EXTRACHROMOSOMAL ELEMENTS

该项目的广泛和长期目标是研究 亚微观结构、组织和复制机制 含有扩增的染色体外元件（称为扩增体） 人甲氨蝶呤（MTX）中的二氢叶酸还原酶（DHFR）基因 抗性细胞系这些扩增体是独特的，因为它们以 线性和圆形形式。小型（约650平方米） 对）的这些扩增体为我们提供了一个很好的系统来研究 详细地介绍了染色体外的结构和复制起点， 元素 本项目的具体目标是：1）克隆扩增的DNA 扩增体上存在的序列，2）确定 扩增体上的DNA序列，3）确定 除了DHFR基因之外的扩增体DNA序列。转录DNA 序列将被分离和测序，和4）以确定 扩增体的复制机制。 我们计划通过以下方法来实现这些目标：1）粘粒 和酵母人工染色体（YAC）载体和聚合酶链 扩增体DNA克隆反应，2）限制性内切酶 粘粒和YAC克隆的定位和脉冲场凝胶 电泳，以确定结构和组织的 扩增体，3）北方印迹杂交和RNA保护测定， 确定扩增体上转录的DNA序列，和4）脉冲 标记，核酸酶消化和2-D凝胶实验，以确定 复制机制 对这些扩增体的分析可能会更好地了解 基因扩增的一般过程。此外，获得的信息 从这些结构的研究将提供重大进展， 了解癌症产生、肿瘤进展或 以及肿瘤如何产生耐药性。

The broad, long-term objectives of this project are to study the structure, organization and replication mechanism of submicroscopic extrachromosomal elements (termed amplisomes) containing amplified dihydrofolate reductase (DHFR) genes in a human methotrexate (MTX) resistant cell line. These amplisomes are unique since they exist as both linear and circular forms. The small size (about 650 kilobase pairs) of these amplisomes provides us with an excellent system to study in detail the structure and origin of replication of extrachromosomal elements. The specific aims of this project are 1) to clone the amplified DNA sequences present on the amplisomes, 2) to determine the organization of DNA sequences on the amplisomes, 3) to determine the expression of amplisomal DNA sequences other than the DHFR gene. Transcribed DNA sequences will be isolated and sequenced, and 4) to determine the replication mechanism of the amplisomes. We plan to achieve these goals by using the following methods: 1) cosmid and yeast artificial chromosome (YAC) vectors and polymerase chain reaction for the cloning of amplisomes DNAs, 2) restriction enzyme mapping of the cosmid and YAC clones and pulsed field gel electrophoresis to determine the structure and organization of the amplisomes, 3) Northern blot hybridizations and RNA protection assay to determine transcribed DNA sequences on the amplisomes, and 4) pulsed labelling, nuclease digestion and 2-D gel experiments to determine the replication mechanism. Analysis of these amplisomes might provide a better understanding on the general process of gene amplification. In addition, information obtained from studies of these structures will provide major advances in the understanding of the process of cancer generation, tumor progression or both, and of how drug-resistance occurs in tumors.