ORGANIZATION AND EXPRESSION OF CYSTATIN GENES

这项建议的总体目标是了解基因的作用机制。 半胱氨酸氨基转移酶，特别是唾液半胱氨酸氨基转移酶的表达 调节基因转录的顺式和反式作用元件。 半胱氨酸是半胱氨酸蛋白酶抑制剂，被认为是 参与内源性半胱氨酸的保护机制 在炎症或组织坏死过程中释放的蛋白酶。 根据半胱氨酸氨基转移酶的不同，将其分为三个家族 分子结构。家族1主要存在于细胞内 并作为许多大鼠组织的组成成分存在。家庭2 胱抑素主要存在于细胞外液中。Kininogens是 家族3，主要存在于血浆中。而不是家庭1 胱抑素，是大鼠组织的组成成分，家族2 在正常大鼠组织中未检测到半胱氨酸氨基转移酶，但在 β-激动剂异丙肾上腺素对大鼠颌下腺的作用。 尽管家族3半胱氨酸氨基转移酶(激肽原)也起急性期的作用 大鼠血浆中的反应物，它们也存在于正常血浆中。 这些观察表明，三者的生物合成和分泌 半胱胺类药物可能有不同的调节机制。从三点开始 胱抑素家族可能是从一个单一的原始家族进化而来的 序列，似乎它们已经获得了不同的调节器 响应性控制元件。拟议研究的目的是 三种半胱氨酸氨基转移酶在细胞内的差异表达研究 转录水平，并分析顺式作用转录 半胱氨酸氨基转移酶基因元件更准确地说，CDNA克隆对应于 这些半胱氨酸类药物将被分离出来，并用于研究转录 控制力。家系1的基因组克隆(不可诱导的、构成的)和 将使用相应的CDNA分离2族(可诱导的)半胱氨酸菌素 探针、限制性内切酶图谱和相关区域测序。基因组学 半胱氨酸氨基转移酶3家族(激肽原)的结构已基本确定 特色化的。然后，我们打算定位潜在的监管区域 大鼠唾液胱抑素(家族2)基因的顺式作用元件 负责基层和/或高层的推动者 这种基因的转录。这将通过加入5‘- 半胱氨酸氨基转移酶基因侧翼区与一系列缺失突变 一个猫报告基因，并检测报告基因的变化 在多种细胞中表达。最后，我们建议研究DNA- 使用凝胶迁移率变化分析的蛋白质相互作用 确定负责调节老鼠的反式作用因素 唾液胱抑素基因的表达。

The overall goal of this proposal is to understand mechanisms of gene expression of cystatins, particularly salivary cystatins, with emphasis on cis- and trans-acting elements that modulate gene transcription. Cystatins are cysteine proteinase inhibitors that are thought to be involved in the protective mechanisms against endogenous cysteine proteinases that are released during inflammation or tissue necrosis. The cystatins are grouped into three families on the basis of their molecular structure. Family 1 cystatins are found mainly intracellulary and are present as constitutive components of many rat tissues. Family 2 cystatins are mainly present in extracellular fluids. Kininogens are family 3 cystatins, present mainly in the plasma. As opposed to family 1 cystatins, which are constitutive components of rat tissues, family 2 cystatin is not detected in normal rat tissues, but is induced in submandibular glands of rats treated with beta-agonist isoproterenol. Although family 3 cystatins (kininogens) also act as acute-phase reactants in rat plasma, they are also present in the normal plasma. These observations suggest that biosynthesis and secretion of the three cystatins may have different mechanisms for regulation. Since three cystatin families have presumably evolved from a single primordial sequence, it appears that they have acquired distinct modulator responsive control elements. The aim of the proposed research is to study the differential expression of three cystatins at the transcriptional level and to analyze the cis-acting transcriptional elements of cystatin genes. More precisely, CDNA clones corresponding to these cystatins will be isolated and used to study the transcriptional control. Genomic clones for family 1 (non inducible, constitutive) and family 2 (inducible) cystatins will be isolated using respective CDNA probes, restriction mapped and pertinent regions sequenced. Genomic structure of family 3 cystatin (kininogen) has been fairly will characterized. We then intend to locate the potential regulatory regions (cis-acting elements) of the rat salivary cystatin (family 2) gene promotor responsible for either the basal-level and/or high-level transcription of this gene. This will be accomplished by joining the 5'- flanking regions of the cystatin gene and series of deletion mutations to a CAT reporter gene and assaying for changes in reporter gene expression in a variety of cells. Lastly, we propose to study DNA- protein interactions using the gel mobility shift assay in order to identify the trans-acting factors responsible for regulation of rat salivary cystatin gene expression.