LONG RANGE INTERACTIONS IN MU AND BACTERIAL DNA

MU 和细菌 DNA 中的长程相互作用

• 批准号: 2176871
• 负责人: NORMAN P. HIGGINS
• 金额: $ 14.18万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-07-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2176871
• 关键词: DNA gyrase; DNA virus; Salmonella; bacterial DNA; bacterial virus; chromatin; electron microscopy; genetic mapping; genetic recombination; molecular cloning; molecular site; nucleic acid metabolism; nucleic acid structure; site directed mutagenesis; transposon /insertion element

Transposons are ubiquitous DNA elements that vary widely in size and complexity. The smallest and simplest transposons are insertion sequences only a few hundred base pairs long. Larger complex members of the family include eucaryotic RNA tumor viruses (AIDS-related viruses) and procaryotic DNA viruses. A transposons alters chromosome function by moving from site to site in the genome of its host. Impressive advances made in understanding the biochemical mechanics of several specific transposons stand in stark contrast to ignorance about regulatory mechanisms that constrain or activate transposons in genetic populations. One of the most efficient transposons is a large DNA virus--phage Mu--that is capable of transposing 100 times per hour. Our general goal is to understand how Mu organizes its large DNA domain during transposition reactions and how the Mu domain interacts with domains in the host bacterium. DNA domains define a limit for DNA supercoiling and they confine certain classes of DNA-DNA site interactions. The topoisomerases are intimately involved in domain formation in both procaryotes and eucaryotes. There are two general goals in this application. One is to explore the interwound supercoil structure of the bacteriophage Mu domain. This will be carried out by creating a set of modified vires carrying pairs of the gamma-delta transposon's resolution (res) sites. A gyrase anchored at the center of Mu is critical for transposition in vivo. We will test the influence of this single gyrase enzyme on the supercoiled structure of the Mu domain in vivo by measuring its effect on recombination rates of res sites spotted at different points in the Mu genome. The second goal is to compare the supercoil domain structure of phage Mu with a similar sized segment of the bacterial genome. We intend to learn how a Mu domain interacts with a bacterial chromatin during transposition. The bacterial domain we will study is the region from minute 41 to 42 in Salmonella typhimurium, which starts at the his operon and includes two operons that are transcribed under anaerobic growth conditions, pdu and cob. We plan to answer two questions. First, how do the changes in chromatin structure that occur when cells change from aerobic growth to anaerobic growth affect Mu transposition? This study will exploit a new technique called Muprinting, which reveals the detailed integration pattern of Mu as it moves from site to site. The differences between the patterns in aerobic and anaerobic cultures give clues to the location and activities of proteins bound to DNA under both conditions. Second, we will introduce gamma-delta res sites into this region of the bacterial genome and compare the site specific recombination rates of gamma-delta sites in this domain to the data set from Mu.

转座子是普遍存在的DNA元件，其大小变化很大， 复杂性 最小和最简单的转座子是插入序列 只有几百个碱基对家庭中较大的复杂成员 包括真核RNA肿瘤病毒（AIDS相关病毒）， 原核DNA病毒。转座子通过以下方式改变染色体功能 在宿主基因组中从一个位点转移到另一个位点印象深刻的进步 在理解几种特定的生物化学机制方面取得了进展， 转座子与对调控的无知形成鲜明对比 限制或激活遗传群体中转座子的机制。 最有效的转座子之一是一种大型DNA病毒-噬菌体Mu-， 每小时能变换100次。我们的总体目标是 了解Mu在转座过程中如何组织其大DNA结构域 Mu结构域如何与宿主中的结构域相互作用 细菌。 DNA结构域定义了DNA超螺旋的极限，它们限制了某些 DNA-DNA位点相互作用的分类。拓扑异构酶与 在原核生物和真核生物中都参与了结构域的形成。有 本申请中的两个总体目标。一个是探索 噬菌体Mu结构域的超螺旋结构。这将被携带 通过创建一组携带伽马-德尔塔对的修改过的病毒 转座子的分辨率（res）的网站。一个固定在穆中心的回旋体 是体内转座的关键。我们将测试这一影响 单促旋酶对体内Mu结构域超螺旋结构的影响 通过测量其对在 Mu基因组中的不同点。 第二个目标是比较噬菌体Mu的超螺旋结构域结构 有着相似大小的细菌基因组片段。我们打算学习 Mu结构域在转座过程中如何与细菌染色质相互作用。 我们将研究的细菌结构域是从41分钟到42分钟的区域， 鼠伤寒沙门氏菌，它开始于his操纵子， 在厌氧生长条件下转录的操纵子，PDU和 雄天鹅。我们计划回答两个问题。第一，如何改变 当细胞从有氧生长转变为有氧生长时， 厌氧生长影响木转位？这项研究将开发一种新的 一种名为Muprinting的技术，它揭示了 Mu从一个地点移动到另一个地点的模式。之间的差异 有氧和厌氧培养的模式提供了线索， 在两种条件下与DNA结合的蛋白质的活性。二是 将γ-δ res位点引入细菌基因组的该区域 并比较了不同基因组中γ-δ位点的位点特异性重组率， 这个领域的数据集从穆。