Functional analysis of the cell-specific enhancer in the human proopiomelanocortin gene by beta-galactosidase histochemical staining.
Nucleotide sequences responsible for the cell-specific expression of the human proopiomelanocortin (POMC) gene were analyzed by histochemical staining of beta-galactosidase in culture cells transfected with chimeric genes containing the 5'-flanking regions of the human POMC gene fused to the Escherichia coli lacZ gene. The chimeric genes were stably introduced into various culture cells, including AtT-20 cells, which express the endogenous mouse POMC gene. Whereas the control gene containing the cytomegalovirus enhancer was expressed in all cell lines tested, only AtT-20 cells supported the efficient transcription of the gene containing 2.9 kb of the human POMC 5'-flanking region. These results indicate that the stable transfection-expression system utilizing the histochemical detection of the gene expression is a useful method for the analysis of cell-specific gene expression. These results have also confirmed that the trans-acting factors in mouse AtT-20 cells interact with the human POMC gene promoter region and activate the transcription of the gene. Deletion analysis has demonstrated that the profiles of the transcriptional activity of the various human POMC-lacZ fusion genes are similar to those of the rat POMC gene described previously. Comparison of the human and the rat 5'-flanking sequences revealed close homology in several regions, which might be involved in the efficient transcription of the POMC gene in AtT-20 cells.
Tsukada
T
T
Department of Experimental Radiology, Kyoto University Faculty of Medicine, Japan.
Nakai
Y
Y
Fukushima
M
M
Usui
T
T
Imura
H
H
Takebe
H
H
D000818:Animals; D001483:Base Sequence; D002461:Cell Line, Transformed; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006651:Histocytochemistry; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011333:Pro-Opiomelanocortin / Q000096:biosynthesis / Q000235:genetics*; D001616:beta-Galactosidase / Q000032:analysis* / Q000235:genetics
D011333:Pro-Opiomelanocortin; D001616:beta-Galactosidase