[Structural characterization of 5' flanking regulatory region of DNA repair gene Rad51].

OBJECTIVE: To clarify the regulatory elements of Rad51 gene in its 5'flanking region.
METHODS: Various constructs were obtained by cloning different DNA fragments into pGL3 reporter vector. These constructs were then introduced into osteosarcoma cell line U2-OS by calcium phosphate method for transient expression of reporter gene, and luciferase activities were measured by luciferase assay.
RESULTS: Cells transfected with pGL3 constructs containing fragment -964 to +1430 and -733 to +1430 showed high luciferase activities. Obvious elevation of luciferase activities was also observed in cells transfected with pGL3 constructs containing four shorter derivative fragments -964 to -412, -746 to -412, -651 to -412 and -536 to -412. The highest luciferase activities were measured in transfected cells with plasmids containing fragment -964 to -412, and the lowest were in transfected cells with plasmids containing fragment -536 to -412. Luciferase activities in transfected cells with plasmids containing fragment -651 to -412 were higher than that in transfected cells with plasmids containing fragment -746 to -412.
CONCLUSION: It is believable that the basic transcription-promoting element (promoter) for Rad51 gene resides between -536 to -412, and two transcription-enhancing elements (enhancer) or binding sites of positive transcription factors reside between -651 to -536 and -964 to -746, whereas one transcription-inhibiting element (silencer) or binding site of negative transcription factor may reside between -746 to -651.