Isolation and functional characterization of the 5'-upstream region of mouse P/Q-type Ca2+ channel alpha1A subunit gene.
The omega-agatoxin-IVA-sensitive P/Q-type Ca2+ channel is predominantly expressed in the nervous system. To dissect the molecular mechanisms underlying the neuron-specific expression of the P/Q-type channel, we have isolated and characterized the 5'-upstream region of the mouse alpha1A subunit gene. A transcription start site appeared to exist at -269 bp upstream from the start codon as found by 5' RACE analysis. The proximal promoter of the alpha1A subunit gene lacks a typical TATA box, but contains several transcription factor binding sequences, including two Sp1 sites. When linked to a placental alkaline phosphatase (PLAP) reporter gene to examine the promoter activity, the 6.3-kb (-6,273 to +269) 5'-upstream region, but not a smaller 3.0-kb construct (-3, 021 to +269), was able to drive the reporter gene in neuron-like PC12 cells. In contrast, neither of these constructs enhanced the PLAP expression in fibroblast NIH3T3 cells. The sequence between 6.3 and 3.0 kb of the 5'-upstream region did not show promoter activity in either of the cell lines, but enhanced TK promoter activity in PC12 cells, though not in NIH3T3 cells. These results suggest that neuron-specific elements of the alpha1A subunit gene are likely to be located in the distal upstream regions (-6,273 to -3,021) of the 5'-upstream sequence.
Takahashi
E
E
Tsukuba Research Laboratories, Eisai Co. Ltd., 5-1-3, Tokodai, Tsukuba, Ibaraki, 300-2635, Japan. e2-takahashi@eisai.co.jp
Murata
Y
Y
Oki
T
T
Miyamoto
N
N
Mori
Y
Y
Takada
N
N
Wanifuchi
H
H
Wanifuchi
N
N
Yagami
K
K
Niidome
T
T
Tanaka
I
I
Katayama
K
K
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D015220:Calcium Channels / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification*; D020864:Calcium Channels, N-Type; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D051379:Mice; D008969:Molecular Sequence Data; D016716:PC12 Cells; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid
D015220:Calcium Channels; D020864:Calcium Channels, N-Type; D012333:RNA, Messenger