Identifying the Genetic Basis for the DRT/DMC Phenotype

描述（由申请人提供）：拟议研究的目标是了解一种我们认为与癌症相关的基因组不稳定性。某些染色体重排显示整个受影响染色体的延迟复制时间（DRT）和随后有丝分裂中的延迟有丝分裂凝聚（DMC）。在多种人类原发性肿瘤和肿瘤细胞系以及暴露于电离辐射的人类原代细胞中发现了具有DRT/DMC表型的染色体。DRT/DMC染色体与核型异常和涉及DRT/DMC染色体的二次重排相关，这些特征与癌细胞相关。因此，DRT/DMC染色体可能是电离辐射照射下正常细胞和人类癌症中基因组不稳定性的重要来源。利用染色体工程，我们可以在体外可重复地产生显示DRT/DMC的平衡易位。我们的数据表明，控制整个染色体复制时间的基因座的功能中断导致DRT/DMC表型。在这个应用中，我提出了一个体细胞遗传学的方法来确定如何DRT/DMC的监管，并确定和定义特定的染色体位点，隔离与DRT/DMC表型。具体地说，我建议1）确定DRT/DMC表型是显性还是隐性的，通过产生新的易位与现有的loxP标记的染色体，2）表征插入位点在我们现有的工程染色体，导致DRT/DMC，和3）表征顺式作用元件负责调节DRT/DMC。本建议的最终目标是确定这种DRT/DMC表型的分子基础。 有效预防和治疗癌症对公众健康至关重要。我们正在努力了解为什么对一小部分遗传物质的损伤有时会导致整个基因组的稳定性丧失，这可能导致癌症。了解这种不稳定性是如何发生的，有一天可能会导致在早期阶段预防癌症。

DESCRIPTION (provided by applicant): The goal of the proposed research is to understand a form of genomic instability that we believe is relevant to cancer. Certain chromosomal rearrangements show delayed replication timing (DRT) of the entire affected chromosome and delayed mitotic condensation (DMC) in the following mitosis. Chromosomes with the DRT/DMC phenotype are found in a variety of human primary tumors and tumor cell lines, as well as primary human cells exposed to ionizing radiation. DRT/DMC chromosomes are associated with karyotypic abnormalities and secondary rearrangements involving the DRT/DMC chromosome, characteristics which are associated with cancer cells. Therefore, chromosomes with DRT/DMC may be an important source of genomic instability in normal cells exposed to ionizing radiation and in human cancer. Using chromosome engineering, we can reproducibly generate balanced translocations that show DRT/DMC in vitro. Our data suggest that the functional disruption of loci controlling the replication timing of entire chromosomes results in the DRT/DMC phenotype. In this application, I propose a somatic cell genetic approach to determine how DRT/DMC is regulated, and to identify and define specific chromosomal loci that segregate with the DRT/DMC phenotype. Specifically, I propose to 1) determine if the DRT/DMC phenotype is dominant or recessive by generating new translocations with existing loxP tagged chromosomes, 2) characterize the insertion sites in our existing engineered chromosomes that result in DRT/DMC, and 3) characterize the cis-acting elements responsible for regulating DRT/DMC. The ultimate goal of this proposal is to determine the molecular basis for this DRT/DMC phenotype. The effective prevention and treatment of cancer is of immense importance to public health. We are working to understand why damage to a small portion of the genetic material sometimes causes a loss of stability across the entire genome, which may lead to cancer. Understanding how this instability occurs may one day lead to the prevention of cancer at an early stage.