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- Hansson, Maria, et al.
(författare)
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Characterization of two distinct aryl hydrocarbon receptor (AhR2) genes in Atlantic salmon (Salmo salar) and evidence for multiple AhR2 gene lineages in salmonid fish
- 2003
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Ingår i: Gene. - 1879-0038. ; 303, s. 197-206
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Tidskriftsartikel (refereegranskat)abstract
- The aryl hydrocarbon receptor (AhR) mediates the toxicity of several environmental contaminants, e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin, and other halogenated hydrocarbons in vertebrates. This receptor initiates the transcription of several biotransformation enzymes, which in turn are responsible for causing severe harm to biological tissue. Here we describe the isolation and complete characterization of the first two AhR genes from the teleost fish Atlantic salmon (Salmo salar). The predicted amino acid sequences contain regions characteristic of other vertebrate AhRs including basic helix-loop-helix (bHLH) and PER-ARNT-SIM (PAS) domains but show little similarity to other vertebrate AhRs across the C-terminal half. Furthermore, they do not contain distinct Q-rich domains as found in the mammalian AhR, which is in line with previously described fish AhR genes. The salmon cDNAs encode 1106 and 1107 putative residues, respectively, approximately 50 amino acids longer than previously characterized AhR genes. Phylogenetic analyses demonstrated that the two salmon AhR sequences cluster within the AhR subfamily of the bHLH-PAS family, in a clade containing fish AhR2 genes. Although the two AbR2 forms are 92% identical at the amino acid level, the distribution of sequence differences and the presence of both forms in 30 tested individuals suggest that they are not allelic but derived from separate loci. Interestingly, they are not orthologs of the rainbow trout (Oncorhynchus mykiss) AhR2alpha and beta genes and the new salmon loci are therefore here designated AhR2gamma and AhR2delta. In line with this, PCR with DNA from rainbow trout revealed a new trout AhR locus that was more similar to the two salmon genes than to the trout AhR2alpha and beta genes, suggesting that the rainbow trout possesses at least three distinct AhR2 genes. The presence of multiple AhR genes in these species is probably a consequence of the genome duplications that occurred in the early evolution of fish and later also specifically in the salmonid lineage. Reverse transcription-PCR analyses revealed that both AhR2gamma and AhR2delta are transcribed in the liver, spleen and muscles of adult salmon.
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- Hansson, Maria, et al.
(författare)
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Unprecedented genomic diversity of AhR1 and AhR2 genes in Atlantic salmon (Salmo salar L.)
- 2004
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Ingår i: Aquatic Toxicology. - : Elsevier BV. - 1879-1514 .- 0166-445X. ; 68:3, s. 219-232
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Tidskriftsartikel (refereegranskat)abstract
- Aryl hydrocarbon receptor (AhR) genes encode proteins involved in mediating the toxic responses induced by several environmental pollutants. Here, we describe the identification of the first two AhR1 (alpha and beta) genes and two additional AhR2 (alpha and beta) genes in the tetraploid species Atlantic salmon (Salmo salar L.) from a cosmid library screening. Cosmid clones containing genomic salmon AhR sequences were isolated using a cDNA clone containing the coding region of the Atlantic salmon AhR2gamma as a probe. Screening revealed 14 positive clones, from which four were chosen for further analyses. One of the cosmids contained genomic AhR sequences that were highly similar to the rainbow trout (Oncorhynchus mykiss) AhR2alpha and beta genes. SMART RACE amplified two complete, highly similar but not identical AhR type 2 sequences from salmon cDNA, which from phylogenetic analyses were determined as the rainbow trout AhR2alpha and beta orthologs. The salmon AhR2alpha and beta encode proteins of 1071 and 1058 residues, respectively, and encompass characteristic AhR sequence elements like a basic-helix-loop-helix (bHLH) and two PER-ARNT-SIM (PAS) domains. Both genes are transcribed in liver, spleen and muscle tissues of adult salmon. A second cosmid contained partial sequences, which were identical to the previously characterized AhR2gamma gene. The last two cosmids contained partial genomic AhR sequences, which were more similar to other AhR type 1 fish genes than the four characterized salmon AhR2 genes. However, attempts to amplify the corresponding complete cDNA sequences of the inserts proved very difficult, suggesting that these genes are non-functional or very weakly transcribed in the examined tissues. Phylogenetic analyses of the conserved regions did, however, clearly indicate that these two AhRs belong to the AhR type 1 clade and have been assigned as the Atlantic salmon AhR1alpha and AhR1beta genes. Taken together, these findings demonstrate that multiple AhR genes are present in Atlantic salmon genome, which likely is a consequence of previous genome duplications in the evolutionary past of salmonids. Plausible explanations for the high incidence of AhR genes in fish and more specifically in salmortids, like rapid divergences in specialized functions, are discussed.
