cDNA cloning and promoter analysis of rat caspase-9

J. Nishiyama, X. Yi, M. A. Venkatachalam, Z. Dong

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)49-56
Number of pages8
JournalBiochemical Journal
Volume360
Issue number1
DOIs
StatePublished - Nov 15 2001

Fingerprint

Caspase 9
Cloning
Rats
Organism Cloning
Complementary DNA
Genes
5' Flanking Region
Caspases
Walking
CCAAT-Enhancer-Binding Proteins
Hypoxia-Inducible Factor 1
Glucocorticoid Receptors
Transcription
Reporter Genes
Genetic Promoter Regions
Gene expression
Catalytic Domain
Transcription Factors
Apoptosis
Amino Acids

Keywords

  • Apoptosis
  • Gene regulation
  • Hypoxia

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

cDNA cloning and promoter analysis of rat caspase-9. / Nishiyama, J.; Yi, X.; Venkatachalam, M. A.; Dong, Z.

In: Biochemical Journal, Vol. 360, No. 1, 15.11.2001, p. 49-56.

Research output: Contribution to journalArticle

Nishiyama, J. ; Yi, X. ; Venkatachalam, M. A. ; Dong, Z. / cDNA cloning and promoter analysis of rat caspase-9. In: Biochemical Journal. 2001 ; Vol. 360, No. 1. pp. 49-56.
@article{798be50c2a864474847a514ac34def04,
title = "cDNA cloning and promoter analysis of rat caspase-9",
abstract = "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.",
keywords = "Apoptosis, Gene regulation, Hypoxia",
author = "J. Nishiyama and X. Yi and Venkatachalam, {M. A.} and Z. Dong",
year = "2001",
month = "11",
day = "15",
doi = "10.1042/0264-6021:3600049",
language = "English (US)",
volume = "360",
pages = "49--56",
journal = "Biochemical Journal",
issn = "0264-6021",
publisher = "Portland Press Ltd.",
number = "1",

}

TY - JOUR

T1 - cDNA cloning and promoter analysis of rat caspase-9

AU - Nishiyama, J.

AU - Yi, X.

AU - Venkatachalam, M. A.

AU - Dong, Z.

PY - 2001/11/15

Y1 - 2001/11/15

N2 - 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.

AB - 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.

KW - Apoptosis

KW - Gene regulation

KW - Hypoxia

UR - http://www.scopus.com/inward/record.url?scp=0035890467&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0035890467&partnerID=8YFLogxK

U2 - 10.1042/0264-6021:3600049

DO - 10.1042/0264-6021:3600049

M3 - Article

C2 - 11695991

AN - SCOPUS:0035890467

VL - 360

SP - 49

EP - 56

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - 1

ER -