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ANIMAL GENETICS |
Division of Farm Animal Science, School of Clinical Veterinary Science, University of Bristol, Langford, Bristol BS40 5DU, UK
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: breed • 3ß-hydroxysteroid dehydrogenase • gene • pig • polymorphism
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Our recent work has shown that, in pigs, 3ß-HSD also catalyzes the initial step of the hepatic metabolism of the steroid pheromone androstenone, with formation of the product ß-androstenol. Low expression and activity of 3ß-HSD in pig liver is accompanied by a reduced rate of hepatic androstenone clearance and the accumulation of androstenone in pig adipose tissue (Doran et al., 2004). A high level of adipose tissue androstenone is one of the major reasons for "boar taint," an undesirable taste and smell in cooked pork (Bonneau, 1982). At present, there is much interest in identifying genetic polymorphisms that may be causative for or associated with androstenone accumulation in pigs. One of the possibilities could be a polymorphism in the gene coding for hepatic 3ß-HSD.
Pig hepatic, full length 3ß-HSD cDNA has been sequenced (GenBank accession number NM_001004049), and the gene has been assigned to chromosome 4 (Von Teichman et al., 2001). Recently, a sequence of the pig genomic clone containing the entire 3ß-HSD gene has also become available. However, the structure of the pig 3ß-HSD gene has not yet been defined. Whether there are variations in 3ß-HSD DNA sequences from pigs with high and low adipose tissue androstenone levels is also not known.
The aims of this paper were as follows: 1) define the pig 3ß-HSD gene structure using the currently available information; 2) determine whether there are variations in 3ß-HSD DNA sequence among animals of different breeds and among individual animals within a breed; and 3) determine whether androstenone accumulation in pig adipose tissue is related to a polymorphism in the 3ß-HSD gene.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The following entire (i.e., uncastrated) male pigs were used in this study:
The animals were reared on commercial standard pelleted diets (BOCM Pauls Ltd., Portishead, UK) without restriction of feed and water. The diet contained 14 MJ of DE and 10 g of Lys per kg, as-fed basis. The pigs were reared under commercial conditions and slaughtered at the age of 22 to 26 wk in an European Union-approved abattoir at the School of Clinical Veterinary Science, University of Bristol. For carcass weight of individual animals, see Table 1. For RNA isolation and genomic DNA extraction, pig liver samples were collected 5 min after slaughter, immediately snap-frozen in liquid N, and stored at –80°C until further use.
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The primers corresponded to bp 223 to 242 and 1,231 to 1,211 of the published pig 3ß-HSD cDNA sequence (GenBank accession number AF_232699), respectively. Reactions were performed at an annealing temperature of 55°C for 35 cycles using 1 µg of cDNA and High Fidelity Taq (Roche, Welwyn Garden City, UK). The PCR product was ligated into the pGEM-T Easy vector (Promega, Southampton, UK), amplified in Escherichia coli XL Blue (Stratagene, La Jolla, CA), and sequenced with M13 forward and reverse primers. Two different clones were sequenced for each PCR reaction. The sequencing was performed by MWG Biotech Company (Ebersberg, Germany).
PCR, Cloning, and Sequencing of the 3ß-HSD 5'Flanking Region
Genomic DNA was isolated using the Wizard SV Genomic DNA Preparation Kit (Promega). The DNA concentration was determined by measuring absorbance at 260 nm. The PCR reaction was performed at an annealing temperature of 63°C for 35 cycles with the following primers: 5'-CACCTGAGACTTTGGCCG-CAATCAGAG-3'(forward primer), and 5'-CGGATCTC-CTGCAGATCCTTCTCCTCCAG-3'(reverse primer).
The primers correspond to the bases –741 to –715 and +379 to +351 of the sequence presented in Figure 1. Reactions were carried out using 1 µg of genomic DNA. The PCR product was ligated into the pGEM-T Easy vector, amplified in E. coli XL Blue, and sequenced.
