Abstract:

To analyze the regulatory regions upstream of the human PCAN1 gene, a reporter plasmid containing of a 2.6?kb PCAN1 promoter was constructed by the PCR method, and DNA cisacting elements within this promoter were further identified. MethodsA 2.6?kb fragment upstream of the PCAN1 gene was amplified by PCR using human genomic DNA as a template, and it was then inserted into the upstream of the luciferase reporter gene in pGL3basic vector to generate pGL3p2.6kb. Its promoter activity was determined with dualluciferase reporter assay after it has been transfected into prostate cancer LNCaP cells. A series of 5′ deletion mutantluciferase reporter plasmids derived from pGL3p2.6kb were obtained via Eraseabase System. The mutant plasmids were transfected into the LNCaP cells and the promoter activities were determined by dualluciferase reporter assay. ResultsThe sequence of the 2.6?kb promoter fragment proved to be correct by DNA sequecing. Dualluciferase reporter assay showed that the 2.6?kb fragment presented a significant promoter activity with pRLTK into LNCaP cells. Analysis of the mutants showed that PCAN1 promoter activities increased 2.24fold and 2.53fold after the -1?599?bp to -1?541?bp and -347?bp to -84?bp within the promoter were deleted, while the promoter activity decreased 2.11fold after the -1?541?bp to -1?226?bp was deleted. ConclusionA cloned 2.6?kb fragment upstream of the PCAN1 gene presents strong promoter activity. With deletion analysis two negative and one positive regulatory region were identified within the promoter.

Key words: PCAN1 gene, Promoter regions (Genetics), Gene deletion, Gene expression