Recently, this laboratory has been exploring gene expression in the cornea. Particular attention is being given to the corneal epithelial cells because they accumulate very high amounts of soluble enzymes (10 to 30 percent of total soluble protein) reminiscent of lens enzyme crystallins. We have cloned the cDNAs and genes for two of these putative corneal """"""""enzyme crystallins"""""""" [aldehyde dehydrogenase class 3 (ALDH3) and transketolase (TKT)] from the mouse. Full-length cDNAs have been sequenced, and the genes that contain multiple introns have been partially characterized. The ALDH3 and TKT flanking regions have many consensus sequences for control elements that respond to stress induction (i.e., NF-kB, AP-1, NF-1, AP-2, SP-1, GRE, ARE, and XRE). The ALDH3 has a single promoter containing a TATAAA box, but the TKT gene appears to have two promoters. The major promoter lacks a TATAA box and is used in the cornea and other tissues, but the minor promoter (about 650 bp upstream) has a possible TATAA sequence and is used infrequently in liver. The -1103/+96, -515/+96, and -51/+96 TKT promoter fragments fused to the chloramphenicol acetyltransferase gene are functional in transfected lens epithelial cell lines, with higher activities associated with the larger fragments. Although ALDH3 and TKT mRNA are present in the mouse corneal epithelial cells before eye opening 2 weeks after birth, there is a strong increase in their tissue-specific accumulation thereafter, consistent with exposure to light. TKT expression shows the same high level in cornea, localization to the epithelium, and developmental increase in the rat. Transgenic mouse experiments using ALDH promoter (up to 1 kbp)/CAT constructs have failed to identify cornea-specific control elements so far. Cytochemical and in situ hybridization experiments have established the concentrated accumulation of these enzymes in the corneal epithelial cells; enzymatic tests confirmed that these enzymes are active. TKT was also found at moderate levels in other eye tissues. In the healing corneal epithelium, the enriched expression of TKT transcripts in the single-cell layer of the leading edge was suppressed until the cells started to stratify. In situ hybridization experiments have shown that Pax-6 is highly expressed in the stem cells and the basal epithelial cells of the mouse and monkey cornea. Current experiments are aimed at obtaining cultured corneal epithelial cells for transfection studies and identifying transcription factors (possibly Pax-6) that are important for the expression of these corneal """"""""enzyme crystallins.""""""""
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