Functional proteomics of chloroplast nucleoproteins towards an understanding of nucleoid structure, function and dynamics

叶绿体核蛋白的功能蛋白质组学，以了解核结构、功能和动力学

• 批准号: 5402427
• 负责人: Professor Dr. Ralph Bock
• 金额: --
• 依托单位: Max-Planck-Institut für molekulare Pflanzenphysiologie (MPI-MP)
• 依托单位国家: 德国
• 项目类别: Research Units
• 财政年份: 2003
• 资助国家: 德国
• 起止时间: 2002-12-31 至 2007-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5402427?language=en
• 关键词: Functional; proteomics; chloroplast; nucleoproteins; towards

Chloroplast nucleoids contain a set of DNA-binding proteins some of which have architectural function whereas others are likely to act as key regulators of gene expression. We propose to harness the enormous genetic potential of the model organism Chlamydomonas reinhardtii (also referred to as "green yeast") in order to address key questions of chloroplast nucleoid structure and function. The proposed research involves the large-scale purification of chloroplast nucleoids from Chlamydomonas and their systematic proteomics analysis with the ultimate goal to identify the complete set of DNA-binding proteins (structural nucleoid proteins and regulator proteins) associated with the chloroplast genome. We aim to characterize a subset of these factors molecularly and, in addition, we will explore the dynamics of nucleoid architecture by comparatively analyzing the set of DNA-binding proteins associated with chloroplast nucleoids in various environmental conditions and developmental stages. This comparative functional proteomics approach will provide new insights into the regulation of higher-order structures of the chloroplast DNA and the control of chloroplast gene expression through DNA-binding regulator proteins in response to changing environmental conditions and developmental programs. Opinion of the review panel (including one written comment): Prof. Bock wants to isolate nucleoids from Chlamydomonas chloroplasts and identify the proteins contained in these structures. The proteins shall then be checked as GFP fusions for their presumed localisation within the nucleoids. The final aim of this project is to analyse the function of nucleoid proteins by means of the RNAi technology and, furthermore, by comparing the protein composition of nucleoids under different conditions. Prof. Bock is well experienced in genetics and molecular biology of higher plant chloroplasts and has an excellent publication record in this field. The project outlined in the original written proposal was considered very interesting and timely because little is yet known about chloroplast nucleoid proteins and because of the expected impact on understanding prokaryotic nucleoids. However, the reviewers were worried that the set-up was too broad to allow the project to proceed much beyond cataloguing of a large number of candidate proteins. They, therefore, requested that clear selection criteria be given for proteins to be characterized further on a functional level, and thereby the project be more strictly focussed. In addition, a few technical details were asked to be clarified.

叶绿体类核含有一组DNA结合蛋白，其中一些具有结构功能，而另一些则可能作为基因表达的关键调控因子。我们建议利用巨大的遗传潜力的模式生物莱茵衣藻（也被称为“绿色酵母”），以解决关键问题的叶绿体类核的结构和功能。拟议的研究涉及大规模纯化的叶绿体类核衣原体和系统的蛋白质组学分析，最终目标是确定一套完整的DNA结合蛋白（结构类核蛋白和调节蛋白）与叶绿体基因组。我们的目标是表征这些因素的一个子集的分子，此外，我们将探索类核结构的动态比较分析的DNA结合蛋白与叶绿体类核在不同的环境条件和发展阶段。这种比较功能蛋白质组学的方法将提供新的见解，通过DNA结合调节蛋白的叶绿体DNA和叶绿体基因表达的控制，以应对不断变化的环境条件和发展计划的高阶结构的调节。 评审小组的意见（包括一份书面评论）：Bock教授希望从衣原体叶绿体中分离出类核物质，并鉴定这些结构中包含的蛋白质。然后，应将蛋白质作为GFP融合物检查其在类核内的假定定位。本项目的最终目的是通过RNAi技术分析类核蛋白的功能，并进一步通过比较不同条件下类核蛋白的蛋白组成。Bock教授在高等植物叶绿体的遗传学和分子生物学方面具有丰富的经验，并在该领域拥有出色的出版记录。在最初的书面提案中概述的项目被认为是非常有趣和及时的，因为人们对叶绿体类核蛋白知之甚少，并且因为对理解原核类核蛋白的预期影响。然而，评审人员担心，该设置过于宽泛，无法让该项目在对大量候选蛋白质进行编目的基础上继续进行。因此，他们要求为在功能水平上进一步表征的蛋白质提供明确的选择标准，从而更严格地集中该项目。此外，还要求澄清一些技术细节。