Ludwig M Z, Patel N H, Kreitman M
Department of Ecology and Evolution, University of Chicago, Chicago, IL 60637, USA.
Development. 1998 Mar;125(5):949-58. doi: 10.1242/dev.125.5.949.
Experimental investigations of eukaryotic enhancers suggest that multiple binding sites and trans-acting regulatory factors are often required for wild-type enhancer function. Genetic analysis of the stripe 2 enhancer of even-skipped (eve), an important developmental gene in Drosophila, provides support for this view. Given the importance of even-skipped expression in early Drosophila development, it might be predicted that many structural features of the stripe 2 enhancer will be evolutionarily conserved, including the DNA sequences of protein binding sites and the spacing between them. To test this hypothesis, we compared sequences of the stripe 2 enhancer between four species of Drosophila: D. melanogaster, D. yakuba, D. erecta and D. pseudoobscura. Our analysis revealed a large number of nucleotide substitutions in regulatory protein binding sites for bicoid, hunchback, Kruppel and giant, as well as a systematic change in the size of the enhancer. Some of the binding sites in D. melanogaster are either absent or modified in other species. One functionally important bicoid-binding site in D. melanogaster appears to be recently evolved. We, therefore, investigated possible functional consequences of sequence differences among these stripe 2 enhancers by P-element-mediated transformation. This analysis revealed that the eve stripe 2 enhancer from each of the four species drove reporter gene expression at the identical time and location in D. melanogaster embryos. Double staining of native eve protein and transgene mRNA in early embryos showed that the reporter gene mimicked native eve expression and, in every case, produced sharply defined stripes at the blastoderm stage that were coincident with eve stripe 2 protein. We argue that stripe 2 eve expression in Drosophila evolution can be viewed as being under constant stabilizing selection with respect to the location of the anterior and posterior borders of the stripe. We further hypothesize that the stripe 2 enhancer is functionally robust, so that its evolution may be governed by the fixation of both slightly deleterious and adaptive mutations in regulatory protein binding sites as well as in the spacing between binding sites. This view allows for a slow but continual turnover of functionally important changes in the stripe 2 enhancer.
对真核生物增强子的实验研究表明，野生型增强子功能通常需要多个结合位点和反式作用调节因子。果蝇中一个重要的发育基因——even-skipped（eve）条纹2增强子的遗传分析为这一观点提供了支持。鉴于even-skipped在果蝇早期发育中的表达很重要，可以预测条纹2增强子的许多结构特征在进化上是保守的，包括蛋白质结合位点的DNA序列及其间距。为了验证这一假设，我们比较了四种果蝇（黑腹果蝇、雅库布果蝇、直立果蝇和拟暗果蝇）中条纹2增强子的序列。我们的分析揭示了双胸、驼背、克虏伯和巨人等调节蛋白结合位点存在大量核苷酸替换，以及增强子大小的系统性变化。黑腹果蝇中的一些结合位点在其他物种中要么不存在，要么发生了改变。黑腹果蝇中一个功能重要的双胸结合位点似乎是最近进化出来的。因此，我们通过P因子介导的转化研究了这些条纹2增强子之间序列差异可能产生的功能后果。该分析表明，来自这四个物种的eve条纹2增强子在黑腹果蝇胚胎的相同时间和位置驱动报告基因表达。早期胚胎中天然eve蛋白和转基因mRNA的双重染色表明，报告基因模拟了天然eve的表达，并且在每种情况下，在囊胚期都产生了与eve条纹2蛋白重合的清晰界定的条纹。我们认为，果蝇进化过程中条纹2 eve的表达在条纹前后边界的位置方面可被视为处于持续的稳定选择之下。我们进一步假设条纹2增强子在功能上具有稳健性，因此其进化可能受调节蛋白结合位点以及结合位点之间间距中轻微有害和适应性突变的固定所支配。这种观点允许条纹2增强子中功能重要的变化缓慢但持续地更新。
