MOLECULAR ANALYSIS OF CONJUGATION IN TETRAHYMENA

四膜虫接合的分子分析

• 批准号: 3282293
• 负责人: KATHLEEN M KARRER
• 金额: $ 8.88万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-01-01 至 1986-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3282293
• 关键词: DNA; Tetrahymena; alleles; autoradiography; cell nucleus; cell transformation; developmental genetics; endonuclease; gel electrophoresis; gene expression; genetic mapping; genetic recombination; genetic transcription; genetic translation; genome; methylation; microorganism conjugation; molecular cloning; nucleic acid sequence; thin layer chromatography

Tetrahymena is a ciliated protozoan which contains two nuclei, a diploid, germinal micronucleus (mic) and a polyploid macronucleus (mac) which is transcriptionally active in the vegetative cell. At conjugation (sexual reproduction), the macronucleus is degraded and a new macronucleus is made from one of the mitotic products of the zygotic micronucleus. The development of the new macronucleus involves 1.) polyploidization of the bulk of the Tetrahymena genome to the level of 45C 2.) elimination of specific DNA sequences and rearrangement of some others 3.) methylation of the macronuclear DNA and 4.) the initiation of transcription from a genome which was previously silent. We plan to study several aspects of the development of the somatic macronucleus from the germ line micronucleus. We have cloned a fragment of Tetrahymena micronuclear DNA which is rearranged during development of the macronucleus. Molecular cloning techniques will be used to analyze the nature of the rearrangement, to determine whether any of the various DNA sequences which have been implicated in genome rearrangements are in close proximity to this particular rearranged sequence and to probe for transcription of this sequence in vegetative and conjugating cells. Macronuclear DNA in Tetrahymena contains 0.8% methyladenine. Micronuclear DNA is unmethylated. In vivo labelling techniques will be used to determine the developmental stage at which methylation occurs. We have isolated several mic-specific DNA sequences. They will be used to probe RNA preparations to determine whether they are transcribed during conjugation. Several predominant in vivo labelled proteins are specifically synthesized in conjugating cells. Phage wil be selected which contain Tetrahymena DNA sequences homologous to RNAs translated during conjugation. Filter hybridization will be used to determine whether the DNA sequences are mic-specific and/or linked on the Tetrahymena chromosomes and to determine the stage(s) at which the conjugation specific RNAs are transcribed.

四膜虫是一种有纤毛的原生动物，含有两个细胞核，一个二倍体， 生发微核（mic）和多倍体大核（mac） 在营养细胞中具有转录活性。 接合时（有性 繁殖），大核被降解并产生新的大核 来自合子微核的有丝分裂产物之一。 这 新大核的发育涉及 1.) 四膜虫基因组的大部分达到 45C 2.) 消除的水平 特定 DNA 序列和其他一些序列的重排 3.) 甲基化 大核DNA和4.)基因组转录的起始 之前是沉默的。 我们计划从几个方面进行研究 从种系微核发育成体细胞大核。 我们克隆了四膜虫微核 DNA 的片段 在大核发育过程中重新排列。 分子克隆 技术将用于分析重排的性质，以 确定是否有任何不同的 DNA 序列已被 与基因组重排有关的物质与此非常接近 特定的重排序列并探测该序列的转录 营养细胞和接合细胞中的序列。 四膜虫的大核DNA含有0.8%的甲基腺嘌呤。 微核 DNA 未甲基化。 体内标记技术将用于 确定甲基化发生的发育阶段。 我们已经分离出几个麦克风特异性 DNA 序列。 他们将习惯于 探针 RNA 制剂以确定它们是否在过程中被转录 共轭。 几种主要的体内标记蛋白是特异性合成的 在接合细胞中。 将选择含有四膜虫 DNA 的噬菌体 与接合过程中翻译的RNA同源的序列。 筛选 杂交将用于确定 DNA 序列是否 麦克风特异性和/或连接在四膜虫染色体上并确定 接合特异性RNA转录的阶段。