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用性，以及操纵果蝇的遗传，发育和分子工具，使其成为一个非常强大的模型。 昆虫的听觉是由弦音器官介导的，弦音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相同基因的同源物（无调）指定。第一种方法是鉴定特异性破坏弦音器官功能的突变，克隆相应的基因，并阐明其基因产物的细胞定位和分子功能。 将对三个基因中的突变进行分析，这三个基因是bepaly、smetana和触摸不敏感幼虫-B。 第二种方法将利用增强子陷阱菌株，其工程转座子插入物在弦音器官中特异性表达报告基因。 通过该标准鉴定的四种增强子陷阱菌株将用作起始点以克隆侧翼序列以鉴定候选的脊索张力特异性基因。 如果转座子不破坏基因，则将产生不精确的切除衍生物作为在基因中引入突变的方式。 因此，转座子不仅作为报告基因，而且作为分子标签和诱变剂。 第三种方法将使用已知的与耳聋相关的人类基因作为起点来识别果蝇同源基因，然后使用反向遗传学来识别这些基因中的突变以测试功能。 这种反向遗传方法将包括附近的转座子插入或其不精确切除衍生物的表征，以及最近描述的基因置换策略。 通过这些方法中的任何一种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知之甚少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.