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Single nucleotide polymorphism detection in promoter I of the acetyl-CoA carboxylase-α gene in sheep
Small Ruminant Research 59 (2005) 49–53
Single nucleotide polymorphism detection in promoter I of the acetyl-CoA carboxylase-␣ gene in sheep Bianca Moioli∗ , Francesco Napolitano, Luigi Orr`u, Gennaro Catillo Istituto Sperimentale per la Zootecnia, Reproduzioni e Genetica applicata, Via Salaria 31, 00016 Monterotondo, Italy Received 27 January 2004; received in revised form 30 November 2004; accepted 30 November 2004
Abstract During analysis of the genome of four breeds of sheep – Gentile di Puglia, Sopravissana, Sarda and Romanoff – three single nucleotide polymorphisms (SNPs) were detected in the 5 untranslated region of locus OAR292285 encoding the DNA sequence of promoter I of the acetyl-CoA carboxylase-␣ gene. DNA analysis was performed through denaturing high-performance liquid chromatography (DHPLC) using the WAVE® Nucleic Acid Fragment Analysis System. All SNPs were subsequently confirmed by direct sequencing. We demonstrated that the DNA profiles detected through DHPLC allowed discrimination between different genotypes even when multiple SNPs were found within the same amplicon. Allele frequency at each detected SNP was calculated. None of the detected SNPs allowed discrimination between breeds, because at least one individual of each analysed breed possessed each detected allele. However, the Sopravissana breed showed significantly different allele frequencies from the Sarda and the Gentile di Puglia breeds. © 2004 Elsevier B.V. All rights reserved. Keywords: Sheep; Acetyl-CoA carboxylase gene; SNPs; DHPLC
1. Introduction Acetyl-CoA carboxylase (ACACA) is the fluxdetermining enzyme in the regulation of fatty acid synthesis in animal tissues. The structural and functional features of the ACACA-␣ gene were described in rat and sheep (Luo et al., 1989; Barber and Travers, 1998; Travers et al., 2001). Transcription of the ACACA-␣ ∗ Corresponding author. Tel.: +39 06 90090213; fax: +39 06 9061541. E-mail address: [email protected] (B. Moioli).
0921-4488/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2004.11.014
gene is initiated from two promoters, such that the transcripts demonstrate heterogeneity in the 5 untranslated region (UTR) in a tissue specific fashion (Luo et al., 1989; Barber and Travers, 1998). Locus OAR292285 encoding the DNA sequence of promoter I of the ACACA-␣ gene in sheep (accession number AJ292285) was made available by (Travers et al., 2001), while mRNA sequence for the same gene was reported by Barber and Travers (1998) (accession number X80045). The DNA portion, which is common to the two published sequences is the 5 UTR. Although no SNP in the mentioned region in the sheep genome
50
B. Moioli et al. / Small Ruminant Research 59 (2005) 49–53
is referred in literature, the two published sequences show allele differences between each other’s at three sites. The analysis of the 5 UTR region of different breeds of sheep was therefore considered an interesting work for a better knowledge of the genetic diversity of sheep breeds. Several indigenous breeds of sheep exist in Italy; however, their numbers have dramatically declined during the last decades, due to the competition of higher milk yielding breeds, mainly the Sarda breed. The present study was performed in order to analyse the genetic variability of the 5 UTR in sheep of different breeds through the search of single nucleotide polymorphisms (SNPs) to provide solid scientific information to conservation programmes. It was in fact hypothesized that the genetic diversity in the genes responsible of the fatty acid synthesis could elucidate some peculiar characteristics of the endangered breeds that might affect fat yield and quality of their products. Although no causative mutation could be expected in the analysed DNA region, this region includes the ACACA gene promoter, and was therefore selected because genetic variability in promoters can affect gene expression. DNA analysis was performed through denaturing high-performance liquid chromatography (DHPLC) and subsequently by direct sequencing. The analysis of DNA fragments by DHPLC using the WAVE® Nucleic Acid Fragment Analysis System is a versatile and powerful tool allowing the resolution of DNA fragments on the basis of differential retention of doublestranded versus single-stranded DNA (Hecker, 2001). At a given temperature, the difference in the melting between homo- and heteroduplex is revealed by differences in retention times. DHPLC (Oefner and Under hill, 1995) detects heteroduplexes and is used to both identify new polymorphisms as well as genotype new samples. Purpose of the present work was to search for SNPs in the 5 UTR region of the ACACA gene in some Italian local breeds of sheep, using the DHPLC, while verifying the power of this system for mutation discovery, compared to traditional sequencing.
