BEHAVIOR OF P FACTORS--MOVABLE ELEMENTS IN DROSOPHILA

描述：在本申请中，恩格斯博士建议继续他的 P-元件的生物学和缺口修复机制的研究。 七个不同的调查线描述。 第一个是 DNA的合成依赖链退火（SDSA）模型的检验 间隙修复 在这个模型中，差距的两端都侵入了它们的模板 独立地合成互补链。 在这些 在存在两个潜在修复模板的情况下， 模板原则上可用于链合成。 如果 缺口修复的主要机制遵循SDSA途径， 应该可以观察到双模板修复事件， 存在两个潜在的模板。 此外，这种双模板修复 应该以与SDSA模型一致的频率发生。 在第二条研究路线中，恩格斯博士建议进行实验， 旨在了解控制搜索的参数， 同源序列 在以前的研究中，他发现有一个六- 当潜在的模板 序列在同一条染色体上 实验将包括 确定当电势 模板位于附着的X染色体的不同臂上，或位于其它染色体上。 重排的染色体，例如， 异染色质区域以及转换频率是否取决于 根据两个潜在模板相对于差距的位置，即， 是更接近优先使用的差距的模板。 在第三组实验中，恩格斯博士将试图找出 可能参与差距修复过程的基因。 第一基因座 要检查的是拼写检查（spel 1），这是果蝇中酵母的同源物 和人MSH 2错配修复基因。 恩格斯博士将确定这是否 基因是生存所必需的，它的突变是否有任何 对差距修复过程的影响。 他还计划异位表达 另一个物种的MSH 2同源物 其他生物的研究 表明外源MSH 2异位表达可导致 显性突变子表型 在其他实验中，恩格斯博士将 确定已知导致DNA修复缺陷的突变是否也 显示双链断裂修复改变。 这些基因包括 mei-41、mei-9、top1和mus 309。 除了测量频率 在转换事件中，多个其它参数（例如，的长度 转换道，异位模板的使用）将被检查。 在第四组实验中，恩格斯博士将研究 由酵母HO系统产生的DNA断裂。 携带热量的转基因 诱导型HO内切核酸酶基因和诱导型HO内切核酸酶基因的靶序列。 将核酸内切酶引入果蝇。 HO诱导的修复 然后将断裂与由P元素引起的断裂进行比较。 建议的第五部分介绍了继续努力， 优化使用P诱导的缺口修复作为基因修复的方法， 更换. 第六节所述实验的目的是 检查P元件插入的位点特异性，特别是 定义了插入热点。 将引入单碱基变化 以及在插入热点附近， 白色基因。 恩格斯博士将确定这些影响， 核苷酸变化对（体细胞或生殖细胞）P- 元素插入 提案最后一节所述的实验旨在 在理解P-元件转座和 增加重组。 恩格斯博士认为， 这是差距修复过程的结果， 在转化道的边缘有刻痕 一个P元素的插入， 50 C将用于检查重组频率（和转换 道分布）作为距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.