EXTRACHROMOSOMAL ELEMENTS

这项计划广泛而长远的目标是研究 亚微观的结构、组织和复制机制 含有扩增的染色体外元件(称为扩增体) 甲氨蝶呤二氢叶酸还原酶(DHFR)基因 耐药细胞系。这些扩增体是独一无二的，因为它们以 线形和圆形都有。小尺寸(约650千基) 这些扩增体)为我们提供了一个很好的研究系统 详细介绍了染色体外染色体的结构和复制来源 元素。 本项目的具体目标是：1)克隆扩增出的DNA 存在于扩增体上的序列，2)以确定 DNA序列上的扩增体，3)以确定表达 Dhfr基因以外的扩增DNA序列。转录DNA 序列将被分离和测序，以及4)以确定 扩增体的复制机制。 我们计划通过使用以下方法来实现这些目标：1)COSMID 和酵母人工染色体(YAC)载体和聚合酶链 扩增体DNA克隆的反应，2)限制性内切酶 COSMID、YAC克隆和脉冲场凝胶的作图 电泳法测定细胞的结构和组织 3)Northern印迹杂交和RNA保护试验 确定扩增体上转录的DNA序列，以及4)脉冲 标记、核酸酶消化和二维凝胶实验确定 复制机制。 对这些扩增体的分析可能会更好地理解 基因扩增的一般过程。此外，获得的信息 对这些结构的研究将在以下方面提供重大进展 了解癌症的发生、发展或发展过程 以及肿瘤中耐药性是如何发生的。

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.