LONG RANGE INTERACTIONS IN MU AND BACTERIAL DNA

转座子是普遍存在的DNA元件，其大小和 复杂性。最小和最简单的转座子是插入序列 只有几百个碱基对长。家庭中较大的复杂成员 包括真核RNA肿瘤病毒(艾滋病相关病毒)和 原核生物DNA病毒。转座子通过以下方式改变染色体功能 在宿主的基因组中从一个位置移动到另一个位置。令人印象深刻的进步 在了解生化机理方面提出了几个具体的 转座子与对监管的无知形成了鲜明的对比 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-- 每小时可以换位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的DNA结构域 反应以及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超卷曲的极限，它们限制了某些 DNA-DNA位点相互作用的类别。拓扑异构酶是密切相关的 参与原核生物和真核生物的结构域形成。确实有 这个应用程序有两个总体目标。一是探索交织在一起的 噬菌体Mu结构域的超螺旋结构。这将会被携带 通过创建一组携带伽马-三角洲对的修改后的病毒 转座子的分辨(RES)位点。一个固定在Mu中心的陀螺仪 是体内转座的关键。我们将测试这一影响 单旋转酶对体内Mu结构域超螺旋结构的影响 通过测量其对发现的RES位点的重组率的影响 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 具有相似大小的细菌基因组片段。我们打算学习 在转座过程中，Mu结构域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，始于His操纵子，包括两个 在厌氧生长条件下转录的操纵子，PDU和 COB。我们计划回答两个问题。首先，这些变化是如何 染色质结构，当细胞从有氧生长变为 厌氧生长影响牧草转位？这项研究将开发一种新的 技术称为Muprint，它揭示了详细的整合 Mu从一个站点移动到另一个站点的模式。两者之间的差异 需氧和厌氧培养的模式提供了位置和 在这两种情况下，蛋白质与DNA结合的活性。第二，我们将 将Gamma-Delta Res位点引入细菌基因组的这一区域 并比较了伽马三角洲地点的特定重组率 这个域是从Mu.

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.