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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.

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Fluorescent Differential Display. A typical fluorescent image of differentially expressed cDNAs using a 6% denaturing polyacrylamide electrophoresis. Differentially expressed bands obtained from untreated or UVB irradiated cornea epithelial cells are marked by arrows.
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f2-ijerph-02-00051: Fluorescent Differential Display. A typical fluorescent image of differentially expressed cDNAs using a 6% denaturing polyacrylamide electrophoresis. Differentially expressed bands obtained from untreated or UVB irradiated cornea epithelial cells are marked by arrows.

Mentions: The FDD allowed for parallel analysis of four RNA populations. The RNA populations compared were untreated versus UVB irradiated corneal epithelial cells with 2 hours post-treatment and untreated versus UVB irradiated corneal epithelial cells with 4 hours post-treatment. The FDD analysis of the untreated and UVB irradiated corneal epithelial cells indicated a number of similarities in gene expression between untreated and UVB irradiated cells (Figure 2). A large number cDNAs were present in both untreated and UVB irradiated corneal epithelial cells, however, the majority of these genes were not affected by UV radiation exposure. As a result, these cDNAs represent the house keeping genes found in corneal epithelial cells. Figure 2 represents a typical fluorescent image of differentially expressed cDNAs. Several differentially expressed bands were detected in the differential display gels. The eleven differentially expressed bands with the strongest intensities and best resolutions were excised from the gel and re-amplified using the same primer set. Eight of the differentially expressed bands were down-regulated and the other three differentially expressed bands were up-regulated in response to UVB exposure.


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)

Fluorescent Differential Display. A typical fluorescent image of differentially expressed cDNAs using a 6% denaturing polyacrylamide electrophoresis. Differentially expressed bands obtained from untreated or UVB irradiated cornea epithelial cells are marked by arrows.
© Copyright Policy
Related In: Results  -  Collection

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

f2-ijerph-02-00051: Fluorescent Differential Display. A typical fluorescent image of differentially expressed cDNAs using a 6% denaturing polyacrylamide electrophoresis. Differentially expressed bands obtained from untreated or UVB irradiated cornea epithelial cells are marked by arrows.
Mentions: The FDD allowed for parallel analysis of four RNA populations. The RNA populations compared were untreated versus UVB irradiated corneal epithelial cells with 2 hours post-treatment and untreated versus UVB irradiated corneal epithelial cells with 4 hours post-treatment. The FDD analysis of the untreated and UVB irradiated corneal epithelial cells indicated a number of similarities in gene expression between untreated and UVB irradiated cells (Figure 2). A large number cDNAs were present in both untreated and UVB irradiated corneal epithelial cells, however, the majority of these genes were not affected by UV radiation exposure. As a result, these cDNAs represent the house keeping genes found in corneal epithelial cells. Figure 2 represents a typical fluorescent image of differentially expressed cDNAs. Several differentially expressed bands were detected in the differential display gels. The eleven differentially expressed bands with the strongest intensities and best resolutions were excised from the gel and re-amplified using the same primer set. Eight of the differentially expressed bands were down-regulated and the other three differentially expressed bands were up-regulated in response to UVB exposure.

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