GENETICS OF A HORMONE-RESISTANT DROSOPHILA MUTANT

激素抗性果蝇突变体的遗传学

• 批准号: 3283137
• 负责人: Thomas G. Wilson
• 金额: $ 13.84万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-04-01 至 1991-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3283137
• 关键词: Drosophilidae; alleles; biochemical evolution; chlorohydrocarbon insecticide; endonuclease; enzyme mechanism; gene complementation; gene expression; gene frequency; gene mutation; genetic manipulation; genetic mapping; hormone binding protein; hormone receptor; hormone regulation /control mechanism; insecticide resistance; juvenile hormones; molecular cloning; molecular genetics; mutagen testing; nucleic acid hybridization; nucleic acid sequence; point mutation; scintillation counter; temperature sensitive mutant; thin layer chromatography; toxin metabolism; transposon /insertion element

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.

烯虫酯是保幼激素 (JH) 的化学类似物，具有毒性 当局部应用于果蝇时。 我们是 检查对毒性剂量有抵抗力的果蝇突变体 甲氧普林，我们已经确定了一个位点，它赋予了同样多的 对外源性甲氧普林或 JH III 的抵抗力为 100 倍。 这 基因座 Met 已得到遗传和发育特征。 我们相信有统计间接证据表明 Met 控制靶组织中 JH 的接收，可能通过 JH- 受体蛋白。 我们也相信这个轨迹是足够的 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 Met 对 JH 受体蛋白的控制 将通过测量 3H-JH III 结合能力和 无血淋巴幼虫胞浆提取物的结合亲和力 表皮以及成人卵黄发生卵巢组织。 我们期望 观察野生型和突变型 3H-JH III 之间的明显差异 如果 Met 控制 JH 受体蛋白，则具有结合特征。 2）。 Met 的其他等位基因将在 EMS 后恢复， 伽马射线诱变，以及每个的表型特征 将评估等位基因。 我们将允许双方恢复 屏幕中的致死和/或雌性不育等位基因以及 温度敏感等位基因。 这些等位基因将不会有用 不仅用于鉴定克隆的 Met+ 序列，还用于发现 JH 在果蝇中的新作用 Met 表型谱，大概反映了后果 异常的 JH 与靶组织的结合。 3）。 最后，Met+ DNA 序列将被克隆，以便我们可以重建 MET+ 基因 和侧翼序列。 我们从以下位置选择了 Met 等位基因： 杂交-不良基因杂交并期望这些等位基因产生 来自P-元件诱变。 经现场确认后 杂交，我们将使用 P 元素探针来识别 Met 来自果蝇 DNA 构建的基因组文库的序列 携带一种杂种基因缺陷等位基因。 Met 序列 然后，限制性片段将用于探测 Met+ 文库，并且 分离并比对重叠序列以重建 Met+。 Met+ 探针将用于研究 Met+ poly(A)+ 转录本 在果蝇发育过程中。