The work outlined in this proposal includes the building of a differential polarization microscope and the utilization of circular intensity differential scattering (CIDS) methods to study the long-range chiral organization of chromatin in-vivo. During the last three years our laboratory h as developed the technique of CIDS, that is, the preferential scattering of circular polarizations of the light by chiral aggregates. We have shown the sensitivity of this technique to detect long-range chiral structures. One of the by-products of our research has been the development of differential polarization imaging. In this technique images of an object are made through a microscope using two different incident polarizations. The resulting images are subtracted to generate the linear dichroism image of the object (if linear polarization wree used) or the CD/CIDS image if right and left circular polarizations were utilized. The technique allows to spatially resolve in-situ the spectroscopic properties of the object. We are currently developing a new imaging digitization technique that will make possible the generation of CD/CIDS images, not possible previously with the sensitivity of linear arrays. With this microscope we will study the condensation and coiling of DNA in chromosomes. The physical characterization of the higher level organization of chromosomes is essential to understand genetic regulation in eukaryotic systems. Gene regulation has been shown to be related to macroscopic changes in the chromosomes, as visualized by changes in the banding patterns. These structural changes have not been well characterized in-vivo, due to the lack of a noninvasive technique that reports detailed structural information. Differential polarization microscopy, as proposed in this grant, will allow, for the first time, the monitoring of the folding of DNA in chromatin as a function of the cell cycle in-vivo. Such information might also have immediate value for diagnostic use, since it might allow the development of fingerprinting of transformed cells. These studies will be complemented by CD and CIDS measurements of synchronized cell populations and suspensions of mitotic chromosomes. In addition, we will characterize the thermodynamics and kinetics of the condensation process, through parallel studies of in-vitro condensation of DNA.
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