BEHAVIOR OF P FACTORS--MOVABLE ELEMENTS IN DROSOPHILA

P因子的行为——果蝇中的可动元件

• 批准号: 2175955
• 负责人: William R. ENGELS
• 金额: $ 33万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-07-01 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2175955
• 关键词: DNA repair; Drosophilidae; endonuclease; gene expression; gene mutation; gene rearrangement; genetic recombination; heterochromatin; nucleic acid sequence; sex chromosomes; transposon /insertion element

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.

描述：在此应用程序中，恩格斯博士建议继续他的 关于P元素生物学和间隙修复机制的研究。 描述了七种不同的调查线。 第一个是 DNA的合成链退火（SDSA）模型的测试 差距维修。 在此模型中，差距的两端入侵了他们的模板 独立地合成互补链。 在那些 有两个潜在维修模板的情况，两者都 原则上可以将模板用于链合成。 如果是 间隙维修的主要机制遵循此SDSA途径，然后 有可能在存在时观察双板修复事件 存在两个潜在的模板。 此外，这项双板修复 应以与SDSA模型一致的频率发生。 在第二次调查中，恩格斯博士提出了实验 旨在了解搜索搜索的参数 同源序列。 在以前的研究中，他发现有六个 当潜在模板时，转换频率的折叠增加 序列在同一染色体上。 实验将包括 确定当电势时是否也观察到这种顺式效应 模板在附着的X染色体的不同臂上，或其他 重新排列的染色体，例如中间 杂色区域以及转换频率是否取决于 在两个电势模板相对于间隙的位置，即 模板是更靠近差距优先使用的。 在第三组实验中，恩格斯博士将尝试识别 可能参与GAP修复过程的基因。 第一个基因座 要检查的是咒语（Spel1），果蝇的同源物 和人类MSH2不匹配修复基因。 恩格斯博士将确定是否 基因对于生存能力至关重要，并且其中突变是否具有任何 对间隙修复过程的影响。 他还计划异位表达 来自另一物种的MSH2同源物。 其他生物的研究 表明外国MSH2的异位表达可以导致 主要突变器表型。 在其他实验中，恩格斯博士将 确定是否还在DNA修复中引起缺陷的突变 显示双链断裂修复的改变。 这些基因包括 MEI-41，MEI-9，TOP1和MUS309。 除了测量频率 转换事件，许多其他参数（例如，长度 将检查转换区，使用异位模板）。 在第四组实验中，恩格斯博士将检查 酵母HO系统产生的DNA断裂。 携带热的转基因 诱导HO内切核酸内切酶基因和靶序序 核酸内切酶将被引入苍蝇。 HO诱导的修复 然后将与P元素诱导的断裂进行比较。 提案的第五部分描述了不断的努力 优化使用P诱导的间隙修复作为基因的方法 替代品。 第六节中描述的实验的目的是 检查P元素插入的场地特异性，特别是什么 定义一个插入热点。 将引入单基础更改 在插入热点的附近以及附近 白色基因。 然后，恩格斯博士将确定这些影响 核苷酸的变化具有（体细胞或种系）p-的频率 元素插入。 提案的最后一部分中描述的实验是针对的 理解P元素换位与 增加重组。 恩格斯博士建议重组是 差距修复过程的结果，并由无调 在转换区的边缘上有划痕。 p元素插入 50C将用于检查重组频率（和转换 道分布）是距插入位点距离的函数。