Analysis of the 5'-upstream region of mouse P/Q-type Ca2+ channel alpha1A subunit gene for expression in pancreatic islet beta cells using transgenic mice and HIT-T15 cells.
The omega-agatoxin-IVA-sensitive P/Q-type Ca(2+) channel plays a role in insulin release from the pancreatic islets of beta cells. To dissect the molecular mechanisms underlying beta cell expression of the P/Q-type channel, we characterized the 5'-upstream region of the mouse alpha(1A) subunit gene using transgenic mice and HIT insulinoma cells. The E. coli lacZ reporter gene was expressed in pancreatic acini and islets in transgenic mice carrying the 6.3 kb or 3.0 kb of the 5'-upstream region, although those with 1.5 kb or 0. 5 kb of the 5'-upstream region failed to show reporter expression on histological examination. As the expression of alpha(1A)subunit gene could not be detected in acini using RT-PCR analysis, the reporter expression in acini might have been ectopic expression. When linked to the placental alkaline phosphatase reporter gene to examine promoter activity for beta cell expression, the 6.3 kb and 3.0 kb fragment of the 5'-upstream region, but not the smaller 1.5 kb fragment, were able to drive reporter gene expression in HIT cells. The sequence between 3.0 and 1.5 kb upstream of the start codon enhanced thymidine kinase promoter activity in HIT cells, but not in fibroblast NIH3T3 cells. These results suggested that the beta cell-specific elements of the alpha(1A) subunit gene are likely to be located in the distal upstream region (-3021 to-1563) of the 5'-upstream sequence and that the 6.3 kb fragment of the 5'-upstream region alone might be a lack of a negative cis-regulatory element(s) to suppress the alpha(1A) subunit gene expression in acini.
Takahashi
E
E
Laboratory of Seeds Finding Technology, Eisai Co. Ltd, 5-1-3, Tokodai, Tsukuba, Ibaraki, 300-2635, Japan. e2-takahashi@hhc.eisai.co.jp
Miyamoto
N
N
Nagasu
T
T
D000818:Animals; D020865:Calcium Channels, P-Type / Q000737:chemistry / Q000235:genetics*; D020907:Calcium Channels, Q-Type / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D017930:Genes, Reporter; D007340:Insulinoma; D007515:Islets of Langerhans / Q000378:metabolism*; D046911:Macromolecular Substances; D051379:Mice; D008822:Mice, Transgenic; D010179:Pancreas / Q000378:metabolism; D010190:Pancreatic Neoplasms; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014162:Transfection / Q000379:methods; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000235:genetics
D020865:Calcium Channels, P-Type; D020907:Calcium Channels, Q-Type; D046911:Macromolecular Substances; D012333:RNA, Messenger; D001616:beta-Galactosidase