EVOLUTIONARY GENETICS OF BACTERIAL CHROMOSOMES

细菌染色体的进化遗传学

• 批准号: 2370980
• 负责人: Howard Ochman
• 金额: $ 11.6万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2370980
• 关键词: EVOLUTIONARY; GENETICS; BACTERIAL; CHROMOSOMES

DESCRIPTION: Most research on bacterial population genetics has focused nucleotide diversity and allelic variation generated by point mutations and recombination. In this proposal the investigator proposes a series of interesting studies to examine evolution of the genome organization and chromosomal structure in the divergence of bacterial strains. The overall objective is to address adaptive nature of major chromosomal organization and variation at the genomic level. The aims are to determine if bacterial genomes are under stabilizing selection such that bacteria with smaller chromosomes can accommodate greater amounts of extrachromosomal DNA, to study the organizational symmetry of the bacterial chromosome and determine if the occurrence of variable regions maintain the relative positions of the origin and terminus; and finally to examine the occurrence and genomic distribution of large blocks of foreign DNA which encode virulence determinants. To accomplish this the PI proposes to examine variation in the physical map of wild E. coli strains using restrictions digests and pulse gel electrophoresis. In preliminary studies, Dr. Ochman has demonstrated a wide range in genome sizes (4.7 to 5.3 Mb) among 15 natural isolates of E. coli (from the ECOR reference collection). A component of this variation in genome size is due to plasmid DNA and preliminary results suggest that there may be an inverse relationship between chromosome size and plasmid content. The first set of experiments will determine the plasmid content of natural E. coli isolates and separate the chromosome and plasmid components of genome size variation. The experiments involve isolation and digestion of plasmid DNA to identify plasmid fragments that contribute to genome size estimates. To identify parts of the chromosome that are subject to size variation, Dr. Ochman proposes to compare low resolution physical maps based on I-CeuI digestions. This restriction enzyme cleaves E. coli rRNA operons and typically yields 7 fragments from the entire chromosome. By comparing the fragment sizes among natural isolates, Ochman plans to identify regions of the chromosome that are susceptible to length mutations. The largest I-CeuI fragment (which encompasses about 1/2 of the E. coli chromosome) will be further analyzed by mapping BlnI restriction sites. Further delineation of the nature of the length variation will be accomplished through hybridization experiments using I-CeuI fragments (cut from PFG) and filters of the Kohara library. This analysis will identify which Kohara clones contain specific genes and gene regions missing from natural isolates. To test the hypothesis that natural selection maintains symmetry with regard to the origin and terminus of replication, Dr. Ochman proposes to complete studies of 36 E. coli, 2 Escherichia fergusoni strains, and 16 Salmonella strains for which detailed phylogenetic information is available. With the maps in hand, the investigator can statistically test for symmetry in chromosomal changes by locating the origin and terminus (by hybridization). Part of this phase of the work will focus on strains with known pathogenicity islands to see if symmetry has been restored by other chromosomal changes elsewhere in the genome. The study of chromosomal changes among E. coli and Salmonella strains in which phylogenetic information is available will allow this results of these detailed mapping experiments to be analysis in a phylogenetic context. Ochman further proposes to estimate the rates of chromosomal evolution by examining large scale changes in laboratory evolved E. coli that have be serially transferred for more than 10,000 generations (obtained from the laboratory of Richard Lenski).

大多数关于细菌群体遗传学的研究都集中在 点突变产生的核苷酸多样性和等位基因变异， 重组 在这份提案中，研究人员提出了一系列 有趣的研究，以检查基因组组织的进化， 染色体结构的细菌菌株的分歧。 总体目标是解决主要染色体的适应性 基因组水平的组织和变异。 目的是确定 如果细菌基因组处于稳定选择之下， 较小的染色体可以容纳更大量的染色体外DNA， 研究细菌染色体的组织对称性， 确定可变区的出现是否维持相对的 起点和终点的位置;最后检查发生 和基因组分布的大块外来DNA， 毒力决定因子 为了实现这一点，PI建议检查物理地图中的变化 野生E.大肠杆菌菌株，使用限制酶和脉冲凝胶 电泳 在初步研究中，Ochman博士已经证明了广泛的 15个菌株的基因组大小在4.7 ~ 5.3Mb之间。杆菌 (from ECOR参考资料集）。 这种变化的一个组成部分， 基因组大小是由于质粒DNA和初步结果表明， 可能是染色体大小和质粒含量之间的反比关系。 第一组实验将确定天然质粒的质粒含量。 E.分离大肠杆菌的染色体和质粒成分， 基因组大小变异。 实验包括分离和消化 质粒DNA，以鉴定影响基因组大小的质粒片段 估算 为了确定染色体中受大小变化影响的部分， Ochman建议比较基于I-CeuI的低分辨率物理地图 数字化。 该限制性内切酶切割E. coli rRNA操纵子， 通常从整个染色体产生7个片段。 通过比较 片段大小之间的天然分离物，Ochman计划确定的区域， 容易发生长度突变的染色体。 最大的I-CeuI 片段（其包含E.大肠杆菌染色体）将是 通过绘制BlnI限制性位点进一步分析。 将进一步描述长度变化的性质， 通过使用I-CeuI片段（切割）的杂交实验完成 来自PFG）和Kohara图书馆的过滤器。 该分析将确定 哪些克隆体含有特定的基因和基因区域， 天然隔离物 为了验证自然选择在人类的进化过程中 复制的起点和终点，Ochman博士建议完成 36 E.大肠埃希菌2株，沙门氏菌16株 详细的系统发育信息可用的菌株。 与 地图在手，调查人员可以统计测试的对称性， 通过定位起点和终点（通过杂交）来改变染色体。 本阶段工作的一部分将集中在已知的 致病岛，看看是否对称已恢复其他 基因组中其他地方的染色体变化。 对E.大肠杆菌和沙门氏菌菌株 系统发育信息的可用性将使这些结果 详细的绘图实验，以分析在系统发育的背景下。 Ochman进一步提出通过以下方法来估计染色体进化的速率： 研究实验室进化的E.大肠杆菌， 连续转移超过10，000代（从 Richard Lenski的实验室）。