Analysis of human cp 49 gene regulatory elements

Purpose. Lens specific expression of CP 49 is due to transcriptional regulation; Northern blotting reveals no RNA transcripts for BF 49 except in lens RNA samples. We peformed experiments to identify the DNA sequences responsible for this regulation. Methods. We have subcloned human genomic DNA fragments corresponding to the 5' end of the CP 49 gene as well as each intron into Luciferase reporter plasmids. These plasmids {and internal control plasmids) were transfected using Lipofectamine reagents into various cell lines, including the lens derived N1003A and Alpha TN-4. Results. Luciferase assays provide evidence that both a 3.0 kb and 900 bp DNA fragment from the CP 49 upstream region contain DNA sequences sufficient to direct transcription initiation and expression of the Luciferase reporter in both Alpha TN-4 and N1003A cells. Similar experiments reveal no intron located sequence elements with enhancer like activity. Conclusions. Despite the limitation,in vivo, of CP 49 expression to the lens fiber cell we have been able to use lens epithelial derived cell lines to study gene expression. Furthermore, our data do not support the hypothesis that enhancers located in introns are involved in expression.