C 2007) Biochemical Genetics, Vol. 45, Nos. 3/4, April 2007 ( DOI: 10.1007/s10528-006-9072-8
Note
A Novel Nucleotide Sequence Polymorphism in the 5 -Noncoding Region of Bovine Estrogen Receptor α Gene, the RFLP-SnaBI .
T. Szreder,1 B. Zelazowska,1 L. Zwierzchowski,1,3 and Ch. S. Pareek2 Received 17 May 2006—Final 2 October 2006 Published online: 21 February 2007
INTRODUCTION Estrogens influence growth, differentiation, and functioning of many target tissues, including tissues of the male and female reproductive systems, such as the mammary gland, uterus, ovary, testis, and prostate. They play a central role in normal postnatal female physiology, as well as in female pathology. Estrogen receptors α and β (ERα and ERβ) mediate actions of estrogens by regulating transcription of target genes. ERα and ERβ are members of a superfamily of nuclear receptors for diverse hydrophobic ligands. The sequence structure of ERα genes of humans, mice, and rats is known and available in databases (e.g., GenBank). Also known is a partial sequence of the coding regions (cds) and of the 5 region of the ERα gene of sheep and pigs, as well as the sequence of exons 1 and 5–7 of the bovine ERα gene. The typical feature of all genes coding for nuclear receptors, including the ER genes, is a complex structure of their 5 regions. The ERα protein is coded by eight exons. In the 5 region of the ERα gene, however, are additional exons that do not code for protein but have been shown to code for transcripts of different lengths with different 5 UTRs (untranslated regions). In all mRNA variants, the alternative exons are spliced to the + 85 acceptor site located in the coding exon 1. Functions of the different ERα transcripts are not known, but in some cases tissue- or developmental stage-specific expression has been reported.
1 Institute
of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrz˛ebiec, 05-552 W´olka Kosowska, Poland. 2 Department of Animal Genetics, University of Warmia and Mazury, 10-719 Olsztyn-Kortowo, Poland. 3 To whom correspondence should be addressed; e-mail:
[email protected]. 255 C 2007 Springer Science+Business Media, LLC 0006-2928/07/0400-0255/1
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So far, studies on ERα gene polymorphism in farm animals are limited. Rotschild et al. (1996) showed the RFLP PvuII in the noncoding region (an intron) of the ERα gene to be significantly associated with the mean number of piglets born per litter in Meishan pigs. Therefore, ERα was postulated as a candidate gene to study reproductive traits in farm animals. In the previous study, we sequenced a 2853 bp 5 fragment of the bovine ERα gene, and the sequence was deposited in the GenBank database under accession no. AY340597. The sequenced fragment included the noncoding exons A, B, C, their putative promoters, and a part of the protein-coding exon 1. Using this sequence, we identified for the first time a polymorphism within the 5 region of the bovine ERα gene, an A/G transition, which could be recognized with RFLP BglI, lying upstream to exon C. In this study, we identified a new polymorphism in the 5 noncoding region of the bovine ERα gene, an A/G transition at position –1213 relative to the +85 splicing acceptor site in exon 1. This polymorphism is located in the promoter for exon B. The appearance of A and G alleles in small samples of different cattle breeds was estimated.
MATERIALS AND METHODS Animals Polish Black-and-White cattle, with more than 80% Friesian blood (n = 55), Red-and-White (n = 30), Charolaise (n = 18), Limousine (n = 16), Red Angus (n = 10), Hereford (n = 20), Piedmontese (n = 35), and Simmental (n = 11) came from the experimental farm of the Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrz˛ebiec. Cows of Polish local breeds (18 Polish Red and 31 White Back, or Białogrzbietka, cows) were from the Ecological Experimental Farm, PAS, Popielno. Hair samples of 23 Rathi Zebus were brought from India. The 10-mL blood samples were taken from the jugular vein of each animal in test tubes containing K2 EDTA (1.6 mg/mL blood). All procedures involving animals were performed in accordance with the Guiding Principles for the Care and Use of Research Animals and were approved by the Local Ethics Commission (Permission No. 67/2001).
