LASER RAMAN PROBE OF MOLECULAR STRUCTURE IN LIVING CELLS

分子结构的激光拉曼探针 活细胞。 这一提议的前提是， 双螺旋DNA在本质上是多态的，并且可能存在于 不同的构象。 本研究的目的是 确定DNA构象在调节 基因表达。 我们提出两种方法来解决这个问题。 首先，提出直接检测DNA的构象 在某些活细胞中，它发生在不同区域 染色体或染色体器。 我们建议获得 这些测量值是细胞分裂的函数。 我们寻求 确定是否存在构象之间的差异 正在积极转录的DNA和 转录被抑制。 转录的发生 在某些情况下， 染色体区域，例如染色体的蓬松部分， 唾液腺嗜毛蝇 合子转录是已知的 直到海胆的几次细胞分裂后才被抑制 胚胎 我们将能够使用我们最近开发的激光 拉曼显微镜与这些材料，以检查 DNA构象的区域， 转录的区域和转录明显被 压抑 第二种方法的前提是，如果 DNA以不同的构象存在（如A或Z）， 活细胞这些必须发生在特定的碱基序列。 我们 建议继续我们的设计，合成，纯化，和 微结晶的寡核苷酸，以确定 我们称之为DNA构象密码 任一 寡核苷酸双螺旋双链体存在一定的集合， 它将适应的构象状态取决于它的 碱基序列和环境条件。 这 构象与碱基序列对应 代表了一个可以用来预测构象的密码 在活细胞中给定碱基序列的概率。 我们有 发现它是非常容易获得良好的形成，棱镜，光学 10 - 100数量级的寡核苷酸的完美微晶 微米的边缘。 这些微晶太小了， 衍射或常规的拉曼光谱，但它们产生 使用我们的激光拉曼获得清晰漂亮的拉曼光谱 显微镜 从这些光谱中，DNA构象可以是 明确识别。

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.