DEVELOPMENT AND CONTROL OF SYNTHESIS AND SECRETION

本研究的中心重点是了解乳糖酶根皮苷水解酶（LPH），肠道，微绒毛膜糖蛋白膜糖蛋白的哺乳动物新生儿的营养重要的表达控制机制。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基因的DNA序列决定。在上一个资助期间，我们已经确定了2 kb的大鼠LPH启动子在转基因小鼠中的功能，并确定了重要的顺式作用元件在近端启动子，其中之一表现出细胞特异性阻遏活性。mRNA定位的研究表明，特定的模式，这表明LPH mRNA的定位是重要的，其编码的蛋白质的分布。提出的实验假设是：1）正性和负性因子的分级功能调节LPH基因转录，2）顺式作用多态性参与人类乳糖酶持续性的发生，3）LPH mRNA向肠上皮细胞顶极的矢量运动是一种受调节的途径，可能涉及细胞骨架。这些概念形成了本提案的具体目标的基础：I）定义LPH基因表达的转录控制机制：阐明特定转录因子对LPH基因表达的个体和组合效应，鉴定LPH基因的细胞特异性负调控机制，并定义由人LPH基因中决定乳糖酶持久性的DNA多态性鉴定的候选元件。II）鉴定LPH和其他肠细胞mRNA的3 '-UTR中负责其细胞内定位的关键序列元件：通过3 '-UTR的定位和突变分析以及使用腺病毒介导的基因转移技术的细胞内易位研究来确定mRNA定位所必需和足够的序列，表征载体mRNA转运所需的mRNA结合蛋白，并确定细胞骨架在mRNA转运定位中的作用，以及确定细胞骨架在肠细胞中mRNA定位中的作用。总之，本申请中描述的研究应该提供对5'和3'调控元件及其结合蛋白在LPH基因表达的转录和转录后调控中的作用的综合理解。

The central focus of this research is to understand the mechanisms controlling the expression of lactase-phlorizin hydrolase (LPH), an intestinal, microvillus membrane glycoprotein 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 identified important cis-acting elements in the proximal promoter, one of which exhibits cell-specific repressor activity. Studies of mRNA localization demonstrate specific patterns, suggesting that the localization of LPH mRNA is important for the distribution of its encoded protein. The hypothesis underlying the proposed experiments are: 1) that hierarchical function of positive and negative factors regulates LPH gene transcription, 2) that cis-acting polymorphism(s) are involved in the genesis of human lactase persistence, and 3) that vectorial movement of 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 individuals 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 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.