PCR-SSCP Analysis DNA was isolated from blood according to the method of Kanai et al. (1994) or from hair bulbs of Zebu, using the Genomic DNA Extraction Mini prep System (Viogene-Biotek Corp., Taipei County, Taiwan).
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Based on the previously estimated bovine ERα gene sequence (Szreder and Zwierzchowski, 2004a), now available from GenBank (AY332655), and using the Primer3 software (www.genome.wi.mit.edu), the following PCR primers were designed: ERα1F, 5 -GTCAGGTATTCCGTCAGGT-3 , and ERα2R, 5 GCCTTTCTGTTCCTTTGG-3 . These primers were used for PCR amplification of a 340 bp DNA fragment, encompassing a part of the putative promoter for exon B within the 5 region of the bovine ERα gene. The polymerase chain reactions were performed using a PCR mix with primers, both at concentration 5.0 pmol/mL, 1 U Taq polymerase (Qiagen, Germany), 1 µL Taq polymerase buffer, four dNTPs, each at final concentration of 0.2 mM, ca 100 ng genomic DNA, and H2 O up to 10 µL. The following PCR protocol was used: 1 min at 94◦ C, 1 min at 54◦ C, and 1 min at 72◦ C, for 36 cycles. The reactions were performed in an MJ Research Tetrad thermal cycler. The single-strand conformation polymorphism (SSCP) analysis was carried out with a Hoefer SE 600 electrophoresis apparatus (Amersham Biosciences, Piscataway, LA). A thermostatically controlled water circulator maintained the gel at a constant temperature of 24◦ C. The 8% polyacrylamide gel was prepared with a 1 × TBE buffer (0.09 M Tris-boric acid, 0.002 M EDTA). Initial electrophoresis (without samples) was performed for 2 h at 120 V/50 mA. Ten microliters PCR product was mixed with 10 µL denaturation buffer (formamide, 0.25% bromophenol blue, 0.5 M EDTA), denatured for 5 min at 94◦ C, rapidly chilled on ice, and then loaded onto the gel. The electrophoresis was carried out for about 16 h at 80 V, 40 mA, 5 W. The gels were stained using the Silver Staining System (Kucharczyk TE, Warsaw). The patterns of SSCP bands were observed and documented with the Molecular Imager System FX (Bio-Rad, Philadelphia).
DNA Sequencing PCR products of different SSCP patterns (genotypes) of the ERα gene were purified with QIAquick PCR Purification Kit (Qiagen, Germany) and automatically sequenced in an ABJ377 sequencer (Applied Biosystems). The sequences were analyzed using the Sequence Analyzer 2.01 program.
RFLP Analysis The 340 bp PCR product was digested in 10 µL with 10 U SnaBI restriction nuclease (Fermentas UAB, Vilnius, Lithuania) for 3 h at 37◦ C. The restriction fragments were subjected to electrophoresis in 2% agarose/ethidium bromide gels (Invitrogen, Carlsbad, Calif.) in 1 × TBE buffer (0.09 M Tris-boric acid, 0.002 M EDTA). Gels were visualized under UV light and documented in an FX Phosphoimager apparatus (Bio-Rad).
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Fig. 1. Polyacrylamide (8%) gel electrophoresis showing two SSCP patterns of the bovine ERα gene. Lane M, 26-501 bp DNA marker (MspI digest of pUC19).
RESULTS SSCP Polymorphism and Sequencing A PCR-SSCP method was used to identify the polymorphism in the 5 region of the bovine ERα gene. First, a specific PCR product of the desired size of 340 bp was obtained. Then, the PCR product was denatured and subjected to polyacrylamide gel electrophoresis to find an SSCP polymorphism. Results are shown in Fig. 1. The number of bands and their position in the gel clearly show the occurrence of DNA sequence variation. The DNA samples representing different SSCP variants were sequenced (Fig. 2). The nucleotide substitution (the A/G transition) was identified at position –1213 in the bovine ERα gene, relative to the +85 splicing acceptor site in coding exon 1. The polymorphic site was located in the noncoding region of estrogen receptor ERα, in the putative promoter for exon B. Upon sequencing, SSCP variants 1 and 2 appeared to be the AA homozygotes and AG heterozygotes, respectively (Figs. 1 and 2).
