The long term objective is to provide new flow cytometric (FCM) methods for applying multiple DNA base-specific fluorochromes to study cell cycle-related changes in chromatin organization. The approach to achieving this goal requires (a) detailed spectral studies on the various DNA fluorochromes bound to DNA and chromatin, (b) optimizing their FCM application as single or multiple cell staining agents, (c) correlating the differences in accessibility of DNA to different fluorochromes with changes in chromatin structure in viable and fixed cells from cultures of highly, synchronized cells and differentiating cells, and relating the potential changes in accessibility of DNA to different fluorochromes to the cell cycle-related modifications in the nucleoprotein composition in chromatin.
Specific Aim I will be to obtain information from spectrofluorometric studies on DNA-specific dyes relating to energy transfer, extent of fluorescence quenching and other dye-dye interactions that will provide the rationale and feasibility for developing multifluorochrome cell staining techniques and for interpretating the fluorescence patterns obtained from subsequent FCM studies on stained cell populations. Analysis on single and multiple dyes bound to DNA in solution and to chromatin in cells will be made to determine the effects of DNA-dye binding and dye-dye interactions on the fluorescence intensity and the spectral features of dyes with different DNA-dye binding and different spectral characteristics.
Specific Aim 2 is to develop and test the accuracy of single versus multiple DNA fluorochrome labeling protocols in a three-laser excitation flow system that allows sequential excitation and resolution of blue, green, and red fluorescence. A novel Fourier transform FCM will also be used to analyze the emission spectra of each dye and resolve the fluorescence of dyes having overlapping emission with peaks close as 20 nm.
Specific Aim 3 is to assess and correlate differential changes in DNA accessibility to base-specific fluorochromes with the cell cycle-related rearrangements in chromatin orientation of highly synchronized CHO and human diploid cell populations during transition into the various phases of the cell cycle. Similar analyses will be performed on DMSO-induced, differentiating HL60 cells. Potential changes in accessibility of DNA to different fluorochromes will be correlated with data obtained from biochemical studies on the modification of the nucleoproteins in chromatin of cells in various phases of the cell cycle. These correlations will provide a better understanding of the molecular basis for the cell cycle-related alterations in the cytochemical patterns of accessibility to DNA. This is a R24 proposal that will directly support the users of the National Flow Cytometry and Sorting Research Resource (NTCR) at Los Alamos.
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