DEVELOPMENT AND CONTROL OF SYNTHESIS AND SECRETION

这项提案中描述的研究的中心焦点是 对乳糖酶表达调控机制的认识 根皮苷水解酶(L-Ph)在哺乳动物幼体发育中的作用 肠子。这种酶是唯一负责水解酶的 乳糖，牛奶中的主要碳水化合物，对人的生存至关重要 哺乳动物新生儿；成年动物和大多数人类成年人被认为是 缺乏乳糖酶。首席调查员的实验室已经 最近分离并部分测序了一段2.3kb的编码区 小白鼠L-Ph.基于使用推导出的探针获得的最新数据 从这个克隆中，现在看起来很明显，除了预期的高点之外 哺乳动物中L-Ph基因的表达水平，也有显著数量 在成人肠道细胞中持续存在。结合在调查期间获得的数据 上一次授权期展示了L和Ph对糖脂的水解 成人小肠中总L-Ph活性的持续存在， 这些观察结果表明，L-Ph随后的作用更为复杂 到哺乳期的时间比之前所理解的要晚。这些发现改变了 前人对《L》发展史研究的解读 它完全依赖于对特定活动的测量。为了 发育中L-Ph基因表达调控机制的研究 已设计用于：1)确定L-Ph基因的时间模式 测定大鼠全小肠发育过程中的表达 同时测定L-Ph基因、L-Ph酶活性和L-Ph蛋白含量； L-Ph活性改变的细胞学基础 含量采用原位杂交和免疫组织化学方法；3)确定 冬小麦L-Ph酶活性和蛋白质含量的地区差异 与小肠长度方向L-Ph基因表达水平的关系 探讨黄连对L-Ph基因表达的影响机制 激素状态；4)分离L-Ph全长c DNA；5)阐明 正确处理和细胞内处理的结构要求 靶向全长基因和特异性基因的L-Ph细胞 构建或删除；6)确定L-Ph的活性部位及其 通过肽图谱的结构特征，并用这些数据来指导 使用表达的cDNA进行缺失分析。这些加在一起， 在被认为是乳糖酶缺乏的大鼠身上产生的模式 成年期，应该是发生发育变化的代表 在其他哺乳动物和乳糖酶缺乏的人类的肠道中。

The central focus of the research described in this proposal is an understanding of the mechanisms which regulate the expression of lactase- phlorizin hydrolase (L-Ph) during the development of the mammalian small intestine. This enzyme is solely responsible for the hydrolysis of lactose, the primary carbohydrate in milk, and is essential for survival of mammalian neonates; adult animals and most human adults are considered to be lactase-deficient. The laboratory of the principal investigator has recently isolated and partially sequenced a 2.3 kb cDNA encoding a portion of rat L-Ph. On the basis of recent data obtained using probes derived from this clone, it now seems clear that, in addition to the expected high levels of L-Ph mRNA in suckling animals, significant quantities also persist in adult enterocytes. In combination with data obtained during the previous grant period demonstrating hydrolysis of glycolipids by L-Ph and the continued presence of total L-Ph activity in adult small intestine, these observations suggest a more complex function of the L-Ph subsequent to the suckling period than previously understood. These findings alter the interpretation of all previous studies of the development of the L-Ph which relied exclusively on measurements of specific activity. In order to elucidate the mechanisms regulating developmental L-Ph expression studies have been designed to: 1) determine the temporal pattern of L-Ph gene expression during development in the rat total small intestine by measuring concurrently L-Ph mRNA, L-Ph enzyme activity and L-Ph protein content; 2) characterize the cellular basis for alteration sin L-Ph activity and content using in situ hybridization and immunohistochemistry; 3) define regional differences in L-Ph enzyme activity and protein content in relation to L-Ph mRNA levels along the length of the small intestine, and examine the mechanisms by which L-Ph gene expression is influenced by hormonal status; 4) isolate a full-length cDNA encoding L-Ph; 5) elucidate the structural requirements for correct processing and intracellular targeting of L-Ph using transfection of the full length cDNA and specific constructs or deletions; and 6) identify the active sites in L-Ph and their structural features by peptide mapping and use these data to guide deletional analysis using expression cDNAs. Taken together, these patterns, derived in rats which are considered to be lactase deficient in adulthood, should be representative of the developmental changes occurring in other mammals and in the intestine of lactase-deficient humans.