The human salivary glands are specialized tissues that produce a number of different proteins involved in protection of the oral cavity. If we are to fully understand salivary gland function, we need to know the mechanisms of tissue-specific regulation of gene expression involved in the expression of proteins in these glands. At present, little is known of these mechanisms. The long term objective of the proposed research is to develop the cystatin gene family as a model system in which to study the regulation of gene expression in human salivary glands. The type 2 cystatins are a group of cysteine proteinase inhibitors that are major protein constituents of saliva. They are also present in a variety of human tissues and body fluids. Biologically, the type 2 cystatins may be important in the regulation of endogenous cysteine proteinases, and as a defense against tissue penetration by parasites. Further, a defect in cystatin C has been implicated in a fatal hemorrhagic disease in humans. Although closely conserved in sequence, the proportion of the different type 2 cystatins in different locations varies substantially. The multiplicity of type 2 cystatins in humans, their wide tissue distribution, the variation in their relative levels in different sites, and their relative conservation of protein sequence, combine to make the cystatins an excellent model system in which to study differentiation and gene regulation in the human salivary glands. The fundamental hypothesis to be addressed by these studies is that the differential expression of various type 2 cystatins in the human salivary glands is due to tissue-specific molecular mechanisms causing differential transcription of certain cystatin genes, as opposed to differential mRNA stabilities, protein turnover rates, or other mechanisms. The number of different cystatin genes in humans, the tissue distribution of the various forms, and the relative rates of cystatin gene expression are unknown. Therefore, our fundamental hypothesis is presently untested, and the molecular mechanisms which could mediate salivary gland specific regulation are unknown. To test this hypothesis, recombinant DNA techniques will be used to perform experiments aimed at delineating the features of the molecular architecture involved in the expression of cystatin genes.
The specific aims are: 1. To isolate and characterize genomic DNA clones corresponding to the human type 2 cystatins. 2. To sequence the coding and promoter regions of the type 2 cystatin genes. 3. To use gene-specific probes in hybridization analyses (Northern blotting) of RNA from different human tissues. 4. To measure the rates of transcription of type 2 cystatin genes in different tissues. 5. To map the human chromosomal location of the cystatin genes. Information gained through this work will contribute to our understanding of the mechanisms of gene regulation involved in the synthesis of proteins in human salivary glands.
| Dickinson, D P; Thiesse, M; Hicks, M J (2002) Expression of type 2 cystatin genes CST1-CST5 in adult human tissues and the developing submandibular gland. DNA Cell Biol 21:47-65 |
| Thiesse, M; Millar, S J; Dickinson, D P (1994) The human type 2 cystatin gene family consists of eight to nine members, with at least seven genes clustered at a single locus on human chromosome 20. DNA Cell Biol 13:97-116 |
| Dickinson, D P; Zhao, Y; Thiesse, M et al. (1994) Direct mapping of seven genes encoding human type 2 cystatins to a single site located at 20p11.2. Genomics 24:172-5 |
| Dickinson, D P; Thiesse, M; Dempsey, L D et al. (1993) Genomic cloning, physical mapping, and expression of human type 2 cystatin genes. Crit Rev Oral Biol Med 4:573-80 |
| Millar, S J; Dempsey, D; Dickinson, D P (1992) High-yield recovery of recombinant DNA from poorly growing cosmid and lambda genomic clones. Biotechniques 13:554-6, 558-60, 562 |