RFLP-SnaBI Comparison of the restriction maps of both sequence variants revealed that the A → G transition creates a new restriction site for SnaBI nuclease. Thus, digestion with this enzyme enabled the PCR-RFLP analysis of the polymorphism. The nuclease cuts the 340 bp amplification product into 225 and 115 bp fragments for allele G, while allele A remains uncut (Fig. 3). The RFLP-SnaBI analysis enabled identification of one animal, a Charolaise bull, carrying the homozygous
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Fig. 2. Polymorphic variants of the analyzed fragment of the 5 noncoding region of the bovine ERα gene. Sequence of the 38 bp fragment of the bovine ERα gene showing A/G transition at position 1213 upstream of the +85 splicing acceptor site in exon 1.
GG ERα genotype. The GG variant DNA was sequenced to confirm the genotype identity (Fig. 2) Distribution of ERα Genotypes in Cattle Breeds Using the PCR-RFLP method, we studied the A/G polymorphism in representatives of different Bos taurus breeds and one Bos indicus breed, Rathi Zebu. Results are shown in Table I. In Bos taurus cattle, the overall frequency of alleles A and G is 0.92 and 0.08, respectively. Depending on the breed sampled, the estimated frequency of allele A was 1.00 (Limousine, Piedmontese) to 0.81 (Charolaise). In
Fig. 3. RFLP genotyping of the bovine ERα gene. Agarose (2%) gel showing ERα genotypes after digestion of the 340 bp PCR product with SnaBI nuclease. Lane M, 80-1444 bp DNA marker (HaeIII digest of pUC19). Lane PCR, nondigested PCR product. Lanes 3–5, genotypes AA, AG, and GG) as labeled at the top of each lane.
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Table I. Occurrence of A/G Genotypes and Alleles of the 5 Noncoding Region of the ERα Gene Identified by SnaBI RFLP in a Sample of Dairy and Beef Cattle Breeda Genotype and allele BW
RW
WB
Number of animals AA 45 26 22 AG 10 4 7 GG — — — Genotype frequency AA 0.82 0.86 0.76 AG 0.18 0.13 0.24 GG — — — Allele frequency A 0.91 0.93 0.88 G 0.09 0.07 0.12
RA
PR
LI
HE
SIM
CH
PIE
Zebu
9 1 —
13 5 —
18 — —
18 2 —
10 1 —
12 5 1
34
21 2 —
—
0.90 0.10 —
0.72 0.28 —
1.00 — —
0.90 0.10 —
0.91 0.09 —
0.67 0.28 0.05
1.00 — —
0.91 0.09 —
0.95 0.05
0.86 0.14
1.00 —
0.95 0.05
0.95 0.05
0.81 0.19
1.00 —
0.96 0.04
a BW,
Black-and-White; RW, Red-and-White; WB, Whiteback; RA, Red Angus; PR, Polish Red; LI, Limousine; HE, Hereford; SIM, Simmental; CH, Charolaise; PIE, Piedmontese.