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), using a Fisons 8000 series gas chromatograph in hot splitless-split mode (20:1), equipped with a Sil8 capillary column (25 m x 0.25 mm i.d.; Chrompack Ltd., London, UK), He as the carrier gas, and a flame ionization detector. Androstanedione was used as an internal standard. The measurements for each sample were performed in duplicates.
Analysis of Potential Transcription Factor Binding Sites
Analysis of potential transcription factor binding sites was performed by searching the Transfac 4.0 database (http://www.gene-regulation.com/) using Mat-Inspector V2.2 software (Quandt et al., 1995).
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; GenBank accession number NM_001004049), and the gene was mapped to chromosome 4q16–4q21. von Teichman et al. could find no evidence for more than one 3ß-HSD isoform in the pig. Recently the sequence of a 177,720-bp clone derived from a pig genomic library has been reported (clone CH242-150C11, chromosome 4, GenBank accession number CR 938722), and this clone contains the entire pig 3ß-HSD gene. The structure of the gene can be deduced by comparison of the full-length cDNA sequence and the genomic sequence. It should be noted that the 21-base sequence at the extreme 5'end of the 3ß-HSD cDNA does not appear in the genomic sequence. The first point at which the genomic and cDNA sequences coincide is at base 136,333 in the forward sequence of the genomic clone. This sequence starts with CTTGGGCCA; the initial C is assumed to represent the transcription start site and is assigned the number 0 in the remainder of this paper.
The deduced 3ß-HSD gene structure is depicted in Figure 2. The 5'untranslated region (UTR) is composed of exon 1 (bp 0 to +57) and a part of exon 2 (bp +186 to + 415). A 130-bp intron sequence (intron 1) is located between exon 1 and exon 2. The start of the coding region is ATG at +290. The coding region is made up of 3 exons: part of exon 2 (bp +290 to +415), exon 3 (bp +3,915 to + 4,077), and exon 4 (bp +7,359 to +8,602). Exon 4 contains the stop codon at bp +8,170 to +8,172, the 3'UTR, and the polyadenylation signal. Exons 2 and 3 are separated by a 2,500-bp intron (intron 2). Exons 3 and 4 are separated by a 3,282-bp intron (intron 3). There are some discrepancies in the 3'UTR region between the published cDNA and genomic sequences in the region bp +8,281 to +8,323.
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. MatInspector analysis predicts a TATA box at –23 bp and putative binding sites for hepatic transcription factors, such as HNF-1 (bp –132 to –122), HNF-3 (bp –190 to –180), and HNF-4 (bp –480 to –470). It is currently unknown whether these transcription factors actually bind to the 5'flanking region of the pig 3ß-HSD gene. The sequence also contains a series of TTAT repeats starting at bp –537.
Sequencing of Sections of the 3ß-HSD Gene from Pigs of Different Breeds Exhibiting High and Low Backfat Androstenone Levels
Coding Region.
The 3ß-HSD cDNA coding region has been cloned and sequenced for 100% Large White, 40% Large White, 75% Duroc, and 25% Meishan pigs. Animals within each breed varied in the level of backfat androstenone (Table 1). No differences were found in the DNA sequences of 3ß-HSD coding region between animals with high and low androstenone levels in adipose tissue and among animals of different breeds. All the sequences were identical to the cDNA sequence in the database.
5'Flanking Region.
In order to sequence the 3ß-HSD 5'flanking region, genomic DNA was prepared from the same animals, which were used for cDNA sequencing. The DNA region corresponding to bp –741 to +378 was amplified, cloned, and sequenced. This region encompassed both the transcription and translation start sites and also contained the proximal 5'flanking region containing the putative promoter with HNF-1, HNF-3, and HNF-4 binding sites. Differences in the number of TTAT repeats at position –537 were found. The part of the DNA sequence containing TTAT repeats is shown in Figure 3. The sequence in the database contained 10 TTAT repeats starting at this point. Our experiments have established variations in the number of TTAT repeats at the position –537, depending on the breed used. In addition, 3 SNP (at the positions –532, –437, and –180) were found, in which C was sometimes substituted for T (Figure 1). The SNP were associated with the number of TTAT repeats. No other differences in the DNA sequences were observed in the region bp –741 to +378.