2. Materials and methods An amplicon of 388 nucleotides (nt) was obtained by PCR amplification of the DNA of 76 sheep. The
amplified region included the DNA fragment from positions 4263 to 4651 of the locus OAR292285 (Travers et al., 2001, accession number AJ292285) and is located in the 5 UTR of the ACACA-␣ gene, starting 100 nt before the beginning of exon 1, and ending 42 nt after the end of exon 1, so to include both the whole exon 1 and the TATA signal. The sheep belonged to the following breeds: two local endangered breeds, Gentile di Puglia (26 animals) and Sopravissana (32 animals), and one Italian popular and widespread milk breed, the Sarda (15 animals). Three further animals of the Romanoff sheep breed were also added to the analysed sample, simply because they were available at the laboratory. Primers used for the amplification and PCR conditions were as follows: • Forward primer: gtg gca aac gtt gtc ttt ct; • Reverse primer: cgt atg ggc ttc act gac tg; • Thirty-five PCR cycles at annealing temperature of 60 ◦ C for 45 s were performed. The detection of sequence variation was performed on the DHPLC Transgenomic WAVE® system. DNA profiles were compared at the partial denaturation temperature of 59.3 ◦ C. The detected polymorphisms were confirmed through direct sequencing of each sample on the Perkin-Elmer ABI Prism 310 DNA sequencer. Allele frequencies at each detected SNP were calculated by direct counting, and Chi-square analysis of the differences in allele frequency between breed pairs was performed.
3. Results Three SNPs were identified in the analysed animals (Fig. 1): position 4413: G or A; position 4485: G or C; position 4507: T or C. Out of the possible theoretical 27 genotypes, only nine were detected in the analysed sample (Table 1), in detail, three different homozygous genotypes, two genotypes with one heterozygous variant; three genotypes with two heterozygous variants, and one genotype where the three variants were all heterozygous. DHPLC does not allow the detection of different homozygous genotypes, which all show only one peak profile; however, the six genotypes containing either
B. Moioli et al. / Small Ruminant Research 59 (2005) 49–53
51
Table 1 Detected genotypes in the analysed sample Genome position
Fig. 1. DNA sequence of locus OAR292285 from position 4263 to 4651, with indication of SNPs position and limits of exon I through bigger lettering point.
one or two heterozygous alleles were detectable and distinguishable (Fig. 2). Mixture of two different homozygous samples, and subsequent DHPLC analysis, allows to discriminate between the different homozygous genotypes (Fig. 3). All alleles were detected in each of the four breeds. Allele frequency at each locus by breed are presented
Number of animals
4413
4485
4507
GG AA GG GG GA GA GA GG GA
CC GG GG GC CC GG GC GC GC
TT CC TT TT TT TC TT TC TC
32 3 6 14 2 7 2 4 6
in Table 2. Significant differences in allele frequency (P < 0.01) were found between Gentile di Puglia and Sopravissana at all positions, and between Sopravissana and Sarda at position 4485 and 4507.
4. Discussion The three identified SNPs are the same that were found by comparing the two published sequences (Travers et al., 2001; Barber and Travers, 1998), the source of which were crossbred British sheep. In
Fig. 2. Representative DHPLC chromatograms showing resolution of the different genotypes: G1 = GG CC TT; G 2 = AA GG CC; G3 = GG GG TT; G4 = GG GC TT; G5 = GA CC TT; G6 = GA GG TC; G7 = GA GC TT; G8 = GG GC TC; G9 = GA GC TC.