spite of the large panel of the breeds examined, the GG genotype was found in only one Charolaise bull. No significant differences in allele or genotype frequencies were found between cattle breeds and between European and Zebu cattle. DISCUSSION Reproductive traits are of primary interest in livestock because they play a major role in efficiency of production. Selection for increased number of offspring has been employed in model species like mice (Nielsen, 1994) and in farm animals, e.g., pigs (Bidanel et al., 1994; Lamberson et al., 1991; Ollivier and Bolet, 1981) and sheep (Elsen et al., 1994), with only limited success because of the low heritability and the sex-limited nature of reproductive traits. The numerous functions that estrogens play in animals make estrogen receptors and their genes likely candidates for markers of production and functional traits in farm animals. Rotschild et al. (1996) proposed ER genes as candidate markers for prolificacy in pigs. They identified nucleotide sequence polymorphisms in both coding and noncoding regions of the porcine ERα gene. One of these mutations, the RFLP PvuII, was found to have a significant association with the mean number of piglets born per litter. In Meishan sows, the total number of born piglets and the number born alive was 2.3 more for ERα BB females than for AA females. Kmie´c et al. (2002) confirmed these results, showing that allele B in a sow’s genotype was associated with larger litter size. Korwin-Kossakowska (2000), however, in studies carried out with Złotnicka Pstra/Polish Landrace crosses, failed to find any effect of the ERα genotype on litter size. Two additional polymorphisms, RFLP AvaI and RFLP MspA1I, were identified within the coding region of the porcine ERα
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gene, in exon 8: transitions T/C at position +1665 and A/G at position +1754 (Drogemuller et al., 1997). No studies have been performed so far on the effect of these mutations on production or reproductive traits in pigs. In our earlier study (Szreder and Zwierzchowski, 2004a), we sequenced the 2853 bp fragment of the 5 region of the bovine ERα gene (GenBank accession no. AY340597). Moreover, we identified a polymorphism within that sequence, the A/G transition, recognizable by RFLP with BglI restrictase, located upstream to exon C. We also identified a polymorphism in the ERα gene exon 1 that codes for the transactivating domain (Szreder and Zwierzchowski, 2004b). The A/C transversion was found at position 503, recognizable by RFLP-TspRI within the proline codon CCA. Detection of additional polymorphisms is necessary to an investigation of the role of ERα variation in cattle production and functional traits. The A/G polymorphism newly identified in this study in the bovine ERα gene could be a potential genetic marker both for production traits (e.g., lactational performance) and for functional traits (e.g., reproduction). Since the A/G transition is located within the gene region vital for the regulation of its expression, in the putative promoter for noncoding exon B, it might be used in further research on associations between the polymorphism in the regulatory 5 noncoding region of the bovine ERα gene, gene expression, and cattle performance traits. It is noteworthy that the distribution of alleles and genotypes is highly skewed; the allele G is 10 times less frequent than allele A, and the genotype GG must be very rare, as it was found in only one animal out of the 266 sampled from different breeds of cattle. This might decrease the value of this mutation in association studies.
ACKNOWLEDGMENTS The authors thank the Rajastan Go Sewa Sangh Organization, Bikaner,. India, for providing hair samples of Rathi Zebu cattle. We also thank Ms. Beata Zelazowska for technical assistance. This study was funded by the Polish Ministry of Scientific Research and Information Technology Grant 2P06D 015 27.
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Kanai, N., Fujii T., Saito, K., and Yokoyama, T. (1994). Rapid and simple method for preparation of genomic DNA from easily obtainable clotted blood. J. Clin. Pathol. 47:1043–1044. Kmie´c, M., Dvorak, J., and Vrtkova, I. (2002). Study on a relation between estrogen receptor (ESR) gene polymorphism and some pig reproduction performance characters in Polish Landrace breed. Czech J. Anim. Sci. 47:189–193. Korwin-Kossakowska, A. (2000). Geny zwia˛zane z cechami rozrodu s´wi´n. Prace i Materiały Zootechniczne 57:25–36. Lamberson, W. R., Johnson, R. K., Zimmerman, D. R., and Long, T. E. (1991). Direct responses to selection for increased litter size, decreased age at puberty, or random selection following selection for ovulation rate in swine. J. Anim. Sci. 69:3129–3143. Nielsen, M. K. (1994). Selection experiments for reproductive rate in mice. In Proceedings of the Fifth World Congress on Genetics Applied to Livestock Production, vol. 19, University of Guelph, Ontario, Canada, pp. 219–225. Ollivier, L., and Bolet, G. (1981). La selection sus la prolificite chez le porc: Resultats d’une experience de selection sur dix generations. J. Rech. Porcine France 13:261–268. Rotschild, M., Jacobson C., Vaske, D., Tuggle, C., and Wang, L. (1996). The estrogen receptor locus is associated with a major gene influencing litter size in pigs. Proc. Natl. Acad. Sci. U.S.A. 93:201–205. Szreder, T., and Zwierzchowski, L. (2004a). Polymorphism within the bovine estrogen receptor-alpha gene 5’-region. J. Appl. Genet. 45(2):225–236. Szreder, T., and Zwierzchowski, L. (2004b). RFLP-TspRI polymorphism within exon 1 of the bovine estrogen receptor-α (ERα) gene. Anim. Sci. Pap. Rep. 22:543–549.
