THREE DIMENSIONAL RECONSTRUCTIONS OF ARCHAEA

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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。前几年进行的工作导致了样品制备方案的产生，这些方案通常会产生这两种生物的非常高质量的全细胞断层图像。Robertson博士开发的定制软件自动分割沙门氏菌和Sulfolobus中染有重金属(核糖体、细胞膜等)的主要生物分子的断层图像堆栈。自动分割软件提供了在生物体对各种代谢状态做出反应时计数这些生物分子的方法，如热休克、氧化应激(暴露于过氧化氢)或生长介质(如糖/无糖)的变化。自动分割软件还生成细胞的结构生物信息学模型，可用于电子实验。断层扫描工作与Lowicryl包埋Sulfolobus的免疫EM相辅相成，成功地探测到了DNA。

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. Work conducted in previous years has resulted in sample preparation protocols that routinely yield very high quality full-cell tomograms of these two organisms. Custom software created by Dr. Robertson automatically segments the tomographic image stacks for the major biomolecules which stain with heavy metals, (ribosomes, cell membrane, etc) in Salmonella and Sulfolobus. The automatic segmentation software provides the means to enumerate these biomolecules as the organisms respond to a variety of metabolic states such as heat shock, oxidative stress (exposure to hydrogen peroxide), or changes in growth medium (eg sugar/no sugar). The auto-segmentation software also generates structural bioinformatic models of the cells which may be used for in silico experimentation. The tomographic work has been complemented with immuno-EM of Lowicryl embedded Sulfolobus, has been successfully probed for DNA.