REGULATION OF CYSTATIN S GENE EXPRESSION

胱抑素S基因表达的调控

• 批准号: 6379637
• 负责人: PHYLLIS Ann SHAW
• 金额: $ 31.91万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-05-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6379637
• 关键词: animal genetic material tag; cell differentiation; cell growth regulation; developmental genetics; gene expression; gene induction /repression; genetic promoter element; genetically modified animals; glycoproteins; in situ hybridization; laboratory mouse; laboratory rat; nucleic acid sequence; protein structure function; saliva; salivary glands; secretory protein; site directed mutagenesis; tissue /cell culture; transcription factor

Mammalian salivary glands produce secretions that initiate digestion and bathe and protect the oral cavity. Salivary cystatins, proteins secreted by the submandibular and parotid glands, are believed to play a major role in this protection. Cystatins are evolutionarily conserved, naturally occurring cysteine proteinase inhibitors that regulate proteolysis by endogenous cysteine proteinases, as well as by proteinases of microbial pathogens and of some viruses. Although cystatins inhibit cysteine proteinases in vitro, their in vivo functions have not been delineated in detail. Since our long-term goal is to understand the role of cystatins in growth, differentiation, morphogenesis, and function of salivary glands, we propose to examine mechanisms governing expression of the cystatin S gene, which is expressed in rat salivary glands. This proposal will elaborate upon our previous findings that the cystatin S gene is unique in that it is cell type-and salivary gland-specific, it is expressed at a specific stage of posnatal development of the submandibular gland, it is turned off in adult animals, and it can be induced in adult rats by beta-receptor mediated mechanisms. In addition, its 5' flanking sequence has three sequence elements (I, II, and III) that are common to salivary gland-specific genes, and inserted between conserved elements II and III is a GT rich region (GT rich regions are thought to either inhibit or increase transcriptional activity of specific genes). Two major or hypotheses to be tested are that: 1) the conserved sequence elements present in all salivary gland-specific genes are the genetic elements that dictate salivary gland-specific phenotype, and 2) these conserved sequence elements somehow participate in the beta-adrenergic modulation of expression of a salivary gland-specific gene, cystatin S. Our specific aims are to: 1. determine the role of the conserved salivary gland-specific DNA sequence elements in cells in culture 2. identify and delineate potential regulatory elements In the cystatin S promoter that mediate IPR-induced cystatin S gene expression 3. verify that the cis-acting conserved salivary gland-specific regulatory elements, and the regulatory elements that mediate IPR-induced cystatin S gene expression, that were identified in tissue culture act in vivo using transgenic mice 4. determine whether there are changes in trans-acting factors that accompany changes in expression of the cystatin S gene

哺乳动物的唾液腺分泌物，启动消化和洗澡，并保护口腔。 唾液半胱氨酸蛋白酶抑制剂，由下颌下腺和腮腺分泌的蛋白质，被认为在这种保护中起主要作用。胱氨酸蛋白酶抑制剂是进化上保守的天然存在的半胱氨酸蛋白酶抑制剂，其调节内源性半胱氨酸蛋白酶以及微生物病原体和一些病毒的蛋白酶的蛋白水解。 虽然半胱氨酸蛋白酶抑制剂在体外抑制半胱氨酸蛋白酶，但它们在体内的功能尚未详细描述。 由于我们的长期目标是了解胱抑素在唾液腺的生长，分化，形态发生和功能中的作用，我们建议检查机制的胱抑素S基因，这是在大鼠唾液腺中表达的表达。 该建议将详细阐述我们以前的研究结果，半胱氨酸蛋白酶抑制剂S基因是独特的，因为它是细胞类型和唾液腺特异性的，它是在一个特定的阶段表达的出生后发育的下颌下腺，它是关闭的成年动物，它可以在成年大鼠诱导β受体介导的机制。 此外，其5'侧翼序列具有唾液腺特异性基因共有的三个序列元件（I、II和III），并且在保守元件II和III之间插入的是富含GT的区域（富含GT的区域被认为抑制或增加特异性基因的转录活性）。 待检验的两个主要假设是：1）存在于所有唾液腺特异性基因中的保守序列元件是决定唾液腺特异性表型的遗传元件，和2）这些保守序列元件以某种方式参与唾液腺特异性基因半胱氨酸蛋白酶抑制剂S的表达的β-肾上腺素能调节。 我们的具体目标是：1. 确定保守的唾液腺特异性DNA序列元件在培养的细胞中的作用2. 鉴定和描绘介导IPR诱导的半胱氨酸蛋白酶抑制剂S基因表达的半胱氨酸蛋白酶抑制剂S启动子中的潜在调控元件3. 使用转基因小鼠，验证在组织培养中鉴定的顺式作用的保守唾液腺特异性调节元件和介导IPR诱导的胱抑素S基因表达的调节元件在体内起作用4。 确定是否存在伴随胱抑素S基因表达变化的反式作用因子的变化