GENOME BASED ANALYSIS OF METHYLOTROPHY

基于基因组的甲基营养分析

• 批准号: 6351294
• 负责人: Mary E Lidstrom
• 金额: $ 29.29万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-01 至 2003-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6351294
• 关键词: Methanobacteriaceae; bacterial genetics; gene expression; genetic mapping; genetic techniques; genome; methane; microorganism metabolism; nucleic acid sequence; phenotype; polymerase chain reaction

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.

最近获得的关于兼性甲基营养型 细菌，扭脱甲基杆菌AM1表明甲基营养型 这种生物体的新陈代谢是多种代谢程序的混合体 以前被认为是在不同的进化分支中分离的。 为了解决甲基营养作为一个整体在这个有机体，它将 非常有利于进行基因组水平的分析。 M. extorquens AM1基因组全长6Mbp， 会非常昂贵 然而，生理 这种类型的研究不需要完整的基因组序列， 组装成单一重叠群。 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PCR方法的插入失活，和表达 微阵列分析 我们建议在M. extroquens AM1，并使用这些数据来进一步了解 甲基营养 随机测序将进行到3X水平， 它应该能提供大约95%的基因序列数据。 这些数据将被分析为感兴趣的基因，以及基因的亚部分。 这些将被选择用于相邻区域的靶向测序。 基因 感兴趣的突变体将被突变，并且突变体的表型将被 分析了将进行表达阵列分析以鉴定 对常见环境线索作出反应的基因，重点放在 与甲基营养相关的功能。 这四年的最终结果 该项目将是一个代谢框架的发展，其中 我们对甲基营养的理解将被放置。的可用性 大多数基因组的序列数据将为以下方面提供基础： 实验性的方法，应该产生巨大的飞跃， 了解这个基本的代谢系统。 另外这款 该项目将提供一个模型，用于对一些 微生物，代表代谢的广泛多样性， 原核世界，其中完整的基因组测序是不可行的。