IN SITU HYBRIDIZATION VISUALIZED USING BIOTINATED PROBES

这项建议的目标是继续开发和应用 原位杂交方法学用于了解 细胞内核酸序列的组织和表达 手机。原位杂交，特别是与非 同位素检测，是描述分子的有力工具 单个细胞的生物体。由于这项技术的进步， 跨越基础科学和临床科学的重要分支。 在过去的几年中建立了坚实的方法论基础 在授权期内，我们在现场申请方面处于有利地位 以新的方式获得一种生物信息的杂交 本质上是根本的、深远的。异乎寻常的温柔和 我们开发的灵敏的杂交方法已经 使我们能够调查特定的细胞内分布 MRNAs，导致发现不同的mRNAs 细胞骨架蛋白表现出特定和独特的模式 本地化。在这里提出的工作中，我们将扩展这一点 包括多种细胞类型在内的mRNA定位分析 以及其他与细胞骨架相关和非细胞骨架相关的mRNA 细胞骨架蛋白。此外，我们还将雇佣几名 包括光学显微镜和电子显微镜在内的分析方法 信使核糖核酸与细胞骨架结合的机制。 此外，我们实验室的最新发展使我们有可能 以高分辨率和高效率检测单拷贝序列 在间期核内或在染色体上。此方法论在 现在与图像处理相结合使研究成为可能 基因在其功能状态下的组织 间期染色质，使用有序的核阵列 骨骼肌纤维作为一个模型系统。因此，我们可以调查 基因表达的分子细胞学作为一个连续体 核内转录产物的产生与定位 以及细胞质中mRNA的翻译。最后，特别是 杂交和非同位素检测方法学我们继续 进步对几个领域都有重大影响，比如 临床诊断学、人类基因图谱(产前诊断)和 病毒学。

The objective of this proposal is to continue to develop and apply in situ hybridization methodology for an understanding of the organization and expression of nucleic acid sequences within the cell. In situ hybridization, particularly in conjunction with non- isotopic detection, is a powerful tool for describing the molecular biolgoy of a single cell. As such advances in this technology have important ramifications spanning both basic and clinical sciences. Having built a firm methodological foundation during the last grant period, we are in a strong position for applying in situ hybridization in new ways to obtain biological information of a fundamental and far-reaching nature. The unusually gentle and sensitive hybridization methodology we have developed has allowed us to investigate the intracellular distribution of specific mRNAs, leading to the discovery that mRNAs for different cytoskeletal proteins exhibit specific and distinct patterns of localization. In the work proposed here we will extend this analysis of mRNA localization to include a variety of cell types as well as other mRNAs for cytoskeletal-associated and non- cytoskeletal proteins. Moreover, we will employ several approaches including light and electron microscopy to analyze the mechanism of mRNA association with the cytoskeleton. Furthermore, recent developments in our lab make it possible to detect with high resolution and efficiency, a single copy sequence within interphase nuclei or on chromosomes. This methodology in conjunction with image processing now makes it possible to study the organization of genes in their functional state within interphase chromatin, using the ordered array of nuclei within the skeletal myofibre as a model system. Hence, we can investigate the molecular cytology of gene expression as a continuum from the production of transcripts within the nucleus to localization and translation of mRNA in the cytoplasm. Finally, the particular hybridization and non-isotopic detection methodology we continue to advance has significant impact on several fields as diverse as clinical diagnostics, human gene mapping (prenatal diagnosis), and virology.