GENE AMPLIFICATION AND ELIMINATION IN TETRAHYMENA

这里建议进行研究，以调查改变 发育过程中基因的结构和剂量。简单的纤毛虫 四膜虫被选为这些研究的模式系统。四膜虫罐头 在实验室里很容易培养和操纵基因， 生化和发育研究。以前对这一点的研究和 其他实验室已经证明，tRNA基因扩增和特定的DNA 消除发生在体细胞大核的发育过程中。 这种有机体。扩增过程与碎片化相结合 在染色体的特定位置，端粒序列的增加 形成反向重复序列。消除过程是耦合的 DNA的缺失(包括断裂和重新连接) 特定的站点。在这些研究中，我们将尝试确定分子 这些过程的机制和发育调节。DNA位于 染色体断裂和DNA缺失的位置将通过克隆来分离 并通过杂交和测序进行分析。这两种产品的比较 几个不同地点的结构应该揭示出重要的特征 对于特定位点的DNA断裂、端粒DNA添加、DNA消除和 位点特异性DNA缺失。来自几个相同的不同菌株的DNA 相同的遗传来源(核型克隆)将被检查以 确定流程是否在序列级别上是精确的。我们会 在同步发育的大核中直接分析这些过程 确定顺序事件和可能涉及的中间体。 DNA在缺失位点或其附近的转录活性也将 检查以确定DNA缺失和 抄写。要充分了解这些细胞的发育规律 我们将研究DNA断裂和断裂时的染色质结构 核内不同发育阶段的缺失连接。最后，我们 将探索建立体外系统的可能性 使用克隆的DNA和细胞提取液分析这些过程。它是 希望这些研究能够进一步阐明基因的本质 结构和细胞分化，并帮助我们了解 发育受到调控，以及细胞异常生长可能发生的方式。

Studies are proposed here to investigate the various processes which alter the structure and dosage of genes during development. The simple ciliate Tetrahymena is chosen as a model system for these studies. Tetrahymena can be easily cultivated and manipulated in the laboratory for genetic, biochemical and developmental studies. Previous studies from this and other laboratories have shown that tRNA gene amplification and specific DNA elimination occur during the development of the somatic macronucleus of this organism. The amplification process is coupled with the fragmentation of the chromosome at specific sites, the addition of telomeric sequences and the formation of reverse repeats. The elimination process is coupled with the deletion (which involves breakage and rejoining) of DNA at specific sites. In these studies we will try to determine the molecular mechanisms and the developmental regulation of these processes. DNA at sites of chromosomal breakage and DNA deletion will be isolated by cloning and analyzed by hybridization and sequencing. Comparisons of these structures at several different sites should reveal the features important for site specific DNA breakage, telomeric DNA addition, DNA elimination and site specific DNA deletion. DNA from several different strains which share an identical genetic origin (caryonidal clones) will be examined to determine whether the processes are precise at the sequence level. We will analyze these processes directly in synchronously developing macronuclei to determine the sequence events and possible intermediates involved. Transcriptional activities of DNA at or near deletion sites will also be examined to determine the interrelationships between DNA deletion and transcription. To fully understand the developmental regulation of these events we will study the chromatin structure of the DNAs at breakage and deletion junctions in nuclei at various stages of development. Finally, we will explore the possibility of establishing an in vitro system for analyzing these processes using cloned DNAs and cellular extracts. It is hoped that these studies could further elucidate the nature of gene structure and cell differentiation, and help us understand how normal development is regulated and how abnormal cell growth might occur.