DEVELOPMENT AND CONTROL OF SYNTHESIS AND SECRETION

本研究的中心重点是了解控制乳糖酶-芽孢素水解酶（LPH）表达的机制，LPH是一种对哺乳动物新生儿营养重要的肠道、微绒毛膜糖蛋白。LPH的基因表达、酶活性和细胞内加工是肠细胞中关键过程的范例，都是由LPH基因的DNA序列决定的。在上一个资助期内，我们在转基因小鼠中确定了大鼠LPH启动子2 kb的功能，并在近端启动子中确定了重要的顺式作用元件，其中一个表现出细胞特异性抑制活性。mRNA定位的研究显示了特定的模式，这表明LPH mRNA的定位对其编码蛋白的分布很重要。所提出的实验的假设是：1)正负因子的分层功能调节LPH基因的转录，2)顺式作用多态性(s)参与了人类乳糖酶持久性的发生，3)LPH mRNA向肠细胞顶极的载体运动是一个受调节的途径，可能涉及细胞骨架。这些概念构成了本提案具体目标的基础：1)确定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作用，确定LPH基因的细胞特异性负调控机制，并确定由人类LPH基因DNA多态性确定的决定乳糖酶持久性的候选元件。II)确定LPH和其他肠细胞mrna 3'- utr中负责细胞内定位的关键序列元件；通过3'- utr的定位和突变分析，以及腺病毒介导的基因转移技术，确定mRNA定位所需和足够的序列，表征载体mRNA运输所需的mRNA结合蛋白，确定细胞骨架在mRNA运输定位中的作用，并确定细胞骨架在肠细胞中mRNA定位中的作用。综上所述，本申请中描述的研究应提供对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.