Molecular analysis of hybrid incompatibility genes in Drosophila

描述(申请人提供)：已观察到杂交不亲和性(HIS)，包括杂交致死性和不育性，在该门的许多分类群内的种间杂交中被观察到。这些表型背后的分子机制目前尚不清楚，但最近在果蝇中发现的一组快速进化的基因为深入了解这一过程提供了一个令人兴奋的机会。这项建议的目的是详细研究这样一个基因，杂交雄性救援(HMR)，它导致黑腹果蝇/拟果蝇杂交的致命性。HMR基因编码一种含有保守的DNA结合域的蛋白质，但在其他方面在果蝇之间存在着高度的差异。这些特征表明，在杂交动物中，HMR正常发挥物种特异性转录调节因子的功能，并异常结合靶基因导致它们的死亡。此前的遗传学实验表明，HMR还可能与另一种杂交致死基因--母体杂交营救(MHR)相互作用。我将从三个具体目标来检验这些假设。首先，我将通过检测黑腹毛虫中HMR蛋白的DNA结合模式来测试HMR作为转录调节因子的假定作用。多线染色体分析和染色质免疫沉淀(CHIP)将用于确定候选HMR目标基因；这些基因将通过分析HMR缺失背景中的转录效应和与HMR的遗传相互作用进行测试。其次，我将使用类似的方法来确定杂交细胞中的HMR结合是否发生了变化。具体地说，我将测试候选目标基因的一个子集，以确定正常的HMR结合，并分析HMR与整个基因组中不适当的目标位置的异位结合。将对单个靶标进行转录水平改变的测试。第三，我将调查HMR是否与MHR互动。为此，我将检测转基因HMR对依赖于MHR的杂交致死率的影响，并反过来测试MHR突变是否影响依赖于HMR的杂交致死率。这项拟议的工作将是对HI基因的第一次分子研究，并将有助于理解HMR的种内功能与其在杂交致死中的作用之间的关系。 基因组学研究揭示了包括人类在内的许多高等真核生物中存在快速进化的基因。尽管这些基因的生物学作用在很大程度上尚不清楚，但它们可能会影响不同的表型特征，如物种特有的适应能力和对疾病的易感性或抵抗力。对黑腹毛虫HI基因的研究将为理解快速进化基因的功能及其在人类发育和健康中所起的作用提供一个很好的遗传模型。

DESCRIPTION (provided by applicant): Hybrid Incompatibilities (His), including hybrid lethality and sterility, have been observed in interspecies crosses within numerous taxonomic groups across the phyla. The molecular mechanisms underlying these phenotypes are currently not known but a recently identified set of rapidly evolving genes in Drosophila offers an exciting opportunity to gain insight into this process. The goal of this proposal is to examine in detail one such gene, Hybrid male rescue (Hmr), which causes lethality in Drosophila melanogaster/D. simulans hybrids. The Hmr gene encodes a protein that contains conserved DNA-binding domains but is otherwise highly diverged among Drosophila species. These characteristics suggest that HMR normally functions as a species-specific transcriptional regulator and binds target genes abnormally in hybrid animals to cause their death. Previous genetic experiments suggested that Hmr may also interact with another hybrid lethal gene, maternal hybrid rescue (mhr). I will test these hypotheses in three specific aims. First, I will test the putative role of Hmr as a transcriptional regulator by examining the DNA binding pattern of HMR protein in D. melanogaster. Polytene chromosome analysis and chromatin immunoprecipitation (ChIP) will be used to identify candidate HMR target genes; these will be tested by assaying for transcriptional effects in the Hmr-null background and for genetic interactions with Hmr. Second, I will use similar methods to determine whether HMR binding is altered in hybrids. Specifically, I will test a subset of candidate target genes for normal HMR binding and assay for ectopic binding of HMR to inappropriate target sites throughout the genome. Individual targets will be tested for altered transcription levels. Third, I will investigate whether Hmr interacts with mhr. To do this, I will assay for effects of Hmr transgenes on mhr-dependent hybrid lethality and, conversely, I will test if mhr mutations affect Hmr-dependent hybrid lethality. This proposed work will be among the first molecular studies of an HI gene and will help to understand the relationship between the intraspecific function of Hmr and its role in hybrid lethality. Genomic studies have revealed the presence of rapidly evolving genes in a number of higher eukaryotes, including humans. Although the biological roles of these genes are largely unknown, they are likely to influence diverse phenotypic traits such as species-specific adaptations and predisposition or resistance to disease. This study of HI genes in D. melanogaster will provide a good genetic model for understanding the function of rapidly evolving genes and the roles they play in the development and health of humans.