[Cloning and transcriptional regulation analysis of amelogenin promoter in different cells].
OBJECTIVE
To clone different promoter region of amelogenin gene and analyze their transcriptional activity.


METHODS
The upstream regulation sequences of amelogenin gene were retrieved and analyzed. Amplified by PCR from the genomic DNA of mouse C57BL/6J and digested with restriction endonucleases enzyme, different transcriptional regulation sequences of 5'flanking of amelogenin gene including the basal promoter were cloned and ligated with luciferase gene in PGL3-Basic vector. These report vectors were transiently transfected into CHO, Hela and UMR-106 cells and luciferase assay was performed to analyse the transcription activation of these promoters.


RESULTS
6 promoters different in length were cloned. The activity of luciferase was very strong in Hela cells. On the contrary, the CHO and UMR-106 cells showed weak fluorescence. Luciferase activity fluctuated with the different promoter lengths in Hela cell as well as in CHO and UMR-106 cells. 975 bp and 532 bp of amelogenin 5'flanking DNA had a strong transcriptional activation, but 285 bp of amelogenin 5'flanking DNA had a weaker transcriptional activation.


CONCLUSIONS
The sequence of amelogenin promoter can be activated in Hela cell. Hela cell can be used as a good model to study the transcriptional regulation of amelogenin promoter. According to the different activities of different lengths, it is suggested that there were some potential siliencer located between -975 and -532, and some potential activator in the region between -532 and -285.
Zhu
Qing-lin
QL
College of Stomatology, Fourth Military Medical University, Xi'an 710032, Shanxi Province, China.
Wu
Bu-ling
BL
Yu
Qing
Q
Kong
Hui
H
Guo
Ting
T
Fei
Jian
J
Wang
Zhu-gang
ZG
D053523:Amelogenin / Q000235:genetics*; D000818:Animals; D016466:CHO Cells; D003001:Cloning, Molecular; D003412:Cricetulus; D006367:HeLa Cells; D006801:Humans; D008810:Mice, Inbred C57BL; D011401:Promoter Regions, Genetic; D015533:Transcriptional Activation
D053523:Amelogenin