CLONING THE DROSOPHILA RADISH GENE

克隆果蝇萝卜基因

• 批准号: 2689389
• 负责人: WILLIAM G QUINN
• 金额: $ 18.3万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2689389
• 关键词: Drosophilidae; animal genetic material tag; behavioral /social science research tag; behavioral genetics; complementary DNA; enzyme activity; gene induction /repression; gene mutation; genetic promoter element; genetically modified animals; membrane transport proteins; memory; molecular cloning; neurogenetics; nucleic acid sequence; open reading frames; protein kinase A; site directed mutagenesis

DESCRIPTION: Memory of invertebrates and vertebrates can be divided into separate phases: short-term and long-term. Long-term memory has for years been experimentally defined by two operational criteria: (1) it is resistant to agents that disrupt normal brain activity such as anesthesia, electroconvulsive shock or concussion, and (2) it requires new protein synthesis for its establishment. Historically, memories that fit these operational definitions were implicitly considered to be two manifestations of the same molecular event. However, analysis of the Drosophila mutant radish has provided strong evidence that the two criteria define two completely separate components of long-term memory. Mutant radish flies show near-normal initial learning in an olfactory learning paradigm, followed by rapid memory decay. The radish mutation virtually abolishes anesthesia-resistant memory (Folkers et al, Proc. Natl. Acad. Sci. USA 90: 8123-8127, 1993). In striking contrast, this mutation has no detectable effect on protein-synthesis dependent memory (Tully et al., Cell 79: 35-47, 1994). These results with radish mutants clearly indicate that long-term memory consists of two distinct components which appear to be independent and additive on performance (ibid). Currently, the only clue to anesthesia-resistant (consolidated) memory is the radish mutatation. The applicant proposes to clone and identify the radish gene. They have mapped this gene to 140 kb interval on the X-chromosome and have cloned the DNA of this interval by chromosomal walking. They have identified eight transcripts representing five to seven genes. They have isolated and sequenced cDNAs representing five of these transcripts. Homology searches have so far revealed one plausible radish candidate, an ABC1 homology. ABC1 proteins are involved in endocytosis, ion transport or both. The applicant will isolate and sequence wild-type and mutant genomic DNA corresponding to the open reading frame of this and other candidate genes. They will compare these sequences in order to detect the mutation in radish. They will then transform radish flies with a wild-type genomic DNA fragment that includes the indicated transcript, and they will assay these transformants for rescue of memory to definitively identify the radish gene. The identification of the radish gene and its biochemical function will provide information about the molecular mechanism underlying anesthesia-resistant component of long-term memory. Anesthesia-resistant memory in Drosophila almost certainly corresponds to consolidated memory (electroconvulsive-shock-resistant memory) in higher animals. This is the most intensely studied form of long-term memory in vertebrates and corresponds directly to memory in humans that is sensitive to concussion and acute trauma.

描述: 无脊椎动物和脊椎动物的记忆可分为 不同的阶段：短期和长期。 长期记忆已有多年 通过两个操作标准通过实验定义：（1） 对破坏正常大脑活动的药物（例如麻醉剂）有抵抗力， 电休克或脑震荡，以及（2）需要新的蛋白质 综合其成立。 从历史上看，符合这些的记忆 操作定义被隐含地认为是两种表现形式 同一分子事件。 然而，对果蝇突变体的分析 萝卜提供了强有力的证据，证明这两个标准定义了两个 长期记忆的各个组成部分完全独立。 突变的萝卜蝇在嗅觉方面表现出接近正常的初始学习能力 学习范式，随后是记忆快速衰退。 萝卜突变 实际上消除了麻醉抵抗记忆（Folkers 等人，Proc. Natl. 阿卡德。 科学。美国 90：8123-8127，1993）。 与此形成鲜明对比的是，这种突变 对蛋白质合成依赖性记忆没有可检测到的影响（Tully 等 等人，细胞 79：35-47，1994）。 萝卜突变体的这些结果清楚地表明 表明长期记忆由两个不同的部分组成 似乎是独立的并且对性能有附加作用（同上）。 目前，麻醉抵抗（巩固）记忆的唯一线索是 萝卜突变。 申请人提议克隆并鉴定 萝卜基因。 他们将该基因映射到 140 kb 的间隔上 X染色体并通过染色体克隆了该区间的DNA 步行。 他们已经确定了代表五到七人的八份成绩单 基因。 他们分离并测序了代表其中五种的 cDNA 成绩单。 迄今为止，同源搜索发现了一种看似合理的萝卜 候选者，ABC1 同源性。 ABC1 蛋白参与胞吞作用、离子 运输或两者兼而有之。 申请人将分离并测序野生型和 对应于这个和其他的开放阅读框的突变基因组DNA 候选基因。 他们将比较这些序列以检测 萝卜的突变。 然后他们将用野生型改造萝卜蝇 包含指定转录本的基因组 DNA 片段，它们将 分析这些转化体以拯救记忆，以明确识别 萝卜基因。 萝卜基因的鉴定及其生化功能 提供有关潜在分子机制的信息 长期记忆的麻醉抵抗成分。 耐麻醉 果蝇的记忆几乎肯定对应于巩固记忆 （高等动物的抗电惊厥记忆）。 这是 脊椎动物长期记忆最深入研究的形式 直接对应于对脑震荡敏感的人类记忆 急性外伤。