cDNA cloning and promoter analysis of rat caspase-9.
Caspase-9 is the apex caspase of the mitochondrial pathway of apoptosis, which plays a critical role in apoptotic initiation and progression. However, gene regulation of caspase-9 is largely unknown. This is in part due to the lack of information on the gene promoter. Here we have cloned the full-length cDNA of rat caspase-9 and have isolated promoter regions of this gene. The rat caspase-9 cDNA of 2058 bp predicts a protein of 454 amino acids, which contains a caspase-recruitment domain ('CARD') at the N-terminus and enzymic domains at the C-terminus. The enzyme's active site, with a characteristic motif of QACGG, was also identified. Overall, rat and human caspase-9 have 71% identity. With the cDNA sequence, we subsequently isolated the proximal 5'-flanking regions of rat caspase-9 by the procedure of genomic walking. The 2270 bp genomic segment is 'TATA-less', but contains several GC boxes. Elements binding known transcription factors such as Sp-1, Pit-1, CCAAT-enhancer-binding protein (C/EBP), glucocorticoid receptor and hypoxia-inducible factor 1 (HIF-1) were also identified. When cloned into reporter gene vectors, the genomic segment showed significant promoter activity, indicating that the 5'-flanking regions isolated by genomic walking contain the gene promoter of rat caspase-9. Of significance is that the cloned promoter segments were activated by severe hypoxia, conditions inducing caspase-9 transcription. Thus, the genomic sequences reported here contain not only the basal promoter of rat caspase-9 but also regulatory elements responsive to pathophysiological stimuli including hypoxia.
Nishiyama
J
J
Department of Pathology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA.
Yi
X
X
Venkatachalam
M A
MA
Dong
Z
Z
D016475:3T3 Cells; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D053453:Caspase 9; D020169:Caspases / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D000860:Hypoxia; D051379:Mice; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016716:PC12 Cells; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors
D000596:Amino Acids; D018076:DNA, Complementary; C506433:CASP9 protein, human; C506434:Casp9 protein, mouse; C506435:Casp9 protein, rat; D053453:Caspase 9; D020169:Caspases