NUTRITIONAL CONTROL OF ASPARAGINE SYNTHETASE

描述：基因表达的营养控制已在 在细菌和酵母的分子水平上，但在哺乳动物细胞中知之甚少。 申请人已经鉴定了几种蛋白质， 增加的响应AA剥夺的组织或细胞在文化。 一 其中之一是酶AS。 申请人还表明， AS的发生主要或完全是由于转录增强。 整体 该建议的目标是鉴定AS中的顺式作用序列 负责氨基酸依赖性转录的基因 测试假设，增加AS活性在天冬酰胺酶抗性 白血病是AA依赖性转录的结果，可能涉及 AASRE。 在AS基因中寻找顺式作用元件的工作将继续进行 通过三个具体的目标：（1）定位DNA酶I超敏感位点， 或AS基因附近的氨基酸营养和饥饿细胞，（2）进一步在体外 通过使用缺失和突变分析潜在的AASRE 在瞬时表达系统中分析报告构建体，和（3）在 体内足迹分析检测差异表达的位点（AASRE） 与反式作用因子相互作用的细胞。 的 第四个具体目标将测试的假设，天冬酰胺酶抗性的 白血病细胞是由AS基因的转录控制改变引起的。 这将通过比较DNA酶I超敏反应和 来自天冬酰胺酶敏感的和 天冬酰胺酶抗性细胞。

DESCRIPTION: Nutrient control of gene expression has been analyzed at the molecular level in bacteria and yeast, but less is known in mammalian cells. The applicant has identified several proteins for which synthesis is increased in response the AA deprivation of tissue or cells in culture. One of these is the enzyme AS. The applicant has also shown that the induction of AS is largely or entirely due to enhanced transcription. The overall goals of this proposal are to identify the cis-acting sequences in the AS gene that are responsible for the amino acid-dependent transcription and to test the hypothesis that increased AS activity in asparaginase-resistant leukemia is the result of AA-dependent transcription, possibly involving the AASREs. The search for cis-acting elements in the AS gene will proceed through three specific aims: (1) mapping of DNAse I hypersensitive sites in or near the AS gene in aa fed and starved cells, (2) further in vitro analysis of potential AASREs through the use of deletional and mutational analysis of reporter constructs in transient expression systems, and (3) in vivo footprinting analysis to detect sites (AASREs) that are differentially interacting with trans-acting factors in aa fed versus starved cells. The fourth specific aim will test the hypothesis that asparaginase resistance of leukemia cells results from altered transcriptional control of the AS gene. This will be approached by comparing the DNAse I hypersensitivity and in vivo footprinting of chromatin from asparaginase-sensitive and asparaginase-resistant cells.