Investigation of the molecular structure of the soluble corneal proteins and the control mechanisms responsible for their synthesis has been minimal. For example, the major soluble' corneal protein (MCP) which comprises 40% of soluble corneal epithelial protein had undergone only fundamental biochemical characterization until this laboratory, utilizing the mixed oligonucleotide cloning of cDNA (MOPAC) technique, correctly identified its cDNA sequence as encoding a homologue of the unique (class 3) tumor-associated rat aldehyde dehydrogenase (ALDH3) gene. Preliminary survey of members of several other vertebrate classes (bird, reptile, amphibia, bony fish) by Western blotting, enzymatic assay, and direct amino acid sequencing, has not detected the expression of MCP/ALDH3. Our preliminary data also confirms that 1.7 Kb MCP/ALDH3 mRNA transcripts are normally detected by Northern blot analysis only in tissues that include corneal epithelial cells as their major constituent. It has been suggested that MCP's corneal function is derived from its enzymatic activity, although present in amounts that exceed catalytic requirements. Based on our observations we have concluded MCP/ALDH3 is only the second example of the genetic phenomena of 'gene sharing' and its presence in the mammalian cornea arises from a neutral selection process similar to the recruitment of crystallin proteins by the lens. We hypothesize that MCP/ALDH3 transcription is regulated by the utilization of a corneal specific cis-situated regulatory DNA sequence(s) (promoter/enhancer) and trans-acting DNA-binding proteins (DNABP). Delection mutants of putative DNA regulatory elements from 5'- and 3'-flanking regions, and intron sequences of the MCP/ALDH3 gene will be fused to a reporter gene (chloramphenicol acetyl transferase) and analyzed by transient expression assay In primary corneal cultures to Identify the tissue specificity of the MCP/ALDH3 transcriptional cis-control DNA elements. Nuclear extracts containing corneal specific trans-acting proteins that recognize promoter/enhancer DNA regulatory sequences will be characterized by DNAase I footprinting, and the gel retardation assay. Cell-lineage specific transcriptional factors such as the DNABP hypothesized to be present in corneal nuclear extracts likely influence expression of a number of corneal genes. Abundant and moderately abundant corneal cDNA members will be Identified by differential screening of cDNA libraries. Low abundance corneal cDNAs will be Isolated by a modification of the subtraction technique for cloning differentially expressed genes (amplification of 'remainder' cDNA, ARCS). Both classes Include candidate genes that may potentially utilize corneal specific enhancer/promoter element(s) and corneal specific DNA-binding protein(s).
