Purpose. To understand the molecular basis for the high expression of the duck δ2-crystallin gene and the low expression of the chicken δ2 gene in the lens. Methods. Various δ2 promoter fragments were fused to the bacterial CAT reporter gene which was linked to 732 bp of intron 3 containing the chicken 62 enhancer (δ2E) in pSVOCAT. Chicken δEF1 element (or the comparable region from the duck 62 gene) was placed within the chicken αA -148/+77 promoter fragment between positions -96 and -56, which contained a polylinker sequence, and was fused to the CAT gene in p8CAT. Transfection tests were conducted in 14-day-old embryonic chicken lens epithelial cells. Transgenic mice were produced at the NEI Transgenic Facility. Results. The chicken 52 promoter construct comprising the whole spacer (-4.5 kbp/+46) gave about 3 times less CAT activity than the -308/+46 promoter construct in transfected embryonic chicken lens cells. The larger promoter fragment produced 2 lines of trangenic mice with lens-specific transgene expression, while the smaller promoter gave multiple lines with transgene expression in the lens and brain (Purkinje cells). Gel mobility tests showed that the -3509/-2930 and -1710/-1478 sequences in the spacer formed complexes with lens nuclear extract; the former gave 5 DNase I protected regions. Finally, in transfection tests the chicken δEF1 suppressed chicken αA promoter activity, while the comparable duck sequence, which has a single bp difference, did not. Conclusions. These data suggest (1) a lens and brain silencing action located in the spacer upstream of the chicken 62 gene and (2) that a mutation in the negative δEF1 element of the duck δ2E allows high expression in the lens.
|Original language||English (US)|
|Journal||Investigative Ophthalmology and Visual Science|
|State||Published - Feb 15 1996|
ASJC Scopus subject areas
- Sensory Systems
- Cellular and Molecular Neuroscience