Characterization of the rat aflatoxin B1 aldehyde reductase gene, AKR7A1. Structure and chromosomal localization of AKR7A1 as well as identification of antioxidant response elements in the gene promoter

E. Ellis, C.M. Slattery, J.D. Hayes

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Abstract

Rat aflatoxin B-1 aldehyde reductase (called AFAR1 or AKR7A1) is a member of the aldo-keto reductase 7 family, which metabolizes the environmental carcinogen aflatoxin B-1. The expression of this enzyme is markedly increased in rat liver by cancer chemopreventive agents, many of which are believed to regulate gene expression through the antioxidant response element (ARE). In order to understand how this gene is regulated, two overlapping genomic clones have been isolated that contain most of the coding region for the enzyme; together they encompass 14.1 kb of DNA. Characterization of these clones has shown that rat AFAR1 is similar to8 kb long and comprises seven exons and six introns. The seven exons are between 97 and 380 bp in size. The introns range in size from 194 bp to similar to2.9 kb. Fluorescent in situ hybridization localized AFAR1 to rat chromosome 5q36.5, a region that is syntenic with human chromosome 1p35-1p36.1 where AKR7A2 resides. The transcriptional start site (TSS) was determined, using 5'-rapid amplification of cDNA ends, to be an A nucleotide 73 bp upstream from the ATG initiation codon. The 5'-flanking region of AFAR1 was isolated by polymerase chain reaction-based genome walking, and resulted in the isolation of similar to900 bp of genomic DNA upstream from the TSS. Use of a gene expression reporter assay demonstrated that this cloned 5'-flanking region of AFAR1 could support transcription in the rat liver 34 (RL34) epithelial cell line. Within this upstream region of the promoter, a substantial number of sequences were found that are closely similar, but not identical, to the 'core' ARE consensus sequence. Between nucleotides -810 and -106 bp from the TSS 16 ARE-related sequences were identified. Four of these putative enhancers lay between -389 and -355 bp, and the motif 5'-GAGTGAG-3' was repeated three times within the 35 bp region.
LanguageEnglish
Pages727-737
Number of pages10
JournalCarcinogenesis
Volume24
Issue number4
DOIs
Publication statusPublished - Apr 2003

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Antioxidant Response Elements
Aflatoxin B1
Genes
5' Flanking Region
Introns
Exons
Nucleotides
Clone Cells
Environmental Carcinogens
Gene Expression
Aldehyde Reductase
Initiator Codon
DNA
Consensus Sequence
Human Chromosomes
Enzymes
Liver Neoplasms
Fluorescence In Situ Hybridization
Genetic Promoter Regions
Walking

Keywords

  • molecular epidemiology
  • cancer prevention

Cite this

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title = "Characterization of the rat aflatoxin B1 aldehyde reductase gene, AKR7A1. Structure and chromosomal localization of AKR7A1 as well as identification of antioxidant response elements in the gene promoter",
abstract = "Rat aflatoxin B-1 aldehyde reductase (called AFAR1 or AKR7A1) is a member of the aldo-keto reductase 7 family, which metabolizes the environmental carcinogen aflatoxin B-1. The expression of this enzyme is markedly increased in rat liver by cancer chemopreventive agents, many of which are believed to regulate gene expression through the antioxidant response element (ARE). In order to understand how this gene is regulated, two overlapping genomic clones have been isolated that contain most of the coding region for the enzyme; together they encompass 14.1 kb of DNA. Characterization of these clones has shown that rat AFAR1 is similar to8 kb long and comprises seven exons and six introns. The seven exons are between 97 and 380 bp in size. The introns range in size from 194 bp to similar to2.9 kb. Fluorescent in situ hybridization localized AFAR1 to rat chromosome 5q36.5, a region that is syntenic with human chromosome 1p35-1p36.1 where AKR7A2 resides. The transcriptional start site (TSS) was determined, using 5'-rapid amplification of cDNA ends, to be an A nucleotide 73 bp upstream from the ATG initiation codon. The 5'-flanking region of AFAR1 was isolated by polymerase chain reaction-based genome walking, and resulted in the isolation of similar to900 bp of genomic DNA upstream from the TSS. Use of a gene expression reporter assay demonstrated that this cloned 5'-flanking region of AFAR1 could support transcription in the rat liver 34 (RL34) epithelial cell line. Within this upstream region of the promoter, a substantial number of sequences were found that are closely similar, but not identical, to the 'core' ARE consensus sequence. Between nucleotides -810 and -106 bp from the TSS 16 ARE-related sequences were identified. Four of these putative enhancers lay between -389 and -355 bp, and the motif 5'-GAGTGAG-3' was repeated three times within the 35 bp region.",
keywords = "molecular epidemiology, cancer prevention",
author = "E. Ellis and C.M. Slattery and J.D. Hayes",
year = "2003",
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T1 - Characterization of the rat aflatoxin B1 aldehyde reductase gene, AKR7A1. Structure and chromosomal localization of AKR7A1 as well as identification of antioxidant response elements in the gene promoter

AU - Ellis, E.