52
B. Moioli et al. / Small Ruminant Research 59 (2005) 49–53
Fig. 3. Representative DHPLC chromatograms showing resolution of the mixtures of homozygous genotypes: G1 + 2 = GG CC TT + AA GG CC; G1 + 3 = GG CC TT + GG GG TT; G2 + 3 = AA GG CC + GG GG TT. Table 2 Allele frequency at each locus by breed Breed
Allele frequency (%) Genome position 4413
Gentile di Puglia Sarda Sopravissana Romanoff All breeds
Genome position 4485
Genome position 4507
A
G
C
G
C
T
23.08a 16.67 6.25b 33.33 15.13
76.92a 83.33 93.75b 66.67 84.87
46.15a 50.00ac 82.81b 33.33 61.84
53.85a 50.00ac 17.19b 66.67 38.16
28.85a 16.67ac 1.56b 33.33 15.13
71.15a 83.33ac 98.44b 66.67 84.87
Different letters indicate significant differences in allele frequency between breeds at P < 0.01.
the Italian local breeds, the analysed genome region showed high variability; however, genetic variability was found within breed, because at least one individual of each analysed breeds possesses each detected allele. The significant differences in allele frequencies between the Sopravissana breed and the other two Italian breeds confirm the different genetic structure of the Italian local breeds, as reported in previous biodiversity studies (Napolitano et al., 2002). Although this study could not provide SNPs that allow discrimination between breeds, it does provide knowledge of novel SNPs that might be of value in conservation programmes. This work had also the purpose to verify the power of DHPLC for mutation discovery, in particular when multiple SNPs are found in the same amplicon. In fact, we have not found in literature any reference of multiple SNPs detection through DHPLC. In this work, the obtained profiles indicate that it is possible to discriminate between different genotypes also when more than one allele is found at heterozygous status within the same
amplicon. In order to be able to prove this opportunity, all analysed amplicons were also sequenced. In future trials, once different repeatable profiles are identified through DHPLC, only one amplicon for each type of profile will need to be fully sequenced. In order to discriminate between the homozygous genotype profiles, because each of them shows one single peak, it will be opportune to make mixtures of two homozygous samples and sequence only those that make a heteroduplex profile evident. With this system, the number of amplicons that must be sequenced can be highly reduced.
Acknowledgements The authors thank Dr. Stravros Papadimitriou of Trangenomic Ltd. for his assistance in the use of DHPLC and in interpreting DNA chromatograms. This work was supported by the Italian Ministry of Agriculture.
B. Moioli et al. / Small Ruminant Research 59 (2005) 49–53
References Barber, M., Travers, M., 1998. Elucidation of a promoter activity that directs the expression of acetyl-CoA carboxylase ␣ with an alternative N-terminus in a tissue-restricted fashion. Biochem. J. 333, 17–25. Hecker, K.H., 2001. Automated high-accuracy mutation screening with the WAVE® nucleic acid fragment analysis system. In: Proceedings of the SPIE V 4626 on Tools for Molecular Analysis and High-Throughput Screening, p. 57. Luo, X., Park, K., Lopez-Casillas, F., Kim, K.H., 1989. Structural features of the acetyl-CoA carboxylase gene: mechanisms for the generation of mRNAs with 5 end heterogeneity. Proc. Natl. Acad. Sci. U.S.A. 86, 4042–4046.
53
Napolitano, F., Parente, A., Catillo, G., Moioli, B., 2002. In: Proceedings of the 53th EAAP Annual Meeting on Genetic Diversity between Gentile di Puglia, Sopravissana and Sarda breeds, Using Microsatellite Markers, Cairo, September 1–4. Oefner, P.J., Underhill, P.A., 1995. Comparative DNA sequencing by denaturing high-performance liquid chromatography (DHPLC). Am. J. Hum. Genet. 57, 266. Travers, M., Vallance, A.J., Gourlay, H.T., Gill, C.A., Klein, I., Bottema, C.B.K., Barber, M., 2001. Promoter I of the ovine acetyl-CoA carboxylase ␣ gene: an E-box motif at 114 in the proximal promoter binds upstream stimulatory factor (USF)-1 and USF-2 and acts as an insulin-response sequence in differentiating adipocytes. Biochem. J. 359, 273– 284.
