Although the mechanisms involved in dentinogenesis are unknown, it is clear that a unique set of extracellular matrix (ECM) proteins participates in the formation of predentin and its subsequent mineralization to form dentin. Mature odontoblasts secrete collagen at the cell border and non-collagenous proteins (NCPs) at the mineralization front (possibly through odontoblastic processes). Here NCPs form complexes with collagen. Carbonate apatite crystals are formed and this site-specific process of crystal initiation and growth is believed to be controlled by the collagen-NCP complex. In order to elucidate details of dentinogenesis, additional information relative to the NCPs, their metabolism and gene regulation is needed. Of particular interest are proteins found uniquely in dentin ECM. One dentin specific protein, dentin sialoprotein (DSP), a 53 kDa protein, is a sialic acid-rich protein similar in overall properties to cell attachment proteins bone sialoprotein (BSP) and osteopontin (OPN). However, DSP is synthesized only by odontoblasts and dental pulp and not by osteoblasts or other cell types. The sequence of DSP deduced from its cDNA is dissimilar to those of BSP, OPN and other proteins. DSP contains consensus sequences for N- and O-glycosylation sites, as well as casein kinase and 11 phosphorylation sites. Northern blots detected multiple transcripts of approximately 4.6 kb and 1.5 kb. Recent Southern blot analysis of genomic clones strongly suggests the presence of two related DSP genes. We hypothesize that the synthesis and secretion of DSP is crucial to the formation of healthy dentin. In order to test this hypothesis, we will employ molecular biological techniques to elucidate gene structures and their regulation. We propose the following Specific Aims: i. To determine the genomic organization of DSP gene(s). 2. To characterize multiple DSP transcripts and to examine the spatial and temporal expression of these transcripts. 3. To characterize the promoter(s) of DSP gene(s) including DNA sequence, minimal regulatory sequences and nuclear transcription factors and 4. To study the potential regulation of the DSP gene(s) by signal transducing molecules. The studies outlined in this proposal will provide a solid molecular basis for understanding important regulatory events controlling dentinogenesis.