DRUG RESISTANCE AND GENE AMPLICATION IN EUKARYOTES

过去几年的研究表明，酶的过度生产 由基因扩增引起的是一种常见的抗性机制 真核细胞对多种抗增殖剂或选择性 条件。因为基因扩增是在一种高频率下自发发生的 频率，它可能是抗性发展中的一个重大问题 肿瘤对抗肿瘤药物的影响，并可能导致生产过剩 可能导致肿瘤形成的蛋白质。同样重要的是，对 基因如何放大使人们能够深入研究基因组的机制 高等真核生物的重塑。这项建议的具体目的是 继续研究CAD基因扩增的机制。 真核组织培养细胞。该基因编码多功能CAD 催化尿苷生物合成前三步的蛋白质。 经筛选后，获得了含有扩增的CAD基因的细胞 过渡态类似物N-(膦乙酰基)-L-天冬氨酸(PALA)。我们会 扩展我们之前关于基因的染色体位置的研究 影响其所在单元的大小、结构和稳定性 放大了。这些研究将通过转移到不同的 基因组位置我们已经克隆了一个功能性的CAD基因，接下来是 这些基因在新的位置上的扩增。分子，遗传 然后将使用细胞遗传学分析来表征 改变基因扩增中的基因位置。此外，我们还有 最近开发了将其他基因与纯化的CAD联系起来的方法 基因在体内的重组。这使我们能够创造新的基因 可以用来研究CAD基因早期事件的排列 并分析扩增序列是如何丢失的。在 在重组的背景下，我们将研究一些可能是 对于同源重组很重要，我们将尝试靶向基因 通过同源重组进入特定的基因组位置。我们会 也继续我们对CAD基因扩增的后果的研究 它的正常染色体位置。我们将在以前的基础上进行扩展 观察到核糖体序列与CAD序列在 为了确定这些序列是否介导了CAD基因扩增 我们将进一步探索改变CAD记录的机制 是由一些PALA抗性细胞系的CAD基因产生的。

Studies in the past several years have shown that enzyme overproduction resulting from gene amplification is a common mechanism of resistance of eukaryotic cells to a wide range of antiproliferative agents or selective conditions. Since gene amplification occurs spontaneously at a high frequency, it can be a significant problem in the development of resistance of tumors to antineoplastic agents, and possibly lead to the overproduction of proteins which could cause neoplasia. Equally important, the study of how genes amplify enables one to delve into the mechanism of genome remodeling in higher eukaryotes. The specific aim of this proposal is to continue to examine the mechanism of amplification of the CAD gene in eukaryotic tissue culture cells. This gene encodes the multifunctional CAD protein which catalyzes the first three steps of uridine biosynthesis. Cells with amplified CAD genes have been obtained after selection with the transition state analog N- (phosphonacetyl) -L-aspartata (PALA). We will extend our previous studies of how the chromosomal location of a gene affects the size, structure and stability of the units in which it is amplified. These studies will be performed by transferring to different genomic locations a functional CAD gene we have cloned, followed by amplification of these genes in their new positions. Molecular, genetic and cytogenetic analyses will then be used to characterize the effects of changing gene position on gene amplification. In addition, we have recently developed the methodology to link other genes to the purified CAD gene using recombination in vivo. This enables us to create novel gene arrangements which we can use to study the early events in CAD gene amplification and to analyze how amplified sequences can be lost. In the context of recombinations we will study some of the factors which may be important for homologous recombination and we will attempt to target genes into specific genomic locations using homologous recombination. We will also pursue our studies on the consequences of CAD gene amplification in its normal chromosomal location. We will expand upon our previous observation that ribosomal sequences are co-amplified with CAD sequences in order to determine whether such sequences mediate CAD gene amplification and we will explore further the mechanism by which altered CAD transcripts are produced from CAD genes in some PALA-resistant cell lines.