The enhancer of rudimentary gene

基本基因的增强子

基本信息

批准号：
6594306
负责人：
STUART I TSUBOTA
金额：
$ 0.62万
依托单位：
SAINT LOUIS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-03-01 至 2005-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6594306
关键词：
aspartate carbamoyltransferase carbamoylphosphate synthase cell cycle cell differentiation cell growth regulation enzyme activity gene targeting genetic enhancer element hydrolase phosphorylation protein binding protein protein interaction protein structure function regulatory gene yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): Pyrimidine biosynthesis is regulated in a cell-cycle specific manner to ensure sufficient pools for DNA synthesis. At the core of this regulation is the gene CAD in mammals and its homologue, rudimentary (r) in Drosophila melanogaster. These genes encode the first three enzymes in the pyrimidine biosynthetic pathway, including the rate-limiting step, carbamyiphosphate synthetase. The enhancer of rudimentary gene, e(r), was isolated as a regulatory gene of r. It encodes a highly conserved protein, whose function within the cell has not been determined. The maternal and embryonic expression patterns of e(r) suggest an embryonic function in the cell cycle. Other clues to the function and regulation of the e(r) protein (ER) are the presence of three casein kinase II sites on the protein and the binding of the protein to the ribosomal protein S3 (RPS3). The long-term goals of this project are to determine the function of e(r) in growth and development and to elucidate its mode of action in the cell. In this project, the role of the conserved casein kinase II phosphorylation sites in the regulation of the activity of ER will be determined by mutating the sites to either glutamic acid to mimic phosphorylation or to alanine to mimic the unphosphorylated residue. Each mutant gene will then be assayed for activity by its ability to rescue the mutation e(r)pl. Second, the regions of ER and RPS3 that interact will be determined by testing the ability of fragments of each protein to interact in a yeast two-hybrid assay. In this assay, interacting clones will be seen as LEU2 LACZ+ colonies. Finally, null mutations of e(r) will be isolated using a gene-targeting procedure that will utilize a mutant episomal copy of e(r) to recombine with the resident allele. The expression data of e(r) suggest that mutants may be embryonic lethals. If this is the case, the mutants will be analyzed for cellular defects in the embryo that that will help uncover the normal function of e(r).

描述（由申请人提供）：嘧啶生物合成受调节细胞周期特定方式，以确保有足够的 DNA 合成池。在这种调控的核心是哺乳动物的CAD基因及其同源物，果蝇中的初级 (r)。这些基因编码前三个嘧啶生物合成途径中的酶，包括限速酶步骤，氨甲酰磷酸合成酶。基本基因的增强子 e(r) 是分离作为 r 的调节基因。它编码一种高度保守的蛋白质，其在细胞内的功能尚未确定。母亲和 e(r) 的胚胎表达模式表明细胞中的胚胎功能循环。 e(r) 蛋白 (ER) 的功能和调节的其他线索是蛋白质上存在三个酪蛋白激酶 II 位点以及该蛋白转化为核糖体蛋白 S3 (RPS3)。本次活动的长期目标项目的目的是确定 e(r) 在生长和发育中的功能，并阐明其在细胞中的作用方式。在这个项目中，角色保守酪蛋白激酶 II 磷酸化位点的调节 ER 的活性将通过将位点突变为谷氨酸来确定模拟磷酸化或丙氨酸模拟未磷酸化残基。然后将通过其拯救突变体的能力来分析每个突变基因的活性。突变 e(r)pl。其次，ER 和 RPS3 相互作用的区域将是通过测试每种蛋白质片段相互作用的能力来确定酵母二杂交测定。在此测定中，相互作用的克隆将被视为 LEU2 LACZ+菌落。最后，e(r) 的无效突变将使用基因靶向程序将利用 e(r) 的突变附加型副本与常驻等位基因重组。 e(r) 的表达数据表明突变体可能导致胚胎死亡。如果是这样的话，突变体将会分析胚胎中的细胞缺陷，这将有助于揭示 e(r) 的正常函数。