GENE AMPLIFICATION AND ELIMINATION IN TETRAHYMENA

这里提出的研究是调查各种过程，改变 基因在发育过程中的结构和剂量。 简单纤毛虫 四膜虫被选为这些研究的模式系统。 四膜虫罐 在实验室里很容易培养和操作， 生物化学和发育研究。 此前的研究表明， 其他实验室已经表明，tRNA基因扩增和特异性DNA 消除发生在体细胞大核的发育过程中 这个有机体。 扩增过程与裂解过程相结合 染色体在特定位点上，端粒序列的添加 和反向重复序列的形成。 消除过程是耦合的 与DNA的缺失（包括断裂和重新连接）， 具体网站。 在这些研究中，我们将尝试确定分子 机制和这些过程的发展调节。 dna在 通过克隆分离染色体断裂和DNA缺失的位点 并通过杂交和测序进行分析。 这些比较 几个不同地点的结构应该揭示了重要的特征 对于位点特异性DNA断裂、端粒DNA添加、DNA消除和 位点特异性DNA缺失。 几种不同菌株的DNA 将检查相同的遗传起源（核状克隆）， 确定过程在序列级别上是否精确。 我们将 直接在同步发育的大核中分析这些过程， 确定序列事件和可能涉及的中间体。 在缺失位点处或附近的DNA的转录活性也将被检测。 检查以确定DNA缺失和 转录。 为了充分了解这些发育规律， 我们将研究DNA断裂时的染色质结构， 在不同发育阶段的细胞核中缺失连接。 最后我们 将探索建立体外系统的可能性， 使用克隆的DNA和细胞提取物分析这些过程。 是 希望这些研究能进一步阐明基因的本质， 结构和细胞分化，并帮助我们了解如何正常 发育受到调节，以及异常细胞生长可能发生。

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.