Characterization of the mouse aldose reductase gene and promoter in a lens epithelial cell line.
OBJECTIVE
To clone and characterize the mouse aldose reductase (AR) gene and evaluate the functional promoter under basal and hypertonic conditions in mouse lens epithelial cells.


METHODS
The mouse AR gene structure was determined by DNA sequencing, and its chromosomal localization was determined by fluorescent in situ hybridization. A luciferase reporter gene was utilized to assess promoter activities of mouse, rat, and human AR deletion constructs as well as mouse site-directed mutants containing specific deletions of an aldose reductase enhancer element (AEE) or a tonicity response element (TonE). Electrophoretic mobility shift assays were performed to evaluate binding of trans-acting factors to mouse AEE and TonE.


RESULTS
The mouse AR gene (14.2 Kb) is located on chromosome 6. The basal AR promoter activity was greatest for the rat followed by mouse and human. All 3 species demonstrated increased promoter activity under hypertonic conditions. Deletion of TonE decreased mouse AR basal activity 2.5-fold and substantially reduced the osmotic response. Deletion of AEE had only a slight effect on AR promoter activity. Nevertheless, AEE strongly bound multiple trans-acting factors under nonstressed and stressed conditions, while weaker binding was evident for TonE.


CONCLUSIONS
Species-specific differences in AR promoter activities suggest the presence of unique regulatory cis-acting elements. The effects of AEE or TonE on AR transcription appear to involve complex transcriptional regulatory mechanisms.
McGowan
M H
MH
Laboratory of Mechanisms of Ocular Diseases, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Iwata
T
T
Carper
D A
DA
D000449:Aldehyde Reductase / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004848:Epithelium / Q000201:enzymology; D005786:Gene Expression Regulation; D006801:Humans; D006982:Hypertonic Solutions; D017404:In Situ Hybridization, Fluorescence; D007908:Lens, Crystalline / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D051381:Rats; D013045:Species Specificity; D013312:Stress, Physiological / Q000378:metabolism; D015533:Transcriptional Activation
D006982:Hypertonic Solutions; D000449:Aldehyde Reductase