CAREER: Probing the Structure of the Salmonella Chromosome

Segall 9733332许多证据表明E.大肠杆菌和沙门氏菌的细菌染色体影响与各种酶复合物的相互作用。几个结构探针，包括同源重组，转座Tn7和Tn10，和δ γ转座子的解离酶蛋白介导的位点特异性缺失，揭示了染色体的“障碍”的存在。然而，这些障碍的性质及其行动的基础都不得而知。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与染色体外元件（如病毒和转座子）之间的相互作用），因此了解它们的性质和影响以及它们是否以及如何受到调控非常重要。 第一个阶段是明确识别和界定障碍或结构要素。 这项研究以一种比过去更系统和更广泛的方式解决了这个问题。这些实验旨在了解细菌细胞内染色体的三维结构，以及这种结构如何受到细胞生理学的一些基本特征的影响。正在使用的探针是噬菌体λ的位点特异性重组系统，其具有几个特征，使其成为手头任务的灵活和灵敏的工具，并且将产生与先前使用的探针所获得的数据互补的数据。正在解决的问题是：1）所有的染色体序列都有平等的机会彼此？ 2)环状染色体外序列（特别是质粒）对所有染色体位置都有同等的访问权吗？3)染色体外DNA容易接近而其他染色体基因座难以接近的基因座是否定义了限制折叠染色体结构的区域？ 今天教育生物学学生的最限制的方面之一是动手实验室培训不是在经典的课堂环境中，而是在现实生活中的研究实验室。在这一领域具有良好技能的学生非常抢手，但他们中很少有机会进入研究实验室，并接受解决问题技能和熟练掌握实验室方法，实验设计和数据分析的必要培训。为了开始纠正这个问题，正在为本科生组织五个夏季研究讲习班，学生们将在小组中就研究问题的不同方面进行工作。 学生将参与实验的设计，性能和分析，并将学习现代分子技术。讲习班应使学生受益最深刻的暴露他们进行实验下一个更现实的设置比可以复制在较大的实验室课程，学生做设置实验练习。 此外，一些关于染色体结构的实验将被改编并引入一个新的和一个正在进行的实验室课程。

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.