FLOW CYTOMETRIC ANALYSIS OF MULTIPLE DNA FLUOROCHROMES

长期目标是提供新的流式细胞仪(FCM)方法 多种DNA碱基特异性荧光染料在细胞研究中的应用 染色质组织中与周期相关的变化。实现以下目标的方法 这一目标需要(A)对不同的DNA进行详细的光谱研究 与DNA和染色质结合的荧光染料，(B)优化其FCM 作为单细胞或多细胞着色剂的应用，(C)将 DNA对不同荧光染料的可及性随变化的差异 在染色质结构中，活的和固定的细胞来自高度， 同步化细胞和分化细胞，并将潜力 细胞对不同荧光染料的DNA可及性的变化 染色质核蛋白组成中与循环有关的修饰。 具体目标I将是从荧光分光光度法获得信息 与能量转移有关的DNA特异染料的研究，范围 荧光猝灭和其他染料-染料相互作用将提供 发展多荧光细胞染色的理论基础和可行性 以及用于解释从以下获得的荧光图案的技术 随后对染色的细胞群体进行了流式细胞仪研究。对单项和单项的分析 多种染料在溶液中与DNA结合，在细胞中与染色质结合 用于确定DNA与染料结合和染料相互作用的影响 染料的荧光强度和光谱特征 不同的DNA-染料结合和不同的光谱特性。特定的 目标2是开发和测试单个DNA与多个DNA的准确性 三激光激发流动系统中的荧光标记方案 它允许顺序激发和分辨率蓝色、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及对染料荧光的解析 具有重叠发射，峰接近20 nm。具体目标3是 评估和关联DNA可获得性的差异变化 碱基特异性荧光蛋白与细胞周期相关重排 高度同步化的CHO和人二倍体细胞的染色质定位 在过渡到细胞周期的不同阶段期间的种群。 类似的分析将在DMSO诱导的、分化的HL60上进行 细胞。DNA对不同物种的可及性的潜在变化 荧光染料将与从生物化学获得的数据相关联 大鼠细胞染色质核蛋白修饰的研究 细胞周期的不同阶段。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 在接近DNA的细胞化学模式中。这是一辆R24 将直接支持National Flow用户的建议 洛斯阿拉莫斯的细胞学和分选研究资源(NTCR)。

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.