MOLECULAR ANALYSIS OF CONJUGATION IN TETRAHYMENA

四膜虫是一种纤毛原生动物，它含有两个核，一个二倍体， germ微核（MIC）和多倍体大核（MAC）， 在营养细胞中具有转录活性。 在变位时（性 繁殖），大核降解并产生新的大核 来自合子微核的有丝分裂产物。 的 新大核的发育涉及1.）多倍体化 大部分的四膜虫基因组的水平的45 C 2。消除 特定的DNA序列和一些其他的重排3.）甲基化 大核DNA和4.）从基因组开始转录 之前是沉默的。 我们计划从几个方面研究 体细胞大核从生殖细胞微核发育而来。 我们克隆了一个四膜虫微核DNA片段， 在大核发育过程中重新排列。 分子克隆 技术将被用来分析重排的性质， 确定是否有任何不同的DNA序列， 与基因组重排密切相关的基因 特别是重排的序列，并探测这种 在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。 我们已经分离出了几个mic-specific DNA序列。 他们将习惯于 探针RNA制备物，以确定它们是否在 变位 几种主要的体内标记蛋白是特异性合成的 在接合细胞中。 选择含有四膜虫DNA的噬菌体 与接合期间翻译的RNA同源的序列。 滤波器 杂交将用于确定DNA序列是否 mic-specific和/或连锁的四膜虫染色体上，并确定 转录缀合特异性RNA的阶段。

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.