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- Lundell, Kerstin, et al.
(författare)
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Cloning and expression of a pig liver taurochenodeoxycholic acid 6α-hydroxylase (CYP4A21) : a novel member of the CYP4A subfamily
- 2001
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 276:13, s. 9606-9612
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Tidskriftsartikel (refereegranskat)abstract
- A cytochrome P450 expressed in pig liver was cloned by polymerase chain reaction using oligonucleotide primers based on amino acid sequences of the purified taurochenodeoxycholic acid 6 alpha -hydroxylase. This enzyme catalyzes a 6 alpha -hydroxylation of chenodeoxycholic acid, and the product hyocholic acid is considered to be a primary bile acid specific for the pig. The cDNA encodes a protein of 504 amino acids. The primary structure of the porcine taurochenodeoxycholic acid 6 alpha -hydroxylase, designated CYP4A21, shows about 75% identity with known members of the CYP4A subfamily in rabbit and man. Transfection of the cDNA for CYP4A21 into COS cells resulted in the synthesis of an enzyme that was recognized by antibodies raised against the purified pig liver enzyme and catalyzed 6 alpha -hydroxylation of taurochenodeoxycholic acid. The hitherto known CYP4A enzymes catalyze hydroxylation of fatty acids and prostaglandins and have frequently been referred to as fatty acid hydroxylases. A change in substrate specificity from fatty acids or prostaglandins to a steroid nucleus among CYP4A enzymes is notable. The results of mutagenesis experiments indicate that three amino acid substitutions in a region around position 315 which is highly conserved in all previously known CYP4A and CYP4B enzymes could be involved in the altered catalytic activity of CYP4A21.
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- Lundell, Kerstin
(författare)
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Cytochrome P450 Enzymes in Bile Acid Biosynthesis and Fatty Acid Metabolism : Studies on Members of the Porcine CYP4A and CYP8B Subfamilies
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The present investigation is devoted to studies on porcine members of the cytochrome P450 4A (CYP4A) and CYP8B1 subfamilies, which are involved in bile acid biosynthesis and fatty acid metabolism. Hyocholic acid is considered to fulfil the requirements for trihydroxy bile acids in the domestic pig (Sus scrofa) in the absence of cholic acid. Hyocholic acid is a 6α-hydroxylated product of chenodeoxycholic acid and the enzyme catalyzing the 6α-hydroxylation was cloned and found to be an atypical member of the CYP4A subfamily. The primary structure of this porcine enzyme, designated CYP4A21, shows about 75% overall sequence identity to members of the CYP4A subfamily expressed in rabbit and man. Divergent amino acids in a “signature sequence” in the active site of all hitherto known CYP4A fatty acid hydroxylases, were found to be important determinants for the 6α-hydroxylase activity of CYP4A21. Two homologous CYP4A fatty acid hydroxylases, designated CYP4A24 and CYP4A25, expressed in pig liver and kidney were cloned. These two cDNAs encode proteins of 504 amino acids similar to CYP4A21. The overall identity between CYP4A24 and CYP4A25 is 97% compared to 94% identity to CYP4A21. Whereas CYP4A21 clearly deviates regarding structural features and catalytic activity it is more difficult to establish whether CYP4A24 and CYP4A25 are distinct enzymes or allelic variants of a single enzyme. Cloning of the CYP4A21 gene showed a conserved organization compared to CYP4A genes in other species. A segment of the CYP4A24 gene was also cloned and comparison with the CYP4A21 gene revealed an extensive sequence identity also within introns as well as within the proximal promoter regions. This indicates that CYP4A21 and CYP4A fatty acid hydroxylases have a common origin and evolved by gene duplication. The CYP4A21 and CYP4A fatty acid hydroxylases, however, show distinct patterns of expression.The key enzyme in cholic acid biosynthesis, CYP8B1, was markedly expressed in fetal pig liver compared to livers from young pigs. The opposite was shown for the expression of CYP4A21. An apparently conserved pig CYP8B1 gene was cloned and was intronless, similar to CYP8B1 genes from other species. The pig gene encoded a protein of 501 amino acids with 81% identity to CYP8B1 expressed in rabbit and man. Unlike other CYP8B1 genes, the pig promoter lacked a TATA-box. This might offer one explanation for the unusual expression pattern, which appears to be restricted to pig fetal life.
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