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.

甲丁烯，一种少年激素（JH）的化学类似物，是有毒的 当局部应用于果蝇。 我们是 检查果蝇的突变体，这些突变体对有毒剂量有抵抗力 甲丁烯的含量，我们已经确定了一个同样多 作为外源甲酯或JH III的100倍抗性。 这 Met的基因座在遗传和发育上都是表征的。 我们认为有统计间接证据表明 控制目标组织中JH的接收，可能通过JH- 受体蛋白。 我们还认为这个基因座足够 重要性对于保证额外的生化和遗传分析， 该提案中的三个阶段概述了这一点。 1）。 直接确定JH受体蛋白的MET控制 将通过测量3H-JH III结合能力和 胞质提取物的结合亲和力来自无血淋巴幼虫 表皮以及成人卵黄卵巢组织。 我们期望 看到野生型和突变体3H-JH III之间的明显差异 结合特征如果MET控制JH受体蛋白。 2）。 在EMS之后，将回收MET的其他等位基因，并且 伽马射线诱变和每个的表型特征 等位基因将进行评估。 我们将允许两者的恢复 屏幕中的致命和/或女性 - 肌电等位基因以及/或 温度敏感的等位基因。 这些等位基因将不用 仅用于识别克隆的MET+序列，还用于发现 JH在果蝇中的新作用 MET表型的光谱，大概反映了后果 与靶组织的异常JH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。 我们选择了从一个 杂交型杂交交叉，并期望这些等位基因产生 来自P元素诱变。 在原地确认这一点之后 杂交，我们将使用P元素探针来识别MET 来自由蝇的DNA构成的基因组文库的序列 携带一个混合失调等位基因。 MET序列 然后，限制片段将用于探测MET+库，并且 重叠序列隔离并对齐以重建MET+。 Met+的探针将用于研究MET+ Poly（a）+转录本 在果蝇发展期间。