CLONING THE DROSOPHILA RADISH GENE

克隆果蝇萝卜基因

• 批准号: 6019393
• 负责人: WILLIAM G QUINN
• 金额: $ 17.73万
• 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/6019393
• 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以分为 单独的阶段：短期和长期。 长期记忆已经有多年 通过两个操作标准实验定义：（1） 对破坏正常脑活动（例如麻醉）的药物的抵抗力， 电击性休克或脑震荡，（2）需要新的蛋白质 合成其建立。 从历史上看，适合这些的回忆 操作定义被隐式视为两种表现形式 同一分子事件。 但是，分析果蝇突变体 萝卜提供了有力的证据，表明这两个标准定义了两个 长期记忆的完全单独的组成部分。 突变的萝卜蝇在嗅觉中显示出接近正常的初始学习 学习范式，然后是快速记忆衰减。 萝卜突变 实际上消除了防麻醉的记忆（Folkers等，Proc。Natl。 学院。 科学。美国90：8123-8127，1993）。 在显着的对比中，这个突变 对蛋白质合成依赖性记忆没有可检测的影响（Tully et Al。，Cell 79：35-47，1994）。 这些结果与萝卜突变体清楚 表明长期记忆由两个不同的组成部分组成 表现似乎是独立的和添加的（同上）。 当前，抗麻醉（合并）内存的唯一线索是 萝卜变异。 申请人建议克隆并确定 萝卜基因。 他们将此基因映射到140 kb的间隔 X染色体，并通过染色体克隆了此间隔的DNA 步行。 他们已经确定了八个代表五到七的成绩单 基因。 他们具有孤立和测序的cDNA，代表其中五个 成绩单。 到目前为止，同源性搜索显示了一个合理的萝卜 候选人，ABC1同源性。 ABC1蛋白参与内吞作用，离子 运输或两者兼而有之。 申请人将隔离和序列野生型和序列 突变的基因组DNA对应于该和其他的开放阅读框 候选基因。 他们将比较这些序列以检测 萝卜突变。 然后，他们将用野生型转变萝卜苍蝇 基因组DNA片段，包括指示的转录本，它们将 测定这些转化体以拯救记忆，以确定确定 萝卜基因。 萝卜基因及其生化功能的鉴定将 提供有关底层分子机制的信息 长期记忆的抗麻醉组成部分。 抗麻醉 果蝇中的记忆几乎可以肯定对应于合并的记忆 （电击震颤的记忆）在上等动物中。 这是 最深入研究的脊椎动物中的长期记忆形式 直接与对脑震荡敏感的人类的记忆相对应 急性创伤。