IN SITU HYBRIDIZATION VISUALIZED USING BIOTINATED PROBES

本提案的目的是继续发展和应用 原位杂交方法用于了解 组织和表达的核酸序列内 cell. 原位杂交，特别是与非- 同位素检测，是一个强大的工具，用于描述分子 单细胞生物学 随着这项技术的进步， 重要的分支跨越基础和临床科学。 在过去一年中建立了坚实的方法论基础， 赠款期间，我们在一个强大的地位，申请在原地 以新的方式进行杂交，以获得 具有根本性和深远性。 异常温柔， 我们开发的灵敏杂交方法， 使我们能够研究细胞内分布的特定 mRNA，导致发现不同基因的mRNA 细胞骨架蛋白表现出特定的和不同的模式， 本地化 在这里提出的工作中，我们将扩展这个 mRNA定位分析包括多种细胞类型， 以及其他与细胞凋亡相关和非细胞凋亡相关的mRNA， 细胞骨架蛋白 此外，我们还将聘请多名 包括光学和电子显微镜的方法来分析 mRNA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， 在间期核内或染色体上。 这种方法在 与图像处理相结合，现在可以研究 基因在体内的功能状态 间期染色质，使用细胞核内的有序阵列， 骨骼肌纤维作为模型系统。 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， 转录本的产生在细胞核内定位 和mRNA在细胞质中的翻译。 最后，具体 杂交和非同位素检测方法， 对多个领域产生了重大影响， 临床诊断，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.