LASER RAMAN PROBE OF MOLECULAR STRUCTURE IN LIVING CELLS

活细胞分子结构的激光拉曼探针

• 批准号: 3283890
• 负责人: WARNER L PETICOLAS
• 金额: $ 12.37万
• 依托单位: UNIVERSITY OF OREGON
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-08-01 至 1993-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3283890
• 关键词: Chironomidae; DNA; Drosophilidae; Raman spectrometry; chromosomes; conformation; crystallization; genetic transcription; lasers; nucleic acid chemical synthesis; nucleic acid sequence; nucleic acid structure; salivary glands; saltwater environment; sea urchins; ultraviolet microscopy

LASER RAMAN PROBE OF MOLECULAR STRUCTURES IN LIVING CELLS. This proposal begins with the premise that double helical DNA is polymorphic in nature and may exist in different conformations. The purpose of this research is to determine the role played by DNA conformation in regulating gene expression. We propose two approaches to this problem. First, it is proposed to examine directly the conformation of DNA in certain living cells as it occurs in different regions in a chromosome or chromosomal apparatus. We propose to obtain these measurements as a function of cell division. We seek to determine if there is a difference between the conformation of DNA that is actively being transcribed and DNA in which transcription is repressed. The occurrence of transcription has already been determined as a function of position in certain chromosomal regions such as in the puffs of the chromosomes from the salivary gland Drosphilia. Zygotic transcription is known to be suppressed until after several cell divisions in sea urchin embryos. we will be able to use our recently developed laser Raman microscope with these materials to examine the conformation of DNA in regions that are actively being transcribed and regions where transcription is apparently being repressed. The second approach begins with the premise that if DNA exists in different conformations (such as the A or the Z) in living cells these must occur in specific base sequences. We propose to continue our design, synthesis, purification, and microcrystallization of oligonucleotides in order to determine what we will call the conformational code of DNA. To any oligonucleotide double helical duplex there exists a certain set of conformational states to which it will adapt depending upon its base sequence and the environmental conditions. This correspondence between conformation and base sequence represents a code which one can use to predict the conformational probability of a given base sequence in a living cell. We have found it is very easy to obtain well formed, prismatic, optically perfect microcrystals of oligonucleotides of the order of 10 to 100 microns on an edge. These microcrystals are too small for X-ray diffraction or conventional Raman Spectroscopy, but they produce sharp, beautiful Raman spectra using our laser Raman microscope. From these spectra the DNA conformation can be unambiguously identified.

分子结构的激光拉曼探头 活细胞。这项提议的前提是 双螺旋DNA在自然界中是多态的，可能存在于 不同的构象。这项研究的目的是 确定DNA构象在调控中的作用 基因表达。我们提出了两种方法来解决这个问题。 首先，建议直接检查DNA的构象 在某些活细胞中，因为它出现在 染色体或染色体器官。我们建议获得 这些测量作为细胞分裂的函数。我们寻求 确定分子的构象是否存在差异 正在积极转录的DNA和其中的DNA 转录被抑制。转录的发生有 已经被确定为某一位置的函数 染色体区域，如在染色体的泡状物中 来自唾液腺嗜水症。合子转录是已知的 在海胆几次细胞分裂后才被抑制 胚胎。我们将能够使用我们最新研制的激光器 用拉曼显微镜对这些材料进行检测 DNA在活跃区域的构象 转录的和转录明显是 被压抑。第二种方法以这样的前提开始：如果 DNA以不同的构象(如A或Z)存在于 活细胞这些必须出现在特定的碱基序列中。我们 建议继续我们的设计、合成、提纯和 寡聚核苷酸的微晶化研究 我们将称之为DNA的构象密码。给任何人 寡核苷酸双螺旋双链存在一定的一套 它将适应的构象状态取决于其 碱基序列和环境条件。这 构象与碱基序列的对应关系 表示可用于预测构象的代码 活细胞中给定碱基序列的概率。我们有 发现很容易获得成形良好、棱柱状、光学的 10-100量级寡核苷酸的完美微晶 边缘上的微米。这些微晶对X射线来说太小了。 衍射或传统的拉曼光谱，但它们产生 锐利美丽的拉曼光谱使用我们的激光拉曼 显微镜。根据这些光谱，DNA的构象可以是 毫不含糊地确定。