DNA REPLICATION, CELL CYCLE CONTROL AND GENOME FLUIDITY

分子和细胞遗传学研究强烈暗示 癌细胞基因组在恶性进展中的可塑性。 基因扩增是这种遗传学的一个突出例子。 恶性细胞特有的柔韧性。 的 基因扩增介导的原癌基因过表达 现已在多种人类肿瘤中描述， 啮齿类动物的起源，在某些情况下，它已被证明如何， 过度生产可以导致增加的侵入性， 逃避免疫监视，等等。此外，现在有足够的 文献记载在人类肿瘤体内基因扩增 可导致对多种单独的抗微生物剂的抗性， 或组合。 因此，了解 基因扩增可以更好地理解 确保正常遗传稳定性的控制机制 细胞在癌细胞中被废除，并且条件的类型 这有利于放大过程的发生。 等 信息可能导致新的药物输送策略， 通过这种机制产生抗性。 该提案提出了实验，以调查的性质， 基因扩增产生的最早的分子产物。 具体来说，将测试亚显微镜下， 自动复制的圆形元件通常 作为第一步生成。 这一假设是根据 该实验室以前的工作的基础上， 这些元素可能是微小染色体的前体。 这些元素的结构将使用物理和 研究基因区域的分子克隆方法 跨越了数百个酶， 将测试包含功能性复制结构域的元件。 复制起点存在于一个这样的元件中， 将对本实验中以前的样本进行分离和分析， 推断出它的结构是如何影响复制的。 最后，提出了一种基因转移的区域识别方法 人类基因组的高表达能力 基因扩增频率 通过与以前结果的类比 从这个实验室，这种方法可以使人类隔离， 用于进一步分子表征的复制起点。

Molecular and cytogenetic studies strongly implicate the plasticity of the cancer cell genome in malignant progression. Gene amplification is one prominent example of this genetic flexibility which is peculiar to malignant cells. The overexpression of proto oncogenes mediated by gene amplification has now been described in a variety of tumors of human and rodent origin, and in some cases it has been shown how such overproduction can engender increased invasiveness, ability to evade immune surveillance, etc. In addition, there is now ample documentation in human tumors in vivo that gene amplification can lead to resistance to a variety of antineoplastic agents alone, or in combination. Therefore, understanding the mechanisms of gene amplification could lead to a better understanding of how the control mechanisms which insure genetic stability in normal cells are abrogated in cancer cells, and the types of conditions which favor the occurrence of the amplification process. Such information could lead to new strategies of drug delivery to avoid development of resistance by this mechanism. This proposal presents experiments to investigate the nature of the earliest molecular products produced by gene amplification. Specifically, it will be tested whether submicroscopic, autonomously replicating circular elements are commonly generated as a first step. This hypothesis is formulated on the basis of previous work from this laboratory which demonstrated that such elements can be precursors of minute chromosomes. The structure of such elements will be analyzed using physical and molecular cloning methods designed to study genetic regions spanning hundreds of kilobases, and the hypothesis that such elements comprise functional replication domains will be tested. A replication origin present in one such element described previously in this lab will be isolated and analyzed in order to deduce how its structure contributes to regulated replication. Finally, a gene transfer method is proposed for identifying regions of the human genome associated with the capacity for high frequency gene amplication. By analogy with previous results from this lab, this approach could enable isolation of a human replication origin for further molecular characterization.