FLOW CYTOMETRIC ANALYSIS OF MULTIPLE DNA FLUOROCHROMES

DNA结合差异的流式细胞仪(FCM)分析 多个碱基特定的荧光染料可以提供一种独特的方法 检测小鼠染色质天然状态的代谢变化 单个细胞对细胞生理状态的变化作出反应 人口。这一原理是基于这样一个前提，即微妙的 染色质组织中的重排涉及DNA的调制- 核蛋白相互作用导致DNA不成比例的变化 可获得碱基特定的荧光染料，可以有效地 被FCM检测到。这种新技术为以下方面提供了明显的优势 临床、结构和细胞生物学研究包括：(A) DNA荧光染料在相对分子水平下的细胞或染色体的应用 非扰动条件，使染色质保持接近天然状态 构型，(B)快速FCM分析，以关联相对 细胞内多个荧光染料结合的变化和(C)要求 一小部分细胞。我们的长期目标是开发这种新的FCM 提高细胞周期相关检测灵敏度的方法 染色质组织和识别个体的变化 染色体。实现这一目标将需要(1)详细的频谱 精选DNA特异性荧光染料与DNA和DNA结合的研究 染色质，(2)利用独特的FCM光谱和常规 用于量化结合特征的相对变化的仪器 细胞和染色体中多个DNA对染色质的荧光染色，(3) 应用新的计算机分析方法，可以更好地关联绑定 由多个荧光染料组成。具体目的1是执行荧光分光光度法 DNA特异染料的研究，包括新型、超感光染料 荧光染料，例如TOTO和YOYO，以确定荧光寿命， 能量转移，以及其他染料相互作用，将提供 开发多色荧光标记技术的基本原理。特定的 目标2是评估独特分析方法的敏感性。 利用最近开发的FCM仪器，包括我们的阶段 灵敏检测FCM、傅里叶变换FCM以及我们的 多参数三激光FCM系统与双激光、染色体 流式细胞仪分析。具体目标3是应用新的计算机分析方法， 包括IDYLK分析程序、聚类分析、差异分析 本实验室开发的荧光和比率分析 DNA可及性的差异变化与碱基特异性 荧光染料与核蛋白的细胞周期相关修饰 为了更好地了解分子基础， DNA可及性的细胞化学模式的改变。

Flow cytometric (FCM) analyses of the differential in DNA-binding by multiple base-specific fluorochromes can provide a unique approach for detecting metabolic changes in the native state of chromatin in individual cells responding to changes in the physiological state of the population. This rationale is based on the premise that subtle rearrangements in chromatin organization involve modulations in DNA- nucleoprotein interactions resulting in disproportionate changes in DNA accessibility to base-specific fluorochromes that can be efficiently detected by FCM. Such new technology provides distinct advantages for clinical, structural, and cell biology studies including, (a) the application of DNA fluorochromes to cells or chromosomes under relatively non-perturbing conditions so that chromatin remains close to the native configuration, (b) rapid FCM analyses for correlating the relative changes in binding by multiple fluorochromes in cells and (c) requirement of small numbers of cells. The long term goal is to develop this new FCM approach to increase the sensitivity for detecting cell cycle-related changes in chromatin organization and for identifying individual chromosomes. Achieving this goal will require (1) detailed spectral studies on selected, DNA based-specific fluorochromes bound to DNA and chromatin, (2) utilizing unique FCM spectroscopic and conventional instrumentation to quantitate relative changes in binding features of multiple DNA fluorochromes to chromatin in cells and chromosomes, (3) applying new computer analyses methods that can better correlate binding by multiple fluorochromes. Specific Aim 1 is perform spectrofluorometric studies on DNA-specific dyes, including new, ultrasensitive fluorochromes, such as TOTO and YOYO, to determine fluorescence lifetime, energy transfer, and other dye-dye interactions that will provide the rationale for developing multifluorochrome labeling techniques. Specific Aim 2 is to evaluate the sensitivity of unique analytical approaches utilizing recently developed FCM instrumentation, including our phase sensitive detection FCM, the Fourier transform FCM, as well as our multiparameter three laser FCM system and the two laser, chromosome analysis FCM. Specific Aim 3 is to apply new computer analysis methods, including the IDYLK analysis programs, cluster analysis, differential fluorescence and ratio analyses as developed in this laboratory to correlate differential changes in DNA accessibility to base-specific fluorochromes with the cell cycle-related modifications of nucleoproteins to obtain a better understanding of the molecular basis for the alterations in the cytochemical patterns of DNA accessibility.