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(Investigative Ophthalmology and Visual Science. 2003;44:235-243.)
© 2003 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.02-0162
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Isolation and Characterization of the Human CP49 Gene Promoter

Daryle J. DePianto, John F. Hess, Thomas N. Blankenship, and Paul G. FitzGerald

From the Department of Cell Biology and Human Anatomy, School of Medicine, University California, Davis, California.

PURPOSE. This study focuses on the identification of regulatory elements that contribute to lens-specific expression of the human CP49 gene within the 5'-flanking DNA sequences.

METHODS. The DNA sequence upstream of the human CP49 coding region was subcloned as a set of 5' and 3' deletion series. The constructs were transfected into lens (N/N1003A) and nonlens (NIH3T3) cell lines and chicken primary lens cultures, to test for promoter activity and specificity. To further test the specificity, a portion of the 5' flanking DNA sequence was used to drive transgene expression in mice. The flanking DNA sequence was analyzed for potential transcription factor–binding sites.

RESULTS. The 5'-flanking DNA preferentially activated reporter gene expression in a lens-preferred manner when transfected into cultured cells. Transgene expression driven by the CP49 promoter region was lens specific. Analysis of the proximal promoter sequence revealed the presence of potential binding sites for the AP-1, AP-2, and OCT-1 transcription factors and the absence of TATA and CAAT boxes.

CONCLUSIONS. The sequence upstream of the CP49 gene possesses promoter activity and is able to drive lens-preferred expression in both transfection and transgenic experiments. Promoter activity is dependent on the presence of the proximal 300 bp directly upstream of the coding region.






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