Functional Analysis of Even-skipped Stripe 2 Enhancer Evolution

将检查控制配对规则基因even-skipped（eve）表达的调节区的DNA序列中进化变化的表型结果。Eve在早期的果蝇胚胎中表现为一系列的七条横向条纹，这些条纹划分了果蝇未来交替体节的边界。位于eve转录起始位点上游约1.1kb处的671 bp增强子元件调节条带2的表达（s2 e）。四种调节蛋白，激活子bicoid（bcd）和hunchback（hb），以及阻遏子giant（gt）和Kruppel（Kr），每个都在增强子内的多个位点结合，并相互作用以决定发育胚胎中eve stripe 2的表达。通过广泛的定点突变的结合位点和P-介导的转化，一个全面的了解s2 e的结构和功能，最近出现。最近发现广泛的DNA序列变异的s2 e内和果蝇物种之间允许探索的功能进化的调节序列。将研究该序列变异对条纹2表达的时间、空间定位和强度的表型后果。待检验的工作假设是，结合位点（或间距）的个体进化变化将对表达产生可测量的影响，但这些变化的组合将确保每个物种中的近野生型表达模式。将来自几种果蝇的天然存在的s2 e序列各自与报告基因（lacZ）连接并插入D.黑腹果蝇基因组。通过对胚胎进行报道转录物和eve蛋白的双重染色，相对于天然eve表达，发育中胚胎中报道基因的表达模式将在空间和时间上精确定量。将构建嵌合s2 e序列以评估特定进化取代的表型结果，包括调节蛋白结合位点和位点间隔。更复杂的嵌合序列也将被构建和测试，以评估s2 e内代偿性变化的可能性。 非技术部分将研究果蝇偶跳条纹2增强子中调控序列进化的功能后果。这项工作将提供一个基础，了解微进化的力量，调控序列的演变和自然选择在这一过程中发挥的作用。这些研究应阐明基因调控的基本分子机制以及调控序列变异和改变的进化机制。

Kreitman 9604477 Technical section The phenotypic consequences of evolutionary changes in the DNA sequence of a regulatory region controlling expression of the pair-rule gene even-skipped (eve) will be examined. Eve is expressed in early Drosophila embryos as a series of seven transverse stripes which demarcate the boundaries of future alternating segments in the fly. A 671 bp enhancer element located approximately 1.1 kb upstream of the eve transcription start site regulates strip 2 expression (s2e). Four regulatory proteins, the activators bicoid (bcd) and hunchback(hb), and repressors giant (gt) and Kruppel (Kr), each bind at multiple sites within the enhancer and interact to determine the expression of eve stripe 2 in developing embryos. Through extensive site-directed mutagenesis of binding sites and P-mediated transformation, a comprehensive understanding of s2e structure and function has recently emerged. Recently discovered extensive DNA sequence variation in the s2e within and among Drosophila species allows an exploration of the functional evolution of this regulatory sequence. The phenotypic consequences of this sequence variation on the timing, spatial localization and intensity of stripe 2 expression will be investigated. The working hypothesis to be tested is that individual evolutionary changes in binding sites (or in spacing) will have measurable effects on expression, but that combinations of such changes will have occurred to assure a near-wildtype pattern of expression in each species. Naturally occurring s2e sequences from several Drosophila species will each be linked to a reporter gene (lacZ) and inserted into the D. melanogaster genome. The expression pattern of the reporter gene in developing embryos will be precisely quantified both spatially and temporally relative to the native eve expression by doubly staining embryos for both reporter transcript and for eve protein. Chimerical s2e sequences will be constructed to evaluate phenotypic consequences of specific evolut ionary substitutions, both for the regulatory protein binding sites and for the spacing of the sites. More complex chimerical sequences will also be constructed and tested to evaluate the possibility of compensatory changes within a s2e. Non-technical section The functional consequences of regulatory sequence evolution in the even-skipped stripe 2 enhancer of Drosophila will be investigated. The work will provide a foundation for understanding microevolutionary forces governing regulatory sequence evolution and the role natural selection plays in this process. The studies should illuminate basic molecular mechanisms of gene regulation and also evolutionary mechanisms of regulatory sequence variation and change.