FOLATE BINDERS IN HEMATOPOIESIS & CELL REPLICATION

已经描述了许多人类疾病， 由于叶酸的数量不足，但这可能 对大剂量的维生素有反应 这些包括 造血系统疾病，如某些形式的再生障碍性贫血， 遗传性红细胞生成障碍，神经系统疾病， 痴呆症，某些形式的精神分裂症，最近认识到， 脆性X染色体综合征和药物诱导的 叶酸代谢 对大剂量叶酸的反应表明 一个共同的缺陷可能是这些疾病的基础 叶酸 结合蛋白（FBPs），现在已经确定在 膜，在可溶性，最近在核 哺乳动物细胞的一部分可能是共同的联系。 大部分 对这些来自哺乳动物细胞的蛋白质的研究集中在 它们的结构特性，而不是它们的功能， 细胞内的周转。 此外，还没有关于 FBP的分子遗传学来表征结构， 基因的组织和表达。 因此，本项目的主要目的是建立 叶酸结合蛋白（FBP）在肿瘤中的精确功能 叶酸的细胞摄取和细胞内代谢， 充分表征该蛋白质的分子遗传学。 的 实验将旨在实现以下目标： 1)定义FBP合成和降解的动力学 并研究影响这些细胞的因素 使用（35S）蛋氨酸2）图谱进行内在标记的参数 KB细胞中FBP的细胞分布和局部分布 利用电镜免疫细胞学技术，研究 利用单克隆抗体3）克隆 从KB细胞中分离出FBP基因，然后对其进行表征， 组织和核苷酸序列，确定其染色体 定位，并检查不同的人类细胞系和组织， 表达和限制性片段长度多态性 基因4）确定是否将功能性FBP基因转染入 表达很少或不表达FBP的细胞提高了 这些细胞在叶酸缺乏的培养基中。 这项研究可以提供 明确的证据表明，FBP提供了一个关键的功能， 细胞代谢

A number of human diseases have been described which are not due to a quantitative deficiency of folic acid but which may respond to large doses of the vitamin. These include hematopoietic disorders, such as some forms of aplastic anemia, a hereditary form of dyserythropoiesis, neurologic disorders such as dementia, some forms of schizophrenia, the recently recognized fragile X chromosome syndrome, and drug induced disorders of folate metabosism. The response to large doses of folate suggests that a common defect may underlie these disorders. The folate binding proteins (FBPs) which have now been identified in the membrane, in the soluble, and most recently in the nuclear fraction of mammalian cells could be the common link. Most of the work on these proteins from mammalian cells have focused on their structural properties rather than on their function and turnover in the cell. In addition, there have been no studies on the molecular genetics of the FBP to characterize the structure, organization and expression of the gene. The primary aims of this project, therefore, are to establish the precise function(s) of the folate binding proteins (FBPs) in the cellular uptake and intracellular metabolism of folate and to characterize fully the molecular genetics of this protein(s). The experiments will be designed to achieve the following objectives: 1) define the kinetics for the synthesis and degradation of the FBP in human KB cells and study the factors that affect these parameters using intrinsic labeling with (35S)methionine 2) map the cellular and topographic distribution of the FBP in the KB cell using electron microscopy immunocytology and study the factors that modify this distribution using monoclonal antibodies 3) clone the FBP gene from KB cells and then characterize its organization and nucleotide sequence, determine its chromosomal location, and examine different human cell lines and tissues for expression and restriction fragment length polymorphism of the gene 4) determine if transfection of the functional FBP gene into cells which express little or no FBP improves the viability of these cells in folate deficient medium. The study could provide definitive evidence that the FBP provides a critical function in cellular metabolism.