Functional Analysis of Even-skipped Stripe 2 Enhancer Evolution

控制偶规则基因even-skip （eve）表达的调控区域的DNA序列的进化变化的表型后果将被检查。Eve在早期果蝇胚胎中表现为一系列七个横条纹，这些横条纹划定了果蝇未来交替片段的边界。一个671 bp的增强子元件位于eve转录起始位点上游约1.1 kb处，调控条带2的表达（s2e）。四种调节蛋白，激活因子bicoid （bcd）和hunchback(hb)，抑制因子giant （gt）和Kruppel (Kr)，各自在增强子内的多个位点结合并相互作用，决定发育中的胚胎中eve stripe 2的表达。通过广泛的结合位点定向突变和p介导的转化，人们对s2e的结构和功能有了全面的了解。最近在果蝇物种内部和物种之间发现了广泛的DNA序列变异，这使得探索这一调控序列的功能进化成为可能。该序列变异对条带2表达的时间、空间定位和强度的表型影响将被研究。有待验证的工作假设是，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会发生，以确保每个物种的表达模式接近野生型。来自几个果蝇物种的自然产生的s2e序列将分别与一个报告基因（lacZ）连接，并插入到D. melanogaster基因组中。通过对胚胎的报告转录物和eve蛋白进行双重染色，可以精确地量化报告基因在发育胚胎中相对于原生eve表达的空间和时间模式。将构建化学s2e序列来评估特定进化取代的表型后果，包括调节蛋白结合位点和位点间距。更复杂的嵌合序列也将被构建和测试，以评估一个细胞内代偿性变化的可能性。将研究果蝇均匀跳过条纹2增强子的调控序列进化的功能后果。这项工作将为理解调控序列进化的微观进化力量和自然选择在这一过程中所起的作用提供基础。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.