BEHAVIOR OF P FACTORS--MOVABLE ELEMENTS IN DROSOPHILA

描述：在这份申请中，恩格斯博士提议继续他的 P元素生物学及缺口修复机制的研究。 描述了七种不同的调查路线。第一个是一个 DNA合成依赖链退火(SDSA)模型的检验 缝隙修复。在此模型中，缺口的两端都会侵入它们的模板 以独立合成互补链。穿着那些 存在两个潜在修复模板的情况，两者均为 原则上，模板可用于链合成。如果 缺口修复的主要机制是遵循SDSA途径，然后它 应该可以观察双模板修复事件，当存在 目前存在两个潜在的模板。此外，这种双模板修复 应以与SDSA模型一致的频率发生。 在二线调查中，恩格斯博士提出了实验 旨在了解管理搜索的参数 同源序列。在之前的研究中，他发现有六个- 当潜在模板的转换频率成倍增加时 该序列位于同一染色体上。实验将包括 确定是否也观察到这种顺式效应 模板位于附加的X染色体的不同臂上，或位于其他 重新排列的染色体，例如，包含介入的 异染色区和转换的频率是否取决于 根据两个潜在模板相对于间隙的位置，即， 模板是否更接近优先使用的间隙。 在第三组实验中，恩格斯博士将尝试确定 可能参与缺口修复过程的基因。第一个轨迹 需要检查的是拼写检查(Spel1)，这是一种与酵母同源的果蝇 和人类MSH2错配修复基因。恩格斯博士将确定这是否 基因对于生存是必不可少的，它的突变是否会有任何 对缝隙修复过程的影响。他还计划异地表达 来自另一个物种的MSH2同源物。对其他生物体的研究 表明外来MSH2异位表达可能会导致 显性突变子表型。在其他实验中，恩格斯博士将 确定已知的导致DNA修复缺陷的突变是否也 显示双链断裂修复中的变化。这些基因包括 梅-41、梅9、托普1和音乐309。除了测量频率之外 在转换事件中，多个其他参数(例如 转换区域、异位模板的使用)将被检查。 在第四组实验中，恩格斯博士将检查缝隙修复 酵母HO系统产生的DNA断裂。转基因带来了热量 诱导型HO内切酶基因及其靶序列 核酸内切酶将被引入苍蝇体内。血红素氧合酶诱导的修复 然后，我们将把这些突变与P元素所致的突变进行比较。 提案的第五部分描述了继续努力 优化利用P诱导的缝隙修复作为基因检测方法 替补。第六节中描述的实验的目的是 检查P元件插入的位点特异性，特别是什么 定义插入热点。将引入单一基数变化 以及在插入热点的紧邻位置 白人基因。然后，恩格斯博士将确定这些影响 核苷酸变化对(体细胞或生殖系)P- 元素插入。 提案最后一节所描述的实验旨在 理解P-元件转位与基因表达的关系 增加了重组。恩格斯博士认为，这种重组是 缝隙修复过程的结果，由未连接的 尼克斯在转换区域的边缘。P元素插入位置 将使用50C来检查复合频率(和转换 轨迹分布)作为到插入部位的距离的函数。

DESCRIPTION: In this application, Dr. Engels proposes to continue his studies on the biology of P-elements and the mechanisms of gap repair. Seven different lines of investigation are described. The first is a test of the synthesis dependent strand annealing (SDSA) model for DNA gap repair. In this model, both ends of the gap invade their templates independently to synthesize complementary strands. In those circumstances in which there are two potential repair templates, both templates could in principle be used for strand synthesis. If the primary mechanism for gap repair follows this SDSA pathway, then it should be possible to observe bi-template repair events when there are two potential templates present. Moreover, this bi-template repair should occur at a frequency consistent with the SDSA model. In the second line of investigation, Dr. Engels proposes experiments aimed at understanding the parameters governing the search for homologous sequences. In previous studies he found that there is a six- fold increase in the conversion frequency when the potential template sequence was on the same chromosome. Experiments will include determining whether this cis effect is also observed when the potential templates are on different arms of an attached-X chromosome, or on other rearranged chromosomes containing, for example, intervening heterochromatic regions and whether the frequency of conversion depends upon the position of two potential template relative to the gap, i.e., is the template closer to the gap preferentially used. In the third set of experiments, Dr. Engels will attempt to identify genes that may participate in the gap repair process. The first locus to be examined is spellcheck (spel1), a Drosophila homolog for the yeast and human MSH2 mismatch repair genes. Dr. Engels will determine if this gene is essential for viability, and whether mutations in it have any effect on the gap repair process. He also plans to ectopically express a MSH2 homolog from another species. Studies in other organisms indicate that ectopic expression of a foreign MSH2 can result in a dominant mutator phenotype. In other experiments Dr. Engels will determine whether mutations known to cause defects in DNA repair also show alterations in double-strand break repair. These genes include mei-41, mei-9, top1, and mus309. In addition to measuring the frequency of conversion events, a number of other parameters (e.g., the length of the conversion tracts, the use of ectopic templates) will be examined. In the fourth set of experiments, Dr. Engels will examine gap repair of DNA breaks generated by the yeast HO system. Transgenes carrying a heat inducible HO endonuclease gene and the target sequence for the endonuclease will be introduced into flies. The repair of HO-induced breaks will then be compared with those induced by P-elements. The fifth section of the proposal describes continuing efforts to optimize the use of P-induced gap repair as a method for gene replacement. The aim of the experiments described in section six is to examine the site specificity of P-element insertion, in particular what defines an insertional hot spot. Single base changes will be introduced into as well as in the immediate vicinity of an insertional hot spot in the white gene. Dr. Engels will then determine what effects these nucleotide changes have on the frequency of (somatic or germline) P- element insertion. The experiments described in the last section of the proposal are aimed at understanding the relationship between P-element transposition and increased recombination. Dr. Engels suggests that the recombination is a consequence of the gap repair process and is initiated by unligated nicks at the edges of the conversion tracts. A P-element insertion at 50C will be used to examine the recombination frequency (and conversion tract distribution) as a function of distance from the insertion site.