DEVELOPMENT AND CONTROL OF SYNTHESIS AND SECRETION

太空漫游。 本研究的中心焦点是了解控制乳糖酶表达的机制， 根皮苷水解酶（LPH）是一种肠道微绒毛膜糖蛋白，对营养具有重要意义， 哺乳动物新生儿。LPH的基因表达、酶活性和细胞内加工是 肠上皮细胞中关键过程的范例，并且都由LPH的DNA序列决定 基因在上一个资助期间，我们已经确定了大鼠LPH启动子2kb的功能， 转基因小鼠，并确定了重要的cw-作用元件在近端启动子，其中之一， 表现出细胞特异性阻遏物活性。mRNA定位的研究显示了特定的模式， 提示LPH mRNA的定位对其编码蛋白的分布具有重要意义。的 实验的假设是：1）积极和消极的层次功能 2）cw作用多态性参与LPH基因转录的发生， 人乳糖酶持久性; 3）LPH mRNA向肠上皮细胞顶极矢量运动 是一个受调控的途径，可能涉及细胞骨架。这些概念构成了具体目标的基础 I）定义LPH基因表达的转录控制机制： 阐明特异性转录因子对LPH基因表达的单独和联合作用， 确定LPH基因的细胞特异性负调控机制，并定义候选元件 通过人类LPH基因中决定乳糖酶持久性的DNA多态性来鉴定。（二）确定 LPH和其他肠上皮细胞mRNA的3 '-UTR中的关键序列元件负责它们的 细胞内定位：通过定位确定mRNA定位所必需和充分的序列 3 '-UTR的突变分析和使用腺病毒介导的基因的细胞内易位研究 转移技术，表征载体mRNA转运所需的mRNA结合蛋白，并定义 细胞骨架在肠上皮细胞mRNA定位中的作用。总之， 这种应用应该提供对5'和3'调节元件的作用的综合理解， 它们的结合蛋白在LPH基因表达的转录和转录后调控中的作用。

THE SPACEt^ROVIDED. The central focus of this research is to understand the mechanisms controlling the expression of lactase- phlorizin hydrolase (LPH), an intestinal, microvillus membrane glycoprotein important for the nutrition of mammalian neonates. The genetic expression, enzymatic activity, and intracellular processing of LPH are paradigms for critical processes in the enterocyte, and are all determined by the DNA sequence of the LPH gene. During the last funding period, we have defined the function of 2 kb of the rat LPH promoter in transgenic mice, and have identified important cw-acting elements in the proximal promoter, one of which exhibits cell-specific represser activity. Studies of mRNA localization demonstrate specific patterns, suggesting that the location of LPH mRNA is important for the distribution of its encoded protein. The hypotheses underlying the proposed experiments are: 1) that hierarchical function of positive and negative factors regulates LPH gene transcription, 2) that cw-acting polymorphism(s) are involved in the genesis of human lactase persistence, and 3) that vectorial movementof LPH mRNA to the apical pole of the enterocyte is a regulated pathway, perhaps involving the cytoskeleton. These concepts form the basis of the specific aims of the present proposal: I) Define the mechanisms of transcriptional control of LPH gene expression: elucidate the individual and combined effects of specific transcription factors on LPH gene expression, identify the mechanisms of cell-specific negative regulation of the LPH gene, and define candidate elements identified by DNA polymorphisms in the human LPH gene that determine lactase persistence. II) Identify the critical sequence elements in the 3'-UTRs of LPH and other enterocyte mRNAs responsible for their intracellular localization: define the sequences necessary and sufficient for mRNA localization by mapping and mutational analysis of the 3'-UTRs and intracellular translocation studies using adenovirus-mediated gene transfer techniques, characterize mRNA binding proteins required for vectorial mRNA transport, and define the role of the cytoskeleton in localization of mRNAs in enterocytes. Taken together, the studies described in this application should provide an integrated understanding of the role of 5' and 3' regulatory elements and their binding proteins in the transcriptional and post-transcriptional regulation of LPH gene expression.