EVOLUTIONARY GENETICS OF BACTERIAL CHROMOSOMES

描述：大多数细菌群体遗传学的研究都集中在 点突变和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，调查人员提出了一系列 有趣的研究，以检查基因组组织和 细菌菌株分化过程中的染色体结构。 总体目标是解决主要染色体的适应性 基因组水平上的组织和变异。其目的是确定 如果细菌基因组处于稳定的选择之下，那么具有 较小的染色体可以容纳更多的染色体外DNA， 研究细菌染色体的组织对称性和 确定可变区的出现是否保持相对 起点和终点的位置；最后检查发生的情况 以及编码外源DNA的大片段的基因组分布 毒力决定因素。 为了实现这一点，PI建议检查物理地图中的变化 利用限制性内切酶和脉冲胶对野生大肠杆菌菌株的鉴定 电泳法。在初步研究中，奥赫曼博士展示了广泛的 15株自然分离的大肠杆菌基因组大小范围(4.7-5.3Mb) (来自EcoR参考资料集)。这一变化的一个组成部分 基因组大小是由质粒DNA决定的，初步结果表明 可能与染色体大小和质粒含量成反比。 第一组实验将确定天然的 大肠埃希氏菌的分离及染色体和质粒组分的分离 基因组大小变异。实验包括分离和消化 用于识别决定基因组大小的质粒片段的质粒DNA 估计。 为了识别染色体中受大小变化影响的部分，Dr。 Ochman建议基于I-Ceui比较低分辨率物理地图 摘要。这种限制性内切酶能切割大肠杆菌rRNA操纵子和 通常会从整个染色体中产生7个片段。通过比较 在自然分离株中，Ochman计划确定 易受长度突变影响的染色体。最大的i-Ceui 片段(包含约1/2的大肠杆菌染色体)将是 通过绘制BlnI限制酶切点进行进一步分析。 将进一步描述长度变化的性质 通过使用I-CeuI片段(CUT)进行杂交实验完成 来自PFG)和Kohara库的过滤器。这项分析将确定 哪些Kohara克隆包含特定的基因和缺失的基因区域 天然的分离物。 来检验自然选择保持对称性这一假设 对于复制的起点和终点，奥克曼博士建议完成 36株大肠埃希菌、2株弗氏杆菌和16株沙门氏菌的研究 有详细的系统发育信息的菌株。与 手中的地图，调查员可以统计测试对称性在 染色体通过定位起点和终点(通过杂交)发生变化。 这一阶段的部分工作将集中在已知的菌株上。 致病岛，看看对称性是否已经被其他 基因组中其他地方的染色体变化。 沙门氏菌和大肠埃希氏菌染色体变化的研究 哪些可用的系统发育信息将使这些结果 详细的作图实验将在系统发育的背景下进行分析。 Ochman进一步提出，通过以下方法估计染色体进化的速度 检测实验室进化的大肠杆菌的大规模变化 连续传代超过10,000代(从 理查德·伦斯基的实验室)。

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).