CAREER: Probing the Structure of the Salmonella Chromosome

职业：探索沙门氏菌染色体的结构

• 批准号: 9733332
• 负责人: Anca Segall
• 金额: $ 49.79万
• 依托单位: San Diego State University Foundation
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-03-01 至 2003-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9733332&HistoricalAwards=false
• 关键词: CAREER; Probing; Structure; Salmonella; Chromosome

Segall 9733332 Many lines of evidence have shown that the structure of the E. coli and Salmonella bacterial chromosomes affects interactions with various enzyme complexes. Several probes of structure, including homologous recombination, transposition by Tn7 and Tn 10, and site-specific deletion mediated by the resolvase protein of the delta gamma transposon, have uncovered the presence of chromosomal "barriers". However, neither the nature of these barriers nor the basis of their action is known. Since these barriers are likely to affect all systems that interact with chromosomal DNA (repair, replication, and transcription functions as well as interactions between the chromosome and extrachromosomal elements like viruses and transposons), it is important to understand their nature and effects, and if and how they are regulated. The first stage is to unequivocally identify and define the barriers or structural elements. This research addresses this question in a systematic and more extensive manner than has been attempted in the past. The experiments are geared to understanding the three-dimensional structure of the chromosome within the bacterial cell, and how this structure is affected by some basic features of cell physiology. The probe that is being used is the site-specific recombination system of phage lambda, which has several features that make it a flexible and sensitive tool for the task at hand, and which will yield data complementary to that obtained by the probes used previously. The questions that are being addressed are: 1) Do all chromosomal sequences have equal access to each other? 2) Do circular extrachromosomal sequences (specifically plasmids) have equal access to all chromosomal locations? 3) Do loci that are easily accessible to extrachromosomal DNA but are poorly accessible to other chromosomal loci define regions that constrain the structure of the folded chromosome? One of the most limiting aspects of educating biology students today is hands-on laboratory traini ng not in a classical classroom context, but in a real-life research laboratory. Students with well-developed skills in this area are extremely sought-after, but too few of them have the opportunity to enter research labs and receive the necessary training in problem-solving skills and proficiency in lab methods, experimental design, and data analysis. To begin to correct this problem five summer research workshops are being formed for undergraduates where students will work in teams on different facets of a research problem. Students will be involved in the design, performance, and analysis of the experiments, and will learn modern molecular techniques. The workshops should benefit students most profoundly by exposing them to carrying out experiments under a more realistic setting than can be duplicated in larger laboratory courses in which students do set lab exercises. In addition, some of the experiments on chromosome structure will be adapted and introduced into one new and one ongoing laboratory course.

Segall 9733332许多证据表明，大肠杆菌和沙门氏菌细菌染色体的结构会影响与各种酶复合物的相互作用。几种结构探针，包括同源重组，TN7和TN 10的换位，以及由Delta Gamma Transposon的分解蛋白介导的位点特异性缺失，已经发现了染色体“屏障”的存在。但是，这些障碍的性质和行动的基础都不是已知的。由于这些障碍可能会影响与染色体DNA相互作用的所有系统（修复，复制和转录功能以及染色体和外染色体外元素（如病毒和转座子）之间的相互作用），因此了解其性质和效果以及如何调节以及如何受调节非常重要。 第一阶段是明确识别并定义障碍或结构元素。 这项研究以系统的，更广泛的方式解决了这个问题，而不是过去尝试过的。这些实验旨在了解细菌细胞内染色体的三维结构，以及该结构如何受细胞生理的某些基本特征的影响。所使用的探针是噬菌体Lambda的站点特异性重组系统，该系统具有多个功能，使其成为手头任务的灵活和敏感工具，并且将产生与先前使用的探针获得的数据互补的。所解决的问题是：1）所有染色体序列是否彼此均等？ 2）圆形外体序列（特别是质粒）是否可以平等地进入所有染色体位置？ 3）在其他染色体基因座定义折叠染色体结构的区域的区域是否易于进入但易于进入的基因座？ 当今的生物学学生教育学生最有限的方面之一是实验室培训不是在古典的课堂背景下，而是在现实生活中的研究实验室中。在这一领域具有良好技能的学生非常受欢迎，但是很少有人有机会进入研究实验室，并接受了解决问题的技能以及实验室方法，实验设计和数据分析的必要培训。为了纠正这个问题，正在为大学生组建五个夏季研究研讨会，学生将在研究问题的不同方面工作的团队工作。 学生将参与实验的设计，性能和分析，并学习现代分子技术。讲习班应通过将他们暴露于在更现实的环境下进行实验来使学生受益最大的学生，而不是在学生进行实验室练习的较大实验室课程中重复的。 此外，将对染色体结构的一些实验进行调整并引入一个新的和一个正在进行的实验室课程。