MAMMALIAN GENOME ORGANIZATION AND GENE AMPLIFICATION

该项目的长期目标是：1)了解 负责DNA序列扩增的分子机制；2) 了解中的核架构和功能之间的关系 哺乳动物基因组；以及3)确定扩增是否干扰 这些关系。我们已经分离出重叠的重组宇宙 代表扩增出的超过500kb的连续DNA序列 抗甲氨蝶呤中国人二氢叶酸还原酶(DHFR)结构域的研究 仓鼠细胞系。我们已经确定并描述了三个起源 复制，三个末端(至少其中两个可能是固定的)，四个 活性基因，以及这个大结构域中的几个基质连接位点。 我们还使用这些克隆来定义DHFR结构域的重排 伴随着DHFR基因在几个独立分离的， 中度耐药的中国仓鼠细胞系。的具体目标 本研究的目的是：1)分离一系列杂交探针 在2号染色体的长臂上以大约20 MB的间隔定位(站点 DHFR基因)；这些探针将用于脉冲场梯度凝胶 电泳法和荧光原位杂交法分析 在目标2和3中概述的研究；2)分析最早可回收的 在选择的和自发的DHFR扩增物中的扩增事件， 并跟踪这些扩增物的克隆衍生物以评估随后的 3)分离出初始的两个子代细胞 扩增事件，以确定这两种细胞类型 显示DHFR基因拷贝数的相加或倒数变化 由过度复制或不相等的姐妹染色单体交换预测 模型；4)精确地映射我们先前拥有的终点站 使用二维凝胶方法定位在500kb的dhfr结构域中；5) 确定终止符是否表示或映射为接近于矩阵连接 地点；以及6)确定这些终点站是否能够逮捕 在自主引入时单向或双向复制 复制质粒。

The long-term objectives of this project are: 1) to understand the molecular mechanisms responsible for DNA sequence amplification; 2) to understand the relationships between nuclear architecture and function in the mammalian genome; and 3) to determine whether amplification disturbs these relationships. We have isolated overlapping recombinant cosmids representing more than 500 kb of contiguous DNA sequence from the amplified dihydrofolate reductase (DHFR) domain of a methotrexate-resistant Chinese hamster cell line. We have identified and characterized three origins of replication, three termini (at least two of which may be fixed) , four active genes, and several matrix attachment sites within this large domain. We have also used these clones to define rearrangements of the DHFR domain that accompany DHFR gene amplification in several independently isolated, moderately drug-resistant Chinese hamster cell lines. The specific aims of the proposed project are to: 1) isolate a series of hybridization probes positioned at about 20 MB intervals on the long arm of chromosome 2 (site of the DHFR locus); these probes will be used in pulse-field gradient gel electrophoretic and fluorescence in situ hybridization analyses in the studies outlined in aims 2&3; 2) analyze the earliest recoverable amplification events in both selected and spontaneous DHFR amplificants, and follow clonal derivatives of these amplificants to assess subsequent rearrangements; 3) isolate the progeny of the two daughter cells of initial amplification events, in order to determine whether these two cell types show the additive or reciprocal changes in DHFR gene copy number that are predicted by the overreplication or unequal sister chromatid exchange models, respectively; 4) precisely map the termini that we have previously localized in the 500 kb DHFR domain using a two-dimensional gel method; 5) determine whether the termini represent or map close to matrix-attachment sites; and 6) determine whether these termini are capable of arresting replication in one or both directions when introduced into an autonomously replicating plasmid.