GENETICS OF A HORMONE-RESISTANT DROSOPHILA MUTANT

甲氧基丁二烯是保幼激素的化学类似物，是有毒的。 当局部应用于果蝇时。我们是 检测耐中毒剂量的果蝇突变体 我们已经确定了一个基因座，它赋予了 对外源甲基戊二烯或JH III的抗药性为100倍。 Met基因座具有遗传和发育特征。 我们认为有统计上的间接证据表明Met 控制JH在靶组织中的接收，可能通过JH- 受体蛋白。我们也认为这个轨迹是足够的 保证额外的生化和遗传分析的重要性， 本提案分三个阶段概述了这一点。 1)。直接测定一种JH受体蛋白的蛋氨酸控制 将通过测量~3H-JH III结合能力和 无血淋巴幼虫胞液提取物的结合亲和力 表皮和成体卵黄生成卵巢组织。我们预计 看到野生型和突变型~3H-JH III的明显区别 Met控制JH受体蛋白的结合特性。2)。 Met的其他等位基因将在EMS和 伽玛射线诱变及其各自的表型特征 等位基因将被评估。我们将考虑到恢复这两个 筛查中的致死和/或雌性不育等位基因以及 温度敏感的等位基因。这些等位基因不会有用处 不仅用于鉴定克隆的Met+序列，还用于揭示 JH在果蝇中的新角色 Met表型的光谱，推测反映了后果 JH与靶组织的异常结合。3)。最后，蛋氨酸+DNA 将克隆序列，这样我们就可以重建Met+基因 和侧翼序列。我们选择了Met等位基因 杂交-不育杂交，并期望这些等位基因导致 来自P元素的突变。经现场确认后 杂交，我们将使用P-元件探针来鉴定蛋氨酸 从苍蝇DNA构建的基因组文库中的序列 携带一种杂交的不育等位基因。一个Met序列 然后，将使用限制片段来探测Met+文库，以及 分离和比对重叠序列以重建Met+。 来自Met+的探针将用于研究Met+聚(A)+转录本 在果蝇发育过程中。

Methoprene, a chemical analogue of juvenile hormone (JH), is toxic when topically applied to Drosophila melanogaster. We are examining mutants of Drosophila which are resistant to toxic dose of methoprene, and we have identified a locus which confers as much as 100-fold resistance to exogenous methoprene or JH III. This locus, Met, has been genetically and developmentally characterized. We believe that there is statistical indirect evidence that Met controls reception of JH in target tissues, possibly through a JH- receptor protein. We also believe that this locus is of sufficient importance to warrant additional biochemical and genetic analysis, which is outlined in three phases in this proposal. 1). A direct determination of Met control of a JH receptor protein will be carried out by measuring 3H-JH III binding capacity and binding affinity in cytosolic extracts from hemolymph-free larval epidermal as well as adult vitellogenic ovary tissue. We expect to see a clear difference between wild-type and mutant 3H-JH III binding characteristics if Met controls a JH receptor protein. 2). Additional alleles of Met will be recovered following EMS and gamma-ray mutagenesis, and the phenotypic characteristics of each allele will be evaluated. We will allow for the recovery of both lethal and/or female-sterile alleles in the screen as well as temperature-sensitive alleles. These alleles will be useful not only for identifying cloned Met+ sequences but also for uncovering novel roles for JH in Drosophila from an examination of the spectrum of Met phenotypes, presumably reflecting the consequences of aberrant JH binding to target tissues. 3). Finally, Met+ DNA sequences will be cloned so that we can reconstruct the MET+ gene and flanking sequences. We have selected Met alleles from a hybrid-dysgenic cross and expect these alleles to have resulted from P-element mutagenesis. After confirming this by in situ hybridization, we will use a P-element probe to identify Met sequences from a genomic library constructed from DNA from flies carrying one of the hybrid dysgenic alleles. A Met sequence restriction fragment will then be used to probe a Met+ library, and overlapping sequences isolated and aligned to reconstruct Met+. Probes from Met+ will be used to study Met+ poly(A)+ transcripts during Drosophila development.