Chromosome breakage and DNA detection in Tetrahymena

描述（由申请人提供）：DNA重排影响大多数生物体的基因组稳定性，并导致包括人类癌症在内的重大疾病。它们也作为程序化过程发生，在许多生物体的细胞分化中发挥特定作用。一类不寻常的DNA重排发生在全基因组范围内，改变染色体的整体结构。虽然已知的世纪，他们的分子性质是知之甚少。这项建议将研究这种类型的DNA重排的模式真核生物嗜热四膜虫。该项目过去的研究表明，这种生物体的体细胞基因组在发育过程中通过两个过程显着重组：染色体断裂和DNA缺失。DNA缺失发生在几千个位点上，以切除不同大小和序列的特定片段，这些片段总共占基因组的约15%。识别DNA缺失涉及两个顺式作用序列：侧翼调控序列和内部启动序列。该提案将通过测试这些序列中具有特定改变的转基因菌株来确定这些序列如何被识别以及它们如何相互作用，并测试DNA缺失作为一种监视机制以去除入侵的遗传元件（如转座子）的想法。虽然不编码蛋白质，但这些序列在核分化期间转录以产生双链RNA。这项研究将确定这种转录如何调节DNA缺失。如果将这些内部启动子序列人工放置在旧体细胞核中，则它们将抑制DNA的缺失。这种抑制在下一代中延续下来，成为一种表观遗传特征。这种抑制的分子基础及其与异常转录的关系将被研究。染色体断裂发生在由15 bp信号标记的大约200个特定位点处。它切割DNA并将新的端粒序列添加到自由末端。 这些微型染色体在营养生长期间无限期地保持在体细胞核中。这项建议将寻找遗传突变体，并确定控制这一过程的基因，使用一种特殊的转基因菌株进行筛选。它还将确定最近发现的归巢核酸内切酶基因F-Tthl编码断裂染色体的酶的可能性。最后，该提案将通过使用一种基于编码蛋白质的位置识别基因的方法来研究参与DNA重排的新基因。通过结合这些方法，该研究希望揭示这些过程的潜在分子机制，揭示它们与其他细胞过程（如染色体浓缩和DNA修复）的关系，并深入了解它们的生物学作用。

DESCRIPTION (provided by applicant): DNA rearrangements affect genome stabilities in most organisms, and cause major diseases including cancer in humans. They also occur as programmed processes that play specific roles in cell differentiation in many organisms. One unusual class of DNA rearrangements occurs genome-wide to alter overall structures of chromosomes. Although known for more than a century, their molecular nature is poorly understood. This proposal will examine this type of DNA rearrangements in the model eukaryote Tetrahymena thermophila. Past studies of this project have shown that the somatic genome of this organism is dramatically re-structured during development through two processes: chromosome breakage and DNA deletion. DNA deletion occurs at several thousand sites to excise specific fragments of diverse sizes and sequences, which together comprise about 15% of the genome. Recognition for DNA deletion involves two cis-acting sequences: flanking regulatory sequences and internal promoting sequences. This proposal will determine how these sequences are recognized and how they interact with each other by testing transgenic strains with specific alterations in these sequences, and test the idea that DNA deletion serves as a surveillance mechanism to remove invading genetic elements such as transposons. Although not coding for proteins, these sequences are transcribed during nuclear differentiation to produce double stranded RNA. This study will determine how this transcription may regulate DNA deletion. The internal promoting sequences will inhibit DNA deletion if they are artificially placed in the old somatic nucleus. This inhibition perpetuates itself in the following generations and becomes an epigenetic trait. The molecular basis of this inhibition and its relationship to the unusual transcription will be examined. Chromosome breakage occurs at approximately 200 specific sites marked by a 15 bp signal. It cleaves DNA and adds new telomeric sequences to the free ends. These minichromosomes are maintained indefinitely in the somatic nucleus during vegetative growth. This proposal will search for genetic mutants and identify genes that control this process using a special transgenic strain for the screen. It will also determine the possibility that a recently discovered homing endonuclease gene, F-Tthl, encodes the enzyme that breaks chromosomes. Finally, the proposal will research for new genes involved in DNA rearrangements by using an approach that identifies genes based on the location of their encoded proteins. By combining these approaches, the study hope to reveal the underlying molecular mechanisms of these processes, uncover their relationships to other cellular processes such as chromosome condensation and DNA repairs and gain insights to their biological roles.