AU - Slattery, C.M.

AU - Hayes, J.D.

PY - 2003/4

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N2 - Rat aflatoxin B-1 aldehyde reductase (called AFAR1 or AKR7A1) is a member of the aldo-keto reductase 7 family, which metabolizes the environmental carcinogen aflatoxin B-1. The expression of this enzyme is markedly increased in rat liver by cancer chemopreventive agents, many of which are believed to regulate gene expression through the antioxidant response element (ARE). In order to understand how this gene is regulated, two overlapping genomic clones have been isolated that contain most of the coding region for the enzyme; together they encompass 14.1 kb of DNA. Characterization of these clones has shown that rat AFAR1 is similar to8 kb long and comprises seven exons and six introns. The seven exons are between 97 and 380 bp in size. The introns range in size from 194 bp to similar to2.9 kb. Fluorescent in situ hybridization localized AFAR1 to rat chromosome 5q36.5, a region that is syntenic with human chromosome 1p35-1p36.1 where AKR7A2 resides. The transcriptional start site (TSS) was determined, using 5'-rapid amplification of cDNA ends, to be an A nucleotide 73 bp upstream from the ATG initiation codon. The 5'-flanking region of AFAR1 was isolated by polymerase chain reaction-based genome walking, and resulted in the isolation of similar to900 bp of genomic DNA upstream from the TSS. Use of a gene expression reporter assay demonstrated that this cloned 5'-flanking region of AFAR1 could support transcription in the rat liver 34 (RL34) epithelial cell line. Within this upstream region of the promoter, a substantial number of sequences were found that are closely similar, but not identical, to the 'core' ARE consensus sequence. Between nucleotides -810 and -106 bp from the TSS 16 ARE-related sequences were identified. Four of these putative enhancers lay between -389 and -355 bp, and the motif 5'-GAGTGAG-3' was repeated three times within the 35 bp region.

AB - Rat aflatoxin B-1 aldehyde reductase (called AFAR1 or AKR7A1) is a member of the aldo-keto reductase 7 family, which metabolizes the environmental carcinogen aflatoxin B-1. The expression of this enzyme is markedly increased in rat liver by cancer chemopreventive agents, many of which are believed to regulate gene expression through the antioxidant response element (ARE). In order to understand how this gene is regulated, two overlapping genomic clones have been isolated that contain most of the coding region for the enzyme; together they encompass 14.1 kb of DNA. Characterization of these clones has shown that rat AFAR1 is similar to8 kb long and comprises seven exons and six introns. The seven exons are between 97 and 380 bp in size. The introns range in size from 194 bp to similar to2.9 kb. Fluorescent in situ hybridization localized AFAR1 to rat chromosome 5q36.5, a region that is syntenic with human chromosome 1p35-1p36.1 where AKR7A2 resides. The transcriptional start site (TSS) was determined, using 5'-rapid amplification of cDNA ends, to be an A nucleotide 73 bp upstream from the ATG initiation codon. The 5'-flanking region of AFAR1 was isolated by polymerase chain reaction-based genome walking, and resulted in the isolation of similar to900 bp of genomic DNA upstream from the TSS. Use of a gene expression reporter assay demonstrated that this cloned 5'-flanking region of AFAR1 could support transcription in the rat liver 34 (RL34) epithelial cell line. Within this upstream region of the promoter, a substantial number of sequences were found that are closely similar, but not identical, to the 'core' ARE consensus sequence. Between nucleotides -810 and -106 bp from the TSS 16 ARE-related sequences were identified. Four of these putative enhancers lay between -389 and -355 bp, and the motif 5'-GAGTGAG-3' was repeated three times within the 35 bp region.

KW - molecular epidemiology

KW - cancer prevention

UR - http://dx.doi.org/10.1093/carcin/bgg016

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DO - 10.1093/carcin/bgg016

M3 - Article

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EP - 737

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T2 - Carcinogenesis

JF - Carcinogenesis

SN - 0143-3334

IS - 4

ER -