Limits...
Analysis of gene regulation in rabbit corneal epithelial cells induced by ultraviolet radiation.

Stevens JJ, Rogers C, Howard CB, Moore C, Chan LM - Int J Environ Res Public Health (2005)

Bottom Line: Ultraviolet (UV)-induced cataracts are becoming a major environmental health concern because of the possible decrease in the stratospheric ozone layer.Complimentary DNA (cDNA) fragments resulting from fluorescent differentially expressed mRNAs were eluted from the gel and re-amplified.The identification of these genes through sequence analysis could lead to a better understanding of cataract formation via DNA damage and mechanisms of prevention.

View Article: PubMed Central - PubMed

Affiliation: 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

ABSTRACT
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.

Show MeSH

Related in: MedlinePlus

Cloning of reamplified PCR products. Four colonies for each differentially selected band reamplified were checked for inserts by colony PCR. 100 bp ladder (NEB) was used as a size standard in lanes 1A, 1B and 1C for gels. JS represents selected differentially bands from normal and UVB irradiated corneal epithelial cells. Gel A: Lanes 2–5 (Inserts from JS #1; Lanes 6–9 (Inserts from JS #2); Lanes 10–13 (Inserts from JS #3); Lanes 14–17 (Inserts from JS # 4). Gel B: Lanes 2–5 (Inserts from JS #5; Lanes 6–9 (Inserts from JS #6); Lanes 10–13 (Inserts from JS # 7); Lanes 14–17 (Inserts from JS #8). Gel C: Lanes 2–5 (Inserts from JS #9; Lanes 6–9 (Inserts from JS #10); Lanes 10–13 (Inserts from JS #11).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3814696&req=5

f4-ijerph-02-00051: Cloning of reamplified PCR products. Four colonies for each differentially selected band reamplified were checked for inserts by colony PCR. 100 bp ladder (NEB) was used as a size standard in lanes 1A, 1B and 1C for gels. JS represents selected differentially bands from normal and UVB irradiated corneal epithelial cells. Gel A: Lanes 2–5 (Inserts from JS #1; Lanes 6–9 (Inserts from JS #2); Lanes 10–13 (Inserts from JS #3); Lanes 14–17 (Inserts from JS # 4). Gel B: Lanes 2–5 (Inserts from JS #5; Lanes 6–9 (Inserts from JS #6); Lanes 10–13 (Inserts from JS # 7); Lanes 14–17 (Inserts from JS #8). Gel C: Lanes 2–5 (Inserts from JS #9; Lanes 6–9 (Inserts from JS #10); Lanes 10–13 (Inserts from JS #11).

Mentions: The sizes of the differentially expressed bands chosen for reamplification were as follows: JS1, 300 bp; JS2, 550 bp; JS3, 400 bp; JS4, 300 bp; JS5, 280 bp; JS6, 300 bp; JS7, 280 bp; JS9, 200 bp; JS10, 800 bp; JS11, 200 bp; and JS12, 300 bp. The eleven re-amplified bands were cloned into the PCR-TRAP Cloning System. Four colonies for each band were checked for inserts by colony-PCR (Figure 4).


Analysis of gene regulation in rabbit corneal epithelial cells induced by ultraviolet radiation.

Stevens JJ, Rogers C, Howard CB, Moore C, Chan LM - Int J Environ Res Public Health (2005)

Cloning of reamplified PCR products. Four colonies for each differentially selected band reamplified were checked for inserts by colony PCR. 100 bp ladder (NEB) was used as a size standard in lanes 1A, 1B and 1C for gels. JS represents selected differentially bands from normal and UVB irradiated corneal epithelial cells. Gel A: Lanes 2–5 (Inserts from JS #1; Lanes 6–9 (Inserts from JS #2); Lanes 10–13 (Inserts from JS #3); Lanes 14–17 (Inserts from JS # 4). Gel B: Lanes 2–5 (Inserts from JS #5; Lanes 6–9 (Inserts from JS #6); Lanes 10–13 (Inserts from JS # 7); Lanes 14–17 (Inserts from JS #8). Gel C: Lanes 2–5 (Inserts from JS #9; Lanes 6–9 (Inserts from JS #10); Lanes 10–13 (Inserts from JS #11).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814696&req=5

f4-ijerph-02-00051: Cloning of reamplified PCR products. Four colonies for each differentially selected band reamplified were checked for inserts by colony PCR. 100 bp ladder (NEB) was used as a size standard in lanes 1A, 1B and 1C for gels. JS represents selected differentially bands from normal and UVB irradiated corneal epithelial cells. Gel A: Lanes 2–5 (Inserts from JS #1; Lanes 6–9 (Inserts from JS #2); Lanes 10–13 (Inserts from JS #3); Lanes 14–17 (Inserts from JS # 4). Gel B: Lanes 2–5 (Inserts from JS #5; Lanes 6–9 (Inserts from JS #6); Lanes 10–13 (Inserts from JS # 7); Lanes 14–17 (Inserts from JS #8). Gel C: Lanes 2–5 (Inserts from JS #9; Lanes 6–9 (Inserts from JS #10); Lanes 10–13 (Inserts from JS #11).
Mentions: The sizes of the differentially expressed bands chosen for reamplification were as follows: JS1, 300 bp; JS2, 550 bp; JS3, 400 bp; JS4, 300 bp; JS5, 280 bp; JS6, 300 bp; JS7, 280 bp; JS9, 200 bp; JS10, 800 bp; JS11, 200 bp; and JS12, 300 bp. The eleven re-amplified bands were cloned into the PCR-TRAP Cloning System. Four colonies for each band were checked for inserts by colony-PCR (Figure 4).

Bottom Line: Ultraviolet (UV)-induced cataracts are becoming a major environmental health concern because of the possible decrease in the stratospheric ozone layer.Complimentary DNA (cDNA) fragments resulting from fluorescent differentially expressed mRNAs were eluted from the gel and re-amplified.The identification of these genes through sequence analysis could lead to a better understanding of cataract formation via DNA damage and mechanisms of prevention.

View Article: PubMed Central - PubMed

Affiliation: 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

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus