriophage transcription system, we investigated the transcription activity of RNA polymerase on combed DNA by direct visualization of newly synthesized fluorescent RNAs. Our experiments show that no transcription activity occurs on overstretched DNA molecules, whereas we observe a transcription activity for nonoverstretched molecules. This activity is observed both in multiple initiation experiments and for one immobilized T7 RNA polymerase per promoter. These results open possibilities for the study of single enzyme actions on combed DNA by optical methods. T ranscription is a fundamental process in gene expression that allows the regulation of cellular adaptation and differentiation. This function is carried out by RNA polymerases (RNAPs) that produce an RNA copy of a given DNA strand (1). The interaction between the DNA and the RNAP is complex because RNAP must recognize a promoter, a sequence-specific region of doublestranded DNA before polymerization. After isomerization of the nucleoprotein complex, resulting in local melting of the double helix, the enzyme transcribes DNA into RNA following the doublestranded DNA until it reaches a terminator (2). Motion is therefore part of the intrinsic activity of RNAP (3). Having the possibility to follow and visualize the movement of an RNAP on the DNA template will open a new area of investigation for understanding the mechanisms of transcription and its regulation.Our goal is to detect the activity of RNAP along a DNA molecule during the transcription process. Several groups have reported single molecule investigation of the transcription process (4-9). Because we use fluorescence microscopy techniques, we cannot use DNA in its normal aqueous solution state, which is a Brownian fluctuating coil. Therefore the first steps we have to perform are to stretch the DNA molecule to avoid conformational fluctuations and to hinder its Brownian motion. Several techniques permit researchers to obtain both immobilized and stretched DNA molecules. The techniques developed so far are micromanipulation with optical or magnetic tweezers (10-14), elongation in a flow (15) or in an electric field (16), and molecular combing (17). Although micromanipulation has provided much insight it has an inherent disadvantage because the observation is limited to one molecule at a time and large statistics are difficult to obtain. By contrast, molecular combing is of particular interest because it allows direct observation of a large array of immobilized and aligned DNA by fluores...
We investigate experimentally, using quasi-elastic light scattering the dynamical properties of highly swollen lamellar and "sponge" phases. In our system the structural sizes smectic period for lamellar phases or characteristic cell size for "sponge" phases may be changed continuously by adding solvent up to scales of the order of optical wavelengths, i.e.several thousand I. We observe the usual hydrodynamic behaviour, with monoexponential relaxation in time and q~s caling in reciprocal space when working with moderately dilute systems. At higher dilutions, the behaviour is no longer hydrodynamic: a q~u niversal scaling is observed for both lamellar and "sponge" samples, with stretched-exponential relaxation in time and universal exponents for unor~ented lamellar or "sponge" samples. The undulation mode is identified in our data on oriented lamellar phases, both in its hydrodynamic limit and beyond, which leads to a measurement of the bilayer bending modulus t~. A tentative interpretation is given for our data on "sponge" phases, also leading to an estimate for t~.
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