Drosophila Deafness Genes: Analysis of Chordotonal Organ

这项建议的长期目标是了解听觉机械感觉的分子和细胞机制。这些目标将通过使用一种新的果蝇听力遗传模型系统来实现。几乎整个基因组序列的公开可获得性，加上操纵果蝇的遗传、发育和分子工具，使这成为一个非常强大的模型。昆虫的听力是由脊索器官介导的，这些器官与脊椎动物的听觉和前庭毛细胞有关，因为它们在发育过程中由同一基因的同源无调性指定。第一种方法是识别特定破坏脊索器官功能的突变，克隆相应的基因，并阐明其基因产物的细胞位置和分子功能。贝多芬、smetana和触觉不敏感幼虫-B三个基因的突变将受到这一分析的影响。第二种方法将利用增强子陷阱菌株，其工程转座子插入片段专门在脉络膜器官中表达报告基因。根据这一标准确定的四个增强子陷阱菌株将作为起点来克隆侧翼序列，以确定候选的节律特异性基因。如果转座子没有干扰基因，就会产生不精确的切除衍生物，作为在基因中引入突变的一种方式。因此，转座子不仅起到报告作用，还起到分子标签和诱变剂的作用。第三种方法将使用与耳聋相关的已知人类基因作为起点来识别果蝇的同源物，然后使用反向遗传学来识别这些基因中的突变，以测试其功能。这种反向遗传方法的方法将包括表征附近的转座子插入或其不精确的切除衍生物，以及最近描述的基因替换策略。通过这些方法中的任何一种来识别听觉基因并阐明其产物的分子作用将为了解机械感觉的基本但鲜为人知的过程提供非常重要的见解。

The long-term objectives of this proposal are to understand the molecular and cellular mechanisms of auditory mechanosensation. These goals will be achieved by using a new genetic model system for hearing in Drosophila. The public availability of almost the entire genome sequence, together with the genetic, developmental and molecular tools for manipulating Drosophila make this a very powerful model. Hearing in insects is mediated by chordotonal organs, which are related to vertebrate auditory and vestibular hair cells because they are developmentally specified by homologs of the same gene, atonal. The first approach will be to identify mutations that specifically disrupt chordotonal organ function, to clone the corresponding genes and to elucidate the cellular location and molecular function of their gene products. Mutations in three genes, beethoven, smetana and touch-insensitive-larva-B will be subjected to this analysis. The second approach will make use of enhancer trap strains, whose engineered transposon inserts express a reporter gene specifically in chordotonal organs. Four enhancer trap strains identified by this criterion will be used as starting points to clone the flanking sequences to identify candidate chordotonal-specific genes. If the transposon does not disrupt the gene, imprecise excision derivatives will be generated as a way to introduce mutations in the gene. The transposons therefore act not only as reporters, but also as molecular tags and as a mutagen. The third approach will use known human genes associated with deafness as a starting point to identify Drosophila homologs and then to use reverse genetics to identify mutations in these genes to test for function. Methods for this reverse genetic approach will include characterization of nearby transposon insertions or their imprecise excision derivatives as well as recently described gene replacement strategies. Identifying auditory genes by any of these approaches and elucidating the molecular roles of their products will provide very important insight into the fundamental but poorly understood process of mechanosensation.