GENOME BASED ANALYSIS OF METHYLOTROPHY

关于兼性甲基营养的最新证据 细菌，甲烷杆菌extorquens AM1表明，甲基营养 这种有机体中的新陈代谢是新陈代谢常规的混合体。 以前被认为是在不同的进化分支中分开的。 为了在这个有机体中作为一个整体解决甲基营养问题，它将 非常有利于进行基因组水平的分析。M. Extorquens AM1的基因组为6Mbp，并有完整的测序项目 对于这种生物体来说将是极其昂贵的。然而，生理上的 这种类型的研究不需要完整的基因组序列， 组装成单一的重叠群。相反，它足以获得 大部分基因的部分序列，适合数据库 搜索、通过聚合酶链式反应方法的插入失活和表达 基于微阵列的分析。 我们建议在M。 勒索AM1并使用这些数据来加深我们对 我的营养。随机排序将进行到3X级别， 它应该提供大约95%的基因的序列数据。 这些数据将被分析为感兴趣的基因，以及 这些将被选择用于相邻区域的有针对性的排序。基因 感兴趣的将被突变，突变的表型将是 分析过了。将执行表达式数组分析以识别 对常见环境线索作出反应的基因，重点放在 关于甲基营养相关的功能。这4年的最终结果是 项目将是开发代谢框架，在此框架内 我们对甲基营养的理解将被放在。是否可以使用 大多数基因组的序列数据将为 实验方法应该会产生巨大的飞跃 对这一基本代谢系统的了解。此外，这一点 该项目将为一些基因组水平的分析提供一个模型 代表新陈代谢多样性的微生物 原核生物世界，对其完全基因组测序是不可行的。

Recent evidence obtained regarding the facultative methylotrophic bacterium, Methylobacterium extorquens AM1 suggests that methylotrophic metabolism in this organism represents an amalgam of metabolic routines formerly thought to be separated in different evolutionary branches. In order to address methylotrophy as a whole in this organism, it would be highly advantageous to carry out genome-level analysis. M. extorquens AM1 has a genome of 6 Mbp, and a complete sequencing project for this organism would be extremely expensive. However, physiological studies of this type do not require the complete genome sequence, assembled in a single contig. Instead, it is sufficient to obtain partial sequence of the majority of genes, suitable for database searches, insertional inactivation by PCR methods, and expression microarray-based analysis. We propose to carry out a "minimal genome sequencing project" in M. extorquens AM1 and use these data to further our understanding of methylotrophy. Random sequencing will be carried out to the 3X level, which should provide sequence data for about 95 percent of the genes. These data will be analyzed for genes of interest, and a subfraction of these will be chosen for targeted sequencing of adjacent areas. Genes of interest will be mutated and the phenotypes of the mutants will be analyzed. Expression array analysis will be carried out to identify genes that respond to common environmental cues, with emphasis placed on methylotrophy-related functions. The end result of this 4-year project will be the development of a metabolic framework within which our understanding of methylotrophy will be placed. The availability of sequence data for the majority of the genome will provide the basis for experimental approaches that should generate great leaps in our knowledge of this fundamental metabolic system. In addition, this project will provide a model for genome-level analysis of a number of microorganisms that represent the broad diversity of metabolism in the prokaryotic world, for which complete genome sequencing is unfeasible.