ISOLATION OF REGULATORY KINASES FROM THE VISUAL SYSTEM OF DROSOPHILA

从果蝇视觉系统中分离调节激酶

• 批准号: 6240209
• 负责人: J AARON CASSILL
• 金额: $ 10.6万
• 依托单位: UNIVERSITY OF TEXAS SAN ANTONIO
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6240209
• 关键词: Drosophilidae; RNase protection assay; electroretinography; eye; gene expression; gene mutation; gene targeting; genetic mapping; in situ hybridization; morphology; nucleic acid chemical synthesis; nucleic acid sequence; oligonucleotides; phosphorylation; polymerase chain reaction; protein kinase; protein sequence; radionuclides; regulatory gene; retina degeneration; subtraction hybridization; vision; visual photoreceptor

We are interested in isolating and characterizing genes which modulate the visual response. Several members of the visual cascade are known to be phosphorylated in response to activation by light. By analogy to other systems, it is thought that this phosphorylation is responsible for a large part of the modulation of the response. We therefore plan to concentrate on the isolation of genes which encode regulatory kinases. Mutations which cause excessive activation of the visual cascade lead to retinal degeneration. Thus, regulation of the level of response of the visual system is critical not only for proper vision but also to maintain viability of the photoreceptor cells. Mutations which affect the level of phosphorylation of rhodopsin are also know to cause degeneration. Therefore, the serine/threonine kinases we are searching for may be key players in retinal degenerative diseases. In order to isolate serine/threonine protein kinase genes, we have designed two degenerate oligonucleotides which encode conserved amino acid domains VI and VIII found in all members of the kinase family. PCR reactions will be done on cDNA which is enriched for eye-specific sequences by multiple subtractive hybridizations using head mRNA from the mutant fly eya which does not develop eyes. Amplified sequences will be isolated and screened for kinases by sequencing. Any kinases found will be further characterized to assess their role in regulating the visual system.

我们对分离和表征调节基因感兴趣 视觉反应。 已知视觉级联的几个成员是 响应于光的激活而磷酸化。 与其他类比 系统，据认为这种磷酸化负责 响应调制的很大一部分。 因此我们计划 专注于编码调节激酶的基因的分离。 导致视觉级联过度激活的突变导致 视网膜变性。 因此，对反应水平的调节 视觉系统不仅对于正常视力至关重要，而且对于维持视力至关重要 感光细胞的活力。 影响水平的突变 视紫红质的磷酸化也已知会导致变性。 因此，我们正在寻找的丝氨酸/苏氨酸激酶可能是关键 视网膜退行性疾病的参与者。 为了分离丝氨酸/苏氨酸蛋白激酶基因，我们设计了 编码保守氨基酸结构域的两个简并寡核苷酸 VI 和 VIII 存在于激酶家族的所有成员中。 PCR 反应将 在 cDNA 上完成，该 cDNA 通过多种方法富集了眼睛特异性序列 使用来自突变果蝇 eya 的头部 mRNA 进行消减杂交， 不发育眼睛。 扩增的序列将被分离和筛选 通过测序检测激酶。 任何发现的激酶都将被进一步表征 评估它们在调节视觉系统中的作用。