CLONING OF UVB-INDUCED GENES BY DIFFERENTIAL DISPLAY PCR
M. Pourmojib, S. Rezaie, F. Trautinger, R. Knobler and P. Neuner. Department of Dermatology, Division of Special and Environmental Dermatology, University of Vienna, Austria.
mRNA differential display (DD-PCR) was used to gain insight into the regulation of UV-light mediated gene expressions in keratinocytes. Human keratinocytes (HaCaT) were irradiated with UVB at 60 and 100 mJ/cm2, respectively. After a culture period of 30 minutes mRNA was extracted and amplified by reverse transcription polymerase chain reaction (RT-PCR) using a combination of one-base anchored oligo-dT and random arbitrary primers. Polyacrylamid gel electrophoresis revealed several cDNA fragments that were differentially displayed in untreated and UVB irradiated cells. cDNA fragments were eluted from the gel, reamplified and used as probes for Northern blot analysis to confirm UV regulated gene expressions in a time and dose dependent manner. Northern blot analysis revealed a time and UV-dose dependent up- or downregulation of mRNAs in HaCaT cells. Sequence analysis revealed that most UVB - regulated genes found by DD-PCR were of unknown origin or demonstrated little homology to recently described genes. However one cDNA clone showed 97% homology to a recently detected gene overexpressed in breast cancer. Further studies might clarify the pathogeneic role of this gene in the response of human skin to UV.
Figure 1) mRNA from untreated (lane 1) and UVB treated (60 mJ/cm², lane2) cells were reverse transcribed and amplified using the primer pair H-T11G and H-AP (Genhunter, Co. USA). cDNA were separeted on a 6% polyacrylamide denatured gel and detected by autoradiography.
mRNA differential display demonstrated a varity of gene expressions which were either up- or downregulated by UVB as indicated by arrows.
K1 and K2 were downregulated and U1 showed an upregulation after UVB treatment.
These effects were confirmed by Northern blot analysis (Fig.2).
Figure 2) Total RNA was extracted from UVB treated and untreated cells after a culture period of 30 minutes, separeted on a 1% agarose gel and transfered on nylon membrans. Blots were hybridized with cDNA fragments eluated from DD-PCR gel (K1, K2 and U1).
Lane1: untreated cells
Lane 2: UVB radiated cells, 60 mJ/cm²
Lane 3: UVB radiated cells, 100mJ/cm²
Sequence homology to other genes
All sequences were compared to sequences in the EMBL gene data bank. K1 and K2 showed no homology to any sequence in the gene bank. U1 showed 97%homology to a recently described gene,which is overexpressed in a variety of tumor cells in particular in breast cancer cells (Table 1).
Table 1) Information about differerntially expressed clones
mRNA differential display demonstrated that a variety of genes which are differentially expressed in UV treated and untreated cells.
Sequence analysis of isolated differentially expressed sequence tags (DEST) may help to characterize UV regulated genes. Determination of their full length sequence and subsequent transfection or gene knock out experiments should bring further insight into their functional role in the UV response