Isolation and characterization of the human CP49 gene promoter.
OBJECTIVE
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.
DePianto
Daryle J
DJ
Department of Cell Biology and Human Anatomy, School of Medicine, University of California-Davis, One Shields Avenue, Davis, CA 95616, USA.
Hess
John F
JF
Blankenship
Thomas N
TN
FitzGerald
Paul G
PG
D016475:3T3 Cells / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002642:Chick Embryo; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D007124:Immunoenzyme Techniques; D007381:Intermediate Filament Proteins / Q000235:genetics* / Q000378:metabolism; D007908:Lens, Crystalline / Q000196:embryology / Q000378:metabolism; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011817:Rabbits; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection
D005136:Eye Proteins; D007381:Intermediate Filament Proteins; D014157:Transcription Factors; C084808:phakinin