THREE DIMENSIONAL RECONSTRUCTIONS OF ARCHAEA

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。所有已知生物的系统发育树包括三个不同的分支：真核生物、细菌和古生菌。古生菌分支直到最近才被认为是一个在系统发育上与细菌不同的连贯的类群。我们对原核细胞(古生菌和细菌)的精细结构的了解明显少于我们对它们的生化和分子组织的了解。这主要是由于保存方法的限制，在某种程度上，也是由于仪器设备的限制。现代快速冷冻和冷冻替代技术的进步克服了其中的一些问题，使一些原核生物得到了优雅的保存。由于分子研究表明，古生物与真核生物的共同点比细菌更多，因此在转录、翻译和其他DNA相关处理机制方面，这些生物的形态可能会产生丰富的生物信息。通过连续切片断层扫描，捕捉到了高压冷冻、冷冻替代的硫磺的全细胞图像。作为对照，沙门氏菌的全细胞图像是以相同的方式制备的。整个细胞断层图像允许列举Sulfolobus中的几种不同的结构，其中至少有一种结构根据细胞生长所在的培养基中是否存在葡萄糖而变化。DNA保存以前是一个问题，现在在冷冻之前将蔗糖聚合物Ficoll-70添加到生长介质中可以很好地保存DNA。Lowicryl包埋的Sulfolobus已被成功地探测到DNA，并通过断层核糖体排斥模型进行了验证。明年将继续研究Sulfolobus和沙门氏菌的核体结构。未来决定本金利率的一步是分段的劳动密集型性质。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The phylogenetic tree of all known organisms comprises three distinct branches: Eucarya, Bacteria and Archaea. The Archaeal branch was only recently recognized as a coherent group that is phylogenetically distinct from Bacteria. Our knowledge of the fine structure of prokaryotic cells (Archaea and Bacteria) is significantly less than what we know about their biochemical and molecular organization. This is due primarily to limitations in preservation methods and, to some extent, in instrumentation. Advances in modern techniques of rapid freezing and freeze-substitution have overcome some of these problems, yielding elegant preservation of some prokaryotic organisms. Since molecular studies suggest that Archaea have more in common with Eukaryotes than with Bacteria, in terms of transcription, translation and other DNA related processing mechanisms, the morphology of these organisms may yield a wealth of biological information. Whole cell images of high-pressure frozen, freeze-substituted Sulfolobus solfataricus have been captured via serial section tomography. As a control, whole cell images of the bacterium Salmonella have been prepared in an identical manner. The whole cell tomograms have allowed the enumeration of several different structures in Sulfolobus, at least one of which varies according to the presence or absence of glucose in the medium in which the cells are grown. DNA preservation, previously a problem, is currently quite good with the addition of the sucrose polymer Ficoll-70 to the growth medium just prior to freezing. Lowicryl embedded Sulfolobus has been successfully probed for DNA and verified by tomographic ribosome exclusion models. Work will continue on the characterization of nuclear body structure of Sulfolobus and Salmonella during the next year. The principal rate determining step going forward is the labor intensive nature of segmentation.