DIRECTED MUTAGENESIS OF A CLONED EYE-SPECIFIC GENE

克隆眼睛特异性基因的定向诱变

• 批准号: 3426544
• 负责人: DENNIS G BALLINGER
• 金额: $ 2.5万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-08-01 至 1990-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3426544
• 关键词: Drosophilidae; compound eye; gene mutation; histogenesis; molecular cloning; natural gene amplification; nucleic acid probes; nucleic acid sequence; transposon /insertion element; visual photoreceptor

It is proposed to develop a novel application of the polymerase chain reaction (PCR) technique to isolate insertion mutations in a gene suspected of playing a role in Drosophila eye development. PCR is a sensitive method used to amplify particular DNA segments flanked by oligonucleotides. A model system has been used to demonstrate the efficacy of using PCR to detect known P-element transposon insertions. It is proposed to adapt this technique to isolate new insertion mutations. Briefly, multiple oligonucleotides complementary to a single strand of the gene of interest would be included with an oligonucleotide that primes synthesis away from the terminal repeats of the P-element transposable element. A substrate suitable for amplification would arise only if a transposition event led to the insertion of a P- element within about 100-1,000 nt of one of the gene oligonucleotides, yielding an appropriate segment of DNA flanked by primers. The subject of this study will be the gene encoding a differentiation antigen of photoreceptor cells, expressed very early in their development. This gene is expressed about thirty hours earlier than previously studied photoreceptor differentiation antigens. It is expressed at about the same time as other early neuronal differentiation antigens, but it is exquisitely specific for photoreceptor cells. Its time of appearance and tissue specificity suggest that the antigen may play an important role in some aspect of photoreceptor cell differentiation, perhaps in distinguishing photoreceptors from other neurons. This gene has been cloned, and partial sequence information is available. Use of the PCR technique to isolate insertion mutations in this gene will aid in the identification of its function during photoreceptor cell development. If successful, this technique should find broad application to the isolation of insertion mutations in Drosophila genes of importance in the development and function of the visual system.

提出了开发聚合酶的新应用 链反应（PCR）技术分离插入突变， 一种被怀疑在果蝇眼睛发育中起作用的基因。 PCR是用于扩增特定DNA片段的敏感方法 两侧是寡核苷酸。 一个模型系统被用来 证明使用PCR检测已知P元件的有效性 转座子插入。 建议采用这种技术， 分离新插入突变。 简而言之，多个 与以下基因的单链互补的寡核苷酸： 感兴趣寡核苷酸将被包括在内， 远离P元件末端重复序列的合成 转座因子 适合于扩增的底物将 只有当一个换位事件导致插入一个P- 在一个基因的约100- 1，000 nt内的元件 寡核苷酸，产生适当的DNA片段， 引物。 本研究的主题将是编码一种 光感受器细胞分化抗原，表达非常高， 在其发展的早期。 这个基因在大约30个 比以前研究的感光细胞分化早20小时 抗原 它的表达时间与其他早期 神经元分化抗原，但它是精致的特异性 感光细胞。 它出现的时间和组织 特异性表明抗原可能在 光感受器细胞分化某些方面，也许在 区分光感受器和其他神经元。 该基因具有 已克隆，部分序列信息可用。 使用 PCR技术来分离该基因中的插入突变 将有助于识别其在光感受器过程中的功能 细胞发育 如果成功，这项技术将广泛应用于 果蝇重要基因插入突变的分离 视觉系统的发展和功能。