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，并导致人类癌症等重大疾病。它们也作为程序化的过程发生，在许多生物体的细胞分化中起着特定的作用。一种不寻常的DNA重排发生在全基因组范围内，改变了染色体的整体结构。尽管人们对它们的认识已经有一个多世纪了，但对它们的分子性质却知之甚少。本提案将研究这种类型的DNA重排在模式真核生物嗜热四膜虫。该项目的既往研究表明，该生物的体细胞基因组在发育过程中通过染色体断裂和DNA缺失两个过程进行了戏剧性的重组。DNA删除发生在数千个位点上，以切除不同大小和序列的特定片段，这些片段加起来约占基因组的15%。DNA缺失的识别涉及两个顺式作用序列：侧翼调控序列和内部促进序列。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，并验证DNA缺失作为一种监视机制来去除入侵遗传元件（如转座子）的想法。虽然这些序列不编码蛋白质，但在细胞核分化过程中转录产生双链RNA。这项研究将确定这种转录如何调节DNA缺失。如果将内部促进序列人工地置于旧体细胞核中，它们将抑制DNA的删除。这种抑制作用在后代中持续存在，并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检查。染色体断裂发生在大约200个特定的位点上，这些位点上有一个15 bp的信号。它切割DNA并在自由端添加新的端粒序列。这些小染色体在营养生长期间无限期地维持在体细胞核中。本提案将寻找基因突变体，并使用一种特殊的转基因菌株来识别控制这一过程的基因。它还将确定最近发现的归巢内切酶基因f - thl是否可能编码破坏染色体的酶。最后，该提案将使用一种基于其编码蛋白质位置识别基因的方法来研究涉及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（如染色体凝聚和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.