PROGRAMMED CELL DEATH IN A HIGH CONNECTIVITY INVERTEBRATE MODEL

高连接性无脊椎动物模型中的程序性细胞死亡

• 批准号: 3735004
• 负责人: RICHARD A LOCKSHIN
• 金额: --
• 依托单位: ST. JOHN'S UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3735004
• 关键词: Drosophilidae; Manduca; RNA biosynthesis; apoptosis; biochemical evolution; complementary DNA; gene expression; genetic library; in situ hybridization; metamorphosis; protein biosynthesis; protein sequence; southern blotting; subtraction hybridization; synthetic peptide

We propose to study mechanisms of programmed cell death (PCD). In insect metamorphosis PCD is synchronous and total. Previous studies of intersegmental muscles and labial gland of the 10 g metamorphosing Manduca sexta show that cell death can require RNA and protein synthesis. We propose to examine the physiology and molecular biology of cell death and to study conserved genes in Drosophila melanogaster. We will: a. Characterize proteins synthesized during PCD in Manduca labial gland and muscle and compare the two tissues and Drosophila salivary gland. Compare common translation products from dying labial glands and muscle. Correlate changes in morphology of nuclei, DNA loss, and protein synthesis. Microsequence proteins of interest, and make synthetic peptides. Localize these proteins in dying and non-dying cells by immunocytochemistry. b. Isolate and characterize common cDNAs activated during involution of Manduca labial gland and muscle. Establish cDNA libraries from muscle and labial gland at comparable stages of involution. Isolate cDNA common to both tissues by subtractive hybridization of amplified PCR cDNA libraries. Characterize specific clones by: sequencing and comparison to known sequences; comparison of expression pattern during involution; localization of expression by in situ hybridization. c. Characterize PCD genes common to Manduca and Drosophila to assess function and mechanisms of action. Characterize temporal and tissue- specific expression of clones during Drosophila development. Southern- screen Drosophila libraries with isolated Manduca cDNA. Alter expression with P-element transformation. Mechanisms of cell death are likely conserved or use the same vulnerabilities. Our understanding should suggest new directions in therapeutics of aging, malignancy, and restoration of function.

我们建议研究程序性细胞死亡（PCD）的机制。在昆虫中 变态PCD是同步的、完全的。以前的研究 10 g 变态曼杜卡的节间肌和唇腺 sexta 表明细胞死亡可能需要 RNA 和蛋白质合成。我们 提议研究细胞死亡的生理学和分子生物学 研究果蝇的保守基因。 我们将： 一个。曼杜卡唇腺 PCD 期间合成的蛋白质特征 和肌肉并比较这两种组织和果蝇唾液腺。 比较来自垂死的唇腺和肌肉的常见翻译产品。 将细胞核形态的变化、DNA 丢失和蛋白质关联起来 合成。对感兴趣的蛋白质进行微测序，并进行合成 肽。通过以下方式将这些蛋白质定位在死亡和非死亡细胞中 免疫细胞化学。 b.分离并表征在退化过程中激活的常见 cDNA 曼杜卡唇腺和肌肉。建立肌肉和组织的 cDNA 文库 处于退化相当阶段的唇腺。分离 cDNA 共有 两种组织均通过扩增 PCR cDNA 文库进行消减杂交。 通过以下方式表征特定克隆：测序并与已知的比较 序列；复合过程中表达模式的比较； 通过原位杂交定位表达。 c.表征曼杜卡和果蝇常见的 PCD 基因以进行评估 功能和作用机制。表征时间和组织 - 果蝇发育过程中克隆的特异性表达。南方- 用分离的曼杜卡 cDNA 筛选果蝇文库。改变表达方式 与 P 元素变换。 细胞死亡机制可能是保守的或使用相同的 漏洞。 我们的理解应该提出新的方向 衰老、恶性肿瘤和功能恢复的治疗。