ORGANIZATION AND EXPRESSION OF CYSTATIN GENES

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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.