IRON REGULATION OF HUMAN TRANSFERRIN SYNTHESIS

描述：需要检验的假设是一种假定的铁反应 通过与其他三个mRNAs中的序列同源性确定的元件 (铁蛋白、转铁蛋白受体和红系氨基乙酰丙酸合成酶) 转铁蛋白mRNA表达抑制中的作用 对铁水平升高的反应。具体目标首先要确立 这种铁的调节在表达突变基因的转基因小鼠中丢失 嵌合型人转铁蛋白氯霉素乙酰转移酶(CAT)转基因 其中IRE的突变导致蛋白结合的丢失 体外；第二，测定在培养的肝癌(HepG2)细胞中测定 转铁蛋白的时间进程和铁的调节；第三，使用 瞬时转染适当突变的转铁蛋白-CAT构建体， 在人体肝细胞模型系统中建立反应；第四， 用电泳法研究蛋白质与RNA的体外相互作用 纯化的IRP(已证实)或细胞质的迁移率变化分析 Hep G2细胞的提取物，第五，扩大转基因小鼠的研究 表达完整野生型人转铁蛋白或产生人转铁蛋白 带有突变的IRP的mRNA，不再对铁有反应，并决定 铁处理对组织和血清转铁蛋白水平的影响。 研究设计合理，采用了人体和小鼠实验。 包括需要突变的复杂技术的系统 转基因猫科动物构建、瞬时转基因、现有菌株 转基因小鼠有可能与不孕症小鼠杂交。这个 这项研究的意义在于，它将最终确立一种 铁和铁下调转铁蛋白表达的机制 提供了有关转铁蛋白合成机制的新信息 然后可以应用于设计更有效的癌症治疗方法，其中 转铁蛋白被用作有毒物质的载体，如镓或 靶向这些药物的共价连接配体，如阿霉素 表达丰富表面转铁蛋白的快速生长的肿瘤细胞 感受器。尽管应用程序或应用程序中的PI并未实际强调 对于未来的研究，事实是该元素位于5‘区域 转铁蛋白mRNA和铁蛋白的调节作用相反 作为对铁的反应。

DESCRIPTION: The hypothesis to be tested is that a putative iron-responsive element identified by sequence homology with those in three other mRNAs (ferritin, transferrin receptor and erythroid amino-levulinate synthase) functions in the suppression of expression of the transferrin mRNA in response to raised iron levels. The specific aims are first to establish that regulation by iron is lost in transgenic mice expressing a mutated chimeric human transferrin chloramphenicol acetyltransferase (CAT) transgene in which the mutation of the IRE results in loss of protein binding in vitro; second, to determine in cultured hepatoma (HepG2) cells determine time course and regulation by iron of transferrin; and third, using transient transfection of appropriately mutated transferrin-CAT constructs, to establish the response in a model system for human liver cells; four, to characterize protein-RNA interactions in vitro using electrophoretic mobility shift assays of purified IRP (already demonstrated) or cytoplasmic extracts of Hep G2 cells, and fifth, to extend studies in transgenic mice which express intact wildtype human transferrin or produce human transferrin mRNA with mutated IRP and are no longer iron-responsive and determine the effect of iron treatment on tissue and serum levels of human transferrin. The research design is appropriate and uses human and murine experimental systems including sophisticated technologies requiring mutation of transgenic CAT constructs, transient transfection, existing strains of transgenic mice with the potential to cross with atransferrinemic mice. The significance of this research is that it will definitively establish a mechanism whereby transferrin expression is down-regulated by iron and provides new information on the mechanisms of transferrin synthesis which can then be applied to design more effective therapies for cancer, in which transferrin is used as a carrier of toxic agents such as gallium or covalently linked ligand such as doxorubicin to target these agents to rapidly growing tumor cells which express abundant surface transferrin receptors. Although not actually stressed by the PI in the application or for future studies, the fact is that the element resides in the 5' region of the transferrin mRNA and of ferritin but has opposite effects on regulation in response to iron.