IN SITU HYBRIDIZATION VISUALIZED USING BIOTINATED PROBES

这一建议研究了mRNAs的空间组织及其 与细胞结构和功能的关系。蛋白质的分选 到它们在单元内的适当目的地的排序可能会得到帮助 因此蛋白质的合成可以在它们的部位发生 功能的一部分。我们在这项提案中的目标有两个：调查 功能相关的和原位改进的mRNAs的关联 杂交方法使低浓度的mRNA分子 在高分辨率的光学和电子显微镜下可检测到的。为 这些目标中的第一个是，将信使核糖核酸的区隔 对活动鸡胚成纤维细胞的板层进行了研究 成肌细胞。肌动蛋白mRNA已被证明定位于该结构中， 以及肌动蛋白结合蛋白的mRNAs的定位 原肌球蛋白、α-肌动蛋白或肌球蛋白I将被研究以确定 它们与肌动蛋白mRNA的空间分布之间的关系。同样的， 发展肌原纤维为研究肌动蛋白结合提供了一个很好的系统 提供肌节组装的蛋白质。肌球蛋白作为 与肌钙蛋白和结蛋白一样，原肌球蛋白和α-肌动蛋白将 通过数字成像和电子显微镜进行了研究。在细胞内， 一项高分辨率研究将揭示mRNAs如何与细胞 结构以及生理上相关的mRNAs是否附着在这些结构上 同样的结构。对于第二个目标，为了研究mrna 分布检测低浓度、高灵敏度的mRNAs方法 将使用数字成像显微镜开发，银色增强 胶体金，或与寡核苷酸一起原位使用的逆转录酶 底漆。最终，我们希望使用荧光共轭探针 显微注射到细胞内，跟踪体内核酸的运动。 这项提案的目标将提供必要的信息，将 特定基因在细胞内的空间间隔中的表达 从而控制细胞的结构、功能和分化。

This proposal investigates the spatial organization of mRNAs and their relationship to cellular structure and function. The sorting of proteins to their proper destinations within the cell may be assisted by the sorting of their cognate mRNA so that synthesis of proteins can occur at their site of function. Our goals in this proposal are twofold: to investigate the association of mRNAs which are functionally related and to improve in situ hybridization methodology so that low concentration mRNA molecules are detectable at the high resolution of light and electron microscopy. For the first of these goals, the compartmentalization of mRNA will be investigated in the lamella of motile chicken embryo fibroblasts and myoblasts. Actin mRNA has been shown to be localized in this structure, and the localization of the mRNAs for actin-binding proteins such as tropomyosin, alpha-actinin or myosin I will be investigated to determine their relationship to the spatial distribution of actin mRNA. Likewise the developing myofibril presents a good system to investigate actin-binding proteins which provide for the assembly of the sarcomere. The myosins as well as troponins and desmin, tropomyosin and alpha-actinin will be investigated by digital imaging and electron microscopy. Within the cell, a high resolution study will reveal how mRNAs associate with cellular structures and whether physiologically related mRNAs are attached to these same structures. For the second goal, in order to investigate mRNA distribution to detect mRNAs of low concentration, high sensitivity methods will be developed using digital imaging microscopy, silver enhanced colloidal gold, or reverse transcriptase used in situ with oligonucleotide primers. Eventually we wish to use fluorochromeconjugated probes microinjected into cells to follow the movement of nucleic acids in vivo. The goals of this proposal would provide needed information linking the expression of specific genes to spatial compartments within the cell and hence to the control of cell structure, function and differentiation.