DEVELOPMENT AND CONTROL OF SYNTHESIS AND SECRETION

本修订申请描述了旨在继续 阐明生物合成、细胞内加工和分子生物学 微绒毛膜（MVM）酶乳糖酶-根皮苷水解酶（EC 3.2.1.23-EC 3.2.1.62）。 利用器官培养和免疫化学技术， 新合成的细胞内和MVM乳糖酶的标记模式 将在发育期间的不同时间在大鼠肠道中进行检查， 成熟 合成前体和MVM乳糖酶的亚基组成 将使用SDS-PAGE和HPLC进行分析。 糖基化在细胞凋亡中的作用 将使用衣霉素B2研究乳糖酶的细胞内加工 和栗精胺。 这些抑制实验也将产生 组成乳糖酶的寡糖链类型的信息 糖蛋白 为了研究乳糖酶的分子生物学， 将继续使用编码从胎儿分离的酶的cDNA克隆， 在表达载体λ gt 11中制备的大鼠肠cDNA文库。 用一组PCR方法对7个cDNA克隆进行了阳性鉴定。 使用碱性磷酸酶ELISA检测乳糖酶的单克隆抗体， 检测阳性噬菌斑。 氨基酸测序将在 从纯化的乳糖酶亚基产生的蛋白水解片段， 用于合成验证的寡核苷酸探针的信息 免疫阳性cDNA克隆的真实性或用于筛选 lambda gt11 library 以这种方式鉴定的cDNA克隆 将被测序并用于从基因组文库中获得和测序， 编码乳糖酶的全长基因 为了确定是否 酶活性水平的发育调节变化是 与特定乳糖酶mRNA水平或合成的变化相关， 通过北方印迹分析定量肠mRNA， 杂交，并且mRNA功能的变化将通过核内杂交来测量。 径流测定。 总之，所描述的研究应该阐明 乳糖酶的发展和分子生物学。 他们将提供新的 关于这种酶的微绒毛形式，其合成 和细胞内加工，以及它的遗传控制。 这些数据应 为乳糖酶基因的进一步研究和分析提供了基础。 人类乳糖酶缺乏症的遗传控制

This revised application describes studies designed to continue to elucidate the biosynthesis, intracellular processing, and molecular biology of the microvillus membrane (MVM) enzyme lactase-phlorizin hydrolase (EC 3.2.1.23-EC 3.2.1.62). Using organ culture and immunochemical techniques, the labeling patterns of newly synthesized intracellular and MVM lactase will be examined in rat intestine at various times during development and maturation. Subunit composition of synthetic precursors and MVM lactase will be analyzed using SDS-PAGE and HPLC. The role of glycosylation in the intracellular processing of lactase will be studied using tunicamycin B2 and castanospermine. These inhibition experiments will also yield information on the type of oligosaccharide chains which make up the lactase glycoprotein. To examine the molecular biology of lactase, experiments will continue using cDNA clones encoding the enzyme isolated from a fetal rat intestinal cDNA library prepared in the expression vector lambda gt11. Positive identification of 7 cDNA clones has been made with a panel of monoclonal antibodies to lactase using an alkaline phosphatase ELISA to detect positive plaques. Amino acid sequencing will be performed on proteolytic fragments generated from purified lactase subunits, and this information utilized to synthesize oligonucleotide probes for verification of the authenticity of the immunopositive cDNA clones or for screening the lambda gt11 library directly. The cDNA clones identified in this fashion will be sequenced and used to obtain and sequence, from a genomic library, the full-length gene encoding lactase. In order to determine whether developmentally regulated changes in levels of enzyme activity are associated with changes in specific lactase mRNA levels or synthesis, intestinal mRNA will be quantified by northern blot analysis and hybridization, and changes in mRNA function will be measured by nuclear run-off assay. Taken together, the studies described should elucidate the development and molecular biology of lactase. They will provide new information regarding the microvillus form of this enzyme, its synthesis and intracellular processing,and its genetic control. These data should provide the basis for further study of the lactase gene and for analysis of the genetic control of human lactase deficiency.