THREE DIMENSIONAL RECONSTRUCTIONS OF ARCHAEA

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 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，细菌和细菌。 直到最近才认识到，肠分支是一个在遗传学上与细菌不同的连贯类群。 我们对原核细胞（细菌和细菌）的精细结构的了解远远少于我们对它们的生物化学和分子组织的了解。 这主要是由于保存方法的局限性，在某种程度上也是由于仪器的局限性。 现代快速冷冻和冷冻替代技术的进步已经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。 由于分子生物学研究表明，在转录、翻译和其他与DNA相关的加工机制方面，古细菌与真核生物比与细菌有更多的共同点，因此这些生物的形态学可能产生丰富的生物学信息。 全细胞图像的高压冷冻，冷冻替代硫磺硫化叶菌已被捕获通过连续切片断层扫描。 作为对照，以相同的方式制备细菌沙门氏菌的全细胞图像。 在过去几年中进行的工作已经导致了样品制备方案，常规产生非常高质量的全细胞断层扫描这两种生物体。Robertson博士创建的定制软件自动分割沙门氏菌和硫化叶菌中重金属染色的主要生物分子（核糖体，细胞膜等）的断层图像堆栈。 自动分割软件提供了对这些生物分子进行计数的方法，因为生物体对各种代谢状态做出反应，例如热休克、氧化应激（暴露于过氧化氢）或生长培养基的变化（例如糖/无糖）。 自动分割软件还生成可用于计算机实验的细胞的结构生物信息学模型。 断层扫描工作已补充Lowicryl包埋Sulfolobus的免疫EM，已成功探测到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.