[Promoter cloning and activity analysis for iroquois homeobox gene 1 in gastric mucosa cell line].
OBJECTIVE
To clone core promoter regions of iroquois homeobox gene 1 (IRX1) gene and evaluate the regulatory mechanism of IRX1 transcription.


METHODS
Upstream sequence from transcriptional start site was predicted using bioinformatics methods. Serial deleted fragments from IRX1 promoter sequences were amplified by PCR and luciferase reporter plasmids were constructed. The luciferase intensity was analyzed after transferring reporters into GES-1 gastric mucosa cell line.


RESULTS
The promoter of IRX1 was predicted to be within 700 bp from the 5'-flanking region of IRX1 gene. Eight serial deleted luciferase reporter plasmids were constructed. The transcriptional activity of different fragments was expressed as following: p-416>p-584>p-715>p-350>p-687>p-320>p-188>p-92. Except p-320 and p-188, the transcriptional activity of other 6 fragments was higher than that of PGL3-basic plasmid. The transcriptional activity was the highest in p-416 and decreased sharply from p-320 to p-188.


CONCLUSIONS
The fragment p-416 shows the strongest promoter activity. The sequence from -320 bp to -188 bp is identified as core promoter region, which is focused as key sequence for further regulatory analysis, since some binding sites for important transcriptional factors such as Sp1 and TFII D are predicted.
Department of Surgery, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai Institute of Digestive Surgery, Shanghai 200025, China.
Xiao-bo
XB
Guo
Lei
L
Guo
Jian-lin
JL
Wu
Wei-ren
WR
Liu
Jun
J
Ji
Jia-nian
JN
Zhang
Bing-ya
BY
Liu
Zheng-gang
ZG
Zhu
Ying-yan
YY
Yu
D002460:Cell Line; D003001:Cloning, Molecular; D005753:Gastric Mucosa / Q000166:cytology; D005801:Genes, Homeobox; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000235:genetics*
D018398:Homeodomain Proteins; C501591:IRX1 protein, human; D014157:Transcription Factors