Molecular cloning and characterization of murine caspase-12 gene promoter.
The activation of caspase-12 is involved in endoplasmic reticulum-mediated apoptosis. To investigate how caspase-12 is transcriptionally and translationally regulated, we isolated and sequenced the 5'-flanking region of mouse caspase-12 gene by a PCR-mediated chromosome-walking technique, using mouse genomic DNA as a template. Two DNA fragments of 3,221 and 800 bp were isolated and cloned into pGL3 promoterless vector upstream of the luciferase gene. The small DNA fragment contains the first intron sequence located downstream of the first exon and 27 bp from the second exon, whereas the large fragment contains the small fragment and the 5'-flanking region. Reporter constructs generated from these DNA fragments showed a substantial promoter activity in mouse NIH 3T3 or human embryonic kidney 293 cells grown in the presence of 10% serum. In the absence of serum, the luciferase activity was drastically reduced. However, the luciferase mRNA was higher in serum-starved cells than in control cells, suggesting that translation of luciferase mRNA was drastically inhibited. However, Western blot analysis revealed that the quantity of procaspase-12 is actually higher in serum-starved cells relative to that cultured in the presence of 10% serum. Progressive deletion analysis of the 3,221-bp sequence revealed that the highest luciferase activity was observed with the construct containing 700 bp upstream of ATG. The transcriptional initiation site was identified by 5' RACE techniques using total RNA from NIH 3T3 cells. Our results should facilitate studies on the mechanism regulating the expression of this important gene.
Oubrahim
Hammou
H
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-8012, USA. oubrahih@nhlbi.nih.gov
Wang
Jun
J
Stadtman
Earl R
ER
Chock
P Boon
PB
D000818:Animals; D001483:Base Sequence; D053454:Caspase 12; D020169:Caspases / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D016386:Chromosome Walking; D003001:Cloning, Molecular; D016895:Culture Media, Serum-Free; D004247:DNA / Q000235:genetics; D004789:Enzyme Activation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D017384:Sequence Deletion; D014158:Transcription, Genetic; D014162:Transfection
D016895:Culture Media, Serum-Free; D004247:DNA; C506444:CASP12 protein, human; C506440:Casp12 protein, mouse; D053454:Caspase 12; D020169:Caspases