Functional Analysis of Even-Skipped Regulatory Evolution

在真核生物调控元件中，均匀跳过条纹2 （even-skip stripe 2，简称eve stripe 2）的结构/功能关系及其进化的研究将继续进行。条带2元件发生了相当大的进化变化，包括反式作用转录因子的重要功能结合位点的变化。可以看到新的结合位点从头出现，并且可以看到先前存在的位点消失。条纹2元件的长度也在进化，从而改变了转录因子结合位点之间的间距。然而，尽管有这些结构上的变化，当来自不同物种的条纹2元件被放置在黑腹龙体内时，在相同且无法区分的时间和位置表达一个报告基因。因此，尽管该元件的结构发生了变化，但功能守恒仍得以维持，这有力地表明，稳定选择是控制该元件进化的主要选择模式。然而，由两个功能保守元件的左右半部分组成的嵌合eve stripe 2元件不能正确表达，这增加了上位性变化参与维持原生元件功能停滞的可能性。这项工作的具体目的是：1.；利用两种不同的“拯救”技术，通过表型分析进一步探讨原生和嵌合eve stripe 2元件在胚胎晚期、幼虫和成虫中的功能差异。启动黑腹亚群物种中bicoid和kruppel结合位点进化差异的功能研究，以确定这些调节位点是否也发生了代偿性进化3。在不同种类果蝇的原生和嵌合eve stripe 2元件驱动下，定量测定lacZ报告基因的表达水平，并确定这些水平进化差异的分子基础。通过对其他果蝇种类以及蚊子和家蝇进行比较序列分析，评价结果的普遍性。本研究的长期目标是了解真核生物的顺式调控序列进化，包括调控进化发生的突变和功能机制，以及在工作中的选择性和非选择性力量。破译调控序列进化的进化规则可能有利于解码包括人类在内的全基因组序列，因此有望对实验和计算基因组学的知识进步产生重大影响。它还将成为这些重要的生物科学新领域的肥沃训练场。

Research into the structure/function relationship and the evolution of the even-skipped stripe two (eve stripe 2) element of Drosophila melanogaster, arguably the best characterized of all eukaryotic regulatory elements will be continued. Considerable evolutionary change has occurred in the eve stripe 2 element including changes in functionally important binding sites for trans-acting transcription factors. Novel binding sites can be seen to emerge de novo and pre-existing sites can be seen to disappear. The length of stripe 2 element also evolves, thus changing the spacing between transcription factor binding sites. Yet despite these structural changes the stripe 2 elements from several different species, when placed in D. melanogaster, express a reporter gene at identical and indistinguishable times and locations as the native eve stripe 2. Thus functional conservation is maintained despite structural change in the architecture of the element, a strong indication that stabilizing selection is the dominant mode of selection governing this element's evolution. However a chimeric eve stripe 2 element composed of the left and right halves of two functionally conserved elements does not express correctly and raises the possibility that epistatic changes are involved in maintaining functional stasis in the native element. The specific aims of this work are:1. To further investigate functional differences in native and chimeric eve stripe 2 elements by phenotypic analysis in late embryos, larvae and adults using two different "rescue" techniques2. To initiate functional studies of evolved differences in bicoid and kruppel binding sites in the melanogaster subgroup species to determine if compensatory evolution has also occurred in these regulatory loci3. To quantitate expression levels of a lacZ reporter gene when driven by native and chimeric eve stripe 2 elements from different species of Drosophila and to determine the molecular basis of evolved differences in those levels 4. To evaluate the generality of results by conducting comparative sequence analysis in other Drosophila species as well as in the mosquito and housefly.The long-term objective of the proposed research is to understand cis-regulatory sequence evolution in eukaryotes, including the mutational and functional mechanisms by which regulatory evolution occurs and the selective and non-selective forces at work. Deciphering the rules governing the evolution of regulatory sequence evolution is likely to benefit efforts to decode whole genome sequences, including humans and therefore is expected to have a major impact on advancing knowledge in experimental and computational genomics. It will also serve as fertile training ground in these important new areas of biological science.