ORGANIZATION AND EXPRESSION OF CYSTATIN GENES

这项提案的总体目标是了解基因的机制， 胱抑素，特别是唾液胱抑素的表达， 调节基因转录的顺式和反式作用元件。 胱抑素是半胱氨酸蛋白酶抑制剂， 是 参与对内源性半胱氨酸的保护机制 在炎症或组织坏死期间释放的蛋白酶。 半胱氨酸蛋白酶抑制剂根据其功能分为三个家族。 分子结构家族1胱抑素主要存在于细胞内 并作为许多大鼠组织的组成成分存在。家族2 胱抑素主要存在于细胞外液中。激肽原是 家族3胱抑素，主要存在于血浆中。相对于家庭1 半胱氨酸蛋白酶抑制剂，其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蛋白酶抑制剂，但在 用β-激动剂异丙肾上腺素处理的大鼠的下颌下腺。 虽然家族3半胱氨酸蛋白酶抑制剂（激肽原）也可作为急性期 反应物在大鼠血浆中，它们也存在于正常血浆中。 这些观察结果表明，这三种细胞的生物合成和分泌 半胱氨酸蛋白酶抑制剂可能具有不同的调节机制。自从三 半胱氨酸蛋白酶抑制剂家族可能是从一个单一的原始 序列，似乎他们已经获得了不同的调制器， 响应控制元件。拟议研究的目的是 研究三种半胱氨酸蛋白酶抑制剂在大鼠肝组织中的差异表达， 转录水平，并分析顺式作用的转录 半胱氨酸蛋白酶抑制剂基因的元件。更准确地说，对应于 这些半胱氨酸蛋白酶抑制剂将被分离并用于研究转录 控制家族1的基因组克隆（非诱导型，组成型）和 家族2（诱导型）半胱氨酸蛋白酶抑制剂将使用各自的cDNA 探针、限制性酶切图谱和相关区域测序。基因组 家族3半胱氨酸蛋白酶抑制剂（激肽原）结构已经相当令人满意 表征了然后，我们打算定位潜在的监管区域， 大鼠唾液半胱氨酸蛋白酶抑制剂（家族2）基因的顺式作用元件 负责基础水平和/或高水平的促进者 这个基因的转录。这将通过加入5 '- 半胱氨酸蛋白酶抑制剂基因的侧翼区域和一系列缺失突变 与CAT报告基因连接并测定报告基因中的变化 在多种细胞中表达。最后，我们建议研究DNA- 使用凝胶迁移率变动测定蛋白质相互作用， 确定负责调节大鼠的反式作用因子 唾液胱抑素基因表达。

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.