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summarizes the data on the number of TTAT repeat, SNP, and androstenone levels for individual animals of different breeds. The 5'flanking region of the 3ß-HSD gene from 75% Duroc, 100% Large White, and 40% Large White pigs consistently exhibit 7 TTAT repeats with a T at –532 and C at –436 and –179. These animals exhibited widely different levels of androsten-one in the backfat (see Table 1 for androstenone values for individual pigs). The mean values for androstenone level and the SEM in the breeds investigated were as follows: 0.76 ± 0.1, 0.76 ± 0.15, 0.89 ± 0.15, and 1.0 ± 0.2 µg/g of fat for 40% Large White, 100% Large White, 75% Duroc, and 25% Meishan pigs, respectively. There was thus no relationship between differences in the flanking sequence and the levels of backfat androsten-one. Meishan pigs consistently showed 9 TTAT repeats with C at position –531 and T at –436 and –179. Again, there was no relationship between differences in the DNA sequence and the backfat androstenone level.
Introns.
Intron 1 of 3ß-HSD was sequenced as a part of the 5'flanking region sequencing. The sequence is presented in Figure 1. No differences in the DNA sequences were found for animals of different breeds or with high and low androstenone levels. Intron 2 was sequenced from representative pigs of 25% Meishan and 100% Large White pigs. The animals within each breed varied in the level of androstenone in adipose tissue. A number of apparent variations in GA repeats in the extended repeated sequence region starting at +902 were identified. The variations in the amount of the GA repeats did not differ among breeds investigated or among animals with variations in adipose tissue an-drostenone levels (data not shown).
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Both the pig and the human 3ß-HSD genes contain a 130-bp intron in the 5'UTR, and, in both cases, the coding region consists of 3 exons. The lengths of introns 2 and 3 in the pig sequence (2,500 and 3,282 bp, respectively) are, however, different from those in the human sequence (3,884 and 2,168 bp, respectively).
The enzyme 3ß-HSD is involved in hepatic androsten-one metabolism in the pig. Low activity and expression of this enzyme has been shown to be associated with accumulation of androstenone in backfat of some pigs (Doran et al., 2004; Nicolau-Solano et al., 2006). One objective of the present work was to determine whether any polymorphisms are present in the coding or 5'flanking region of the pig 3ß-HSD gene and whether such a polymorphism may be associated with a low 3ß-HSD expression in pigs with high androstenone levels. The results have shown variations in the DNA sequences in the 5'flanking but not the coding regions. Nine TTAT repeats in the 5'flanking region were identified for 25% Meishan pigs, whereas only 7 TTAT repeats were present in 100% Large White, 40% Large White, and 75% Duroc animals. The repeats do not serve as potential transcription factor binding sites and therefore it is unlikely that the variation in the number of repeats affects 3ß-HSD expression. The 5'flanking region also contained 3 SNP, which were breed-specific.
The results suggest that the amount of the repeats and the SNP in the 5'flanking region depend on the presence of Meishan genes.
In conclusion, the present work has reported the structure of the pig 3ß-HSD gene and identified for the first time a number of polymorphisms in the pig 3ß-HSD 5'flanking region. These polymorphisms were not associated with the level of androstenone in adipose tissue but were breed-dependent. The physiological role of these polymorphisms needs to be investigated. It is possible that the polymorphisms which we found might be in linkage disequilibrium with alleles outside of the sequenced region, which might affect the androstenone level. The results of this work might be used for detection of the presence of Meishan genes in western pig breeds, especially if phenotype is not clearly established.
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Footnotes |
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2 Corresponding author: e.udovikova{at}bristol.ac.uk
Received for publication June 7, 2006. Accepted for publication October 18, 2006.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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5-4-isomerase gene and its expression in mammalian cells. Biol. Chem. 265:20463–20475.5- 4 isomerase. Anim. Genet. 32:298–302.[CrossRef][Medline]
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