FLOW CYTOMETRIC ANALYSIS OF MULTIPLE DNA FLUOROCHROMES

长期目标是提供新的流式细胞术（FCM）方法， 应用多种DNA碱基特异性荧光染料研究细胞 染色质组织的周期相关变化。 实现的方法 这一目标需要（a）对各种DNA进行详细的光谱研究 与DNA和染色质结合的荧光染料，（B）优化它们的FCM 作为单一或多个细胞染色剂的应用，（c）将所述细胞染色剂与所述细胞染色剂相关联， DNA对不同荧光染料的可及性差异， 在染色质结构中， 同步细胞和分化细胞，并将潜在的 DNA对细胞中不同荧光染料可及性的变化 染色质中核蛋白组成的周期相关修饰。 具体目标I将是从荧光分光光度计获得信息， 与能量转移有关的DNA特异性染料的研究， 荧光猝灭和其他染料-染料相互作用， 建立多荧光染料细胞染色法的原理和可行性 技术，并解释从 随后对染色的细胞群进行FCM研究。 分析了单一和 与溶液中的DNA和细胞中的染色质结合的多种染料将被 用于确定DNA-染料结合和染料-染料相互作用的影响 对染料的荧光强度和光谱特性的影响 不同的DNA-染料结合和不同的光谱特性。 具体 目的2是开发和测试单个与多个DNA的准确性 三激光激发流动系统中的荧光染料标记方案 允许顺序激发和分辨蓝色、绿色和红色 荧光。 一种新的傅里叶变换FCM也将被用来分析 每种染料的发射光谱并分辨染料的荧光 具有峰值接近20 nm的重叠发射。 具体目标3是 评估和关联DNA可及性的差异变化， 碱基特异性荧光染料与细胞周期相关的重排， 高度同步化CHO和人二倍体细胞的染色质定向 在细胞周期的各个阶段的过渡过程中的群体。 将对DMSO诱导的分化HL 60进行类似的分析。 细胞 DNA对不同DNA的可及性的潜在变化 荧光染料将与从生物化学中获得的数据相关联。 细胞核蛋白修饰的研究 细胞周期的各个阶段。 这些相关性将提供更好的 了解细胞周期相关改变的分子基础 DNA的细胞化学模式。 这是R24 该提案将直接支持国家流程的用户 细胞计数和分选研究资源（NTCR）在洛斯阿拉莫斯。

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.