Analysis of gene regulation in rabbit corneal epithelial cells induced by ultraviolet radiation.
Ultraviolet (UV)-induced cataracts are becoming a major environmental health concern because of the possible decrease in the stratospheric ozone layer. Experiments were designed to isolate gene(s) affected by UV irradiation in rabbit cornea tissues using fluorescent differential display-reverse transcription-polymerase chain reaction (FDDRT-PCR). The epithelial cells were grown in standard medium for 2 or 4 hours post treatment. Cornea epithelial cells were irradiated with UVB for 20 minutes. RNA was extracted and amplified by reverse transcriptase-polymerase chain reaction using poly A+ specific anchoring primers and random arbitrary primers. Polyacrylamide gel electrophoresis revealed several differentially expressed genes in untreated versus UV irradiated cells. Complimentary DNA (cDNA) fragments resulting from fluorescent differentially expressed mRNAs were eluted from the gel and re-amplified. The re-amplified PCR products were cloned directly into the PCR-TRAP cloning system. These data showed that FDDRT-PCR is a useful technique to elucidate UV-regulated gene expressions. Future experiments will involve sequence analysis of cloned inserts. The identification of these genes through sequence analysis could lead to a better understanding of cataract formation via DNA damage and mechanisms of prevention.
Stevens
Jacqueline J
JJ
Molecular Biology Research Laboratory, NIH-Center for Environmental Health, College of Science, Engineering and Technology, Jackson State University, 1400 J R Lynch Street, Jackson, Mississippi 39217, USA. jacqueline.j.stevens@jsums.edu
Rogers
Christian
C
Howard
Carolyn B
CB
Moore
Caronda
C
Chan
Lai-Man
LM
D000818:Animals; D002386:Cataract; D002478:Cells, Cultured; D004249:DNA Damage; D004847:Epithelial Cells / Q000378:metabolism / Q000528:radiation effects*; D019573:Epithelium, Corneal / Q000378:metabolism / Q000528:radiation effects*; D005786:Gene Expression Regulation / Q000528:radiation effects; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014466:Ultraviolet Rays
D012333:RNA, Messenger