RESUMO
Egr-1 is a radiation-inducible transcription factor. It was demonstrated that the regulatory sequence in 5' flanking region of Egr-1 gene could confer the radiation inducibility upon a tumoricidal gene to which it was linked, and was consequently used in experiments on the gene-radiotherapy to tumor. As a first step to test the possibility, we have isolated the 445 bp-long regulatory sequence of Egr-1 gene from BALB/c mouse gcnomic DNA with PCR method. Sequence analysis confirmed that nucleotide sequence of this cloned fragment was identical with one reported previously with only one base difference in A to G substitution at residue - 392, and had six CC( A + T)6GG domains which were essential to the radiation-inducible property of Egr-1 gene. This fragment then was inserted into the Mlu I-Bgl II site of pGL3, a reporter vector containing luciferase gene. The constructs were used to transfect mouse malignant melanoma cells (B16) to characterize the regulatory function of this CC ( A + T)_(6)GG-rich sequence after exposure to r-radiation by a ~(60) Co source. The results indicate that the activity of luciferase in transfectant increases by 138% as compared with control levels, demonstrating that r-radiation inducibility of gene expression can be conferred by the regulatory sequence.