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） 了解核结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。我们分离出了重叠重组蛋白 代表来自扩增的DNA的超过500 kb的连续DNA序列。 甲氨蝶呤耐药中国人二氢叶酸还原酶（DHFR）结构域 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（其中至少两个可以是固定的），四个 活性基因，和几个基质附着位点在这个大域。 我们还使用这些克隆来定义DHFR结构域的重排 伴随DHFR基因扩增， 中度耐药中国仓鼠细胞系。的具体目标 拟开展工作是：1）分离一系列杂交探针 位于2号染色体长臂上约20 MB间隔处（位点 DHFR基因座）;这些探针将用于脉冲场梯度凝胶 电泳和荧光原位杂交分析， 目标2&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）分离两个子细胞的后代， 扩增事件，以确定这两种细胞类型是否 显示DHFR基因拷贝数的加性或倒数变化， 通过过度复制或姐妹染色单体交换不均等预测 模型，分别; 4）精确地映射我们以前的终端 使用二维凝胶法定位于500 kb DHFR结构域; 5） 确定末端是否代表或映射接近基质附着 6）确定这些终端是否能够阻止 在一个或两个方向复制时，引入到一个自主 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.