Single nucleotide polymorphism detection in promoter I of the acetyl-CoA carboxylase-␣ gene in sheep Bianca Moioli∗ , Francesco Napolitano, Luigi Orr`u, Gennaro Catillo Istituto Sperimentale per la Zootecnia, Reproduzioni e Genetica applicata, Via Salaria 31, 00016 Monterotondo, Italy Received 27 January 2004; received in revised form 30 November 2004; accepted 30 November 2004
Abstract During analysis of the genome of four breeds of sheep – Gentile di Puglia, Sopravissana, Sarda and Romanoff – three single nucleotide polymorphisms (SNPs) were detected in the 5 untranslated region of locus OAR292285 encoding the DNA sequence of promoter I of the acetyl-CoA carboxylase-␣ gene. DNA analysis was performed through denaturing high-performance liquid chromatography (DHPLC) using the WAVE® Nucleic Acid Fragment Analysis System. All SNPs were subsequently confirmed by direct sequencing. We demonstrated that the DNA profiles detected through DHPLC allowed discrimination between different genotypes even when multiple SNPs were found within the same amplicon. Allele frequency at each detected SNP was calculated. None of the detected SNPs allowed discrimination between breeds, because at least one individual of each analysed breed possessed each detected allele. However, the Sopravissana breed showed significantly different allele frequencies from the Sarda and the Gentile di Puglia breeds. © 2004 Elsevier B.V. All rights reserved. Keywords: Sheep; Acetyl-CoA carboxylase gene; SNPs; DHPLC
1. Introduction Acetyl-CoA carboxylase (ACACA) is the fluxdetermining enzyme in the regulation of fatty acid synthesis in animal tissues. The structural and functional features of the ACACA-␣ gene were described in rat and sheep (Luo et al., 1989; Barber and Travers, 1998; Travers et al., 2001). Transcription of the ACACA-␣ ∗ Corresponding author. Tel.: +39 06 90090213; fax: +39 06 9061541. E-mail address: [email protected] (B. Moioli).
0921-4488/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2004.11.014
gene is initiated from two promoters, such that the transcripts demonstrate heterogeneity in the 5 untranslated region (UTR) in a tissue specific fashion (Luo et al., 1989; Barber and Travers, 1998). Locus OAR292285 encoding the DNA sequence of promoter I of the ACACA-␣ gene in sheep (accession number AJ292285) was made available by (Travers et al., 2001), while mRNA sequence for the same gene was reported by Barber and Travers (1998) (accession number X80045). The DNA portion, which is common to the two published sequences is the 5 UTR. Although no SNP in the mentioned region in the sheep genome
50
B. Moioli et al. / Small Ruminant Research 59 (2005) 49–53
is referred in literature, the two published sequences show allele differences between each other’s at three sites. The analysis of the 5 UTR region of different breeds of sheep was therefore considered an interesting work for a better knowledge of the genetic diversity of sheep breeds. Several indigenous breeds of sheep exist in Italy; however, their numbers have dramatically declined during the last decades, due to the competition of higher milk yielding breeds, mainly the Sarda breed. The present study was performed in order to analyse the genetic variability of the 5 UTR in sheep of different breeds through the search of single nucleotide polymorphisms (SNPs) to provide solid scientific information to conservation programmes. It was in fact hypothesized that the genetic diversity in the genes responsible of the fatty acid synthesis could elucidate some peculiar characteristics of the endangered breeds that might affect fat yield and quality of their products. Although no causative mutation could be expected in the analysed DNA region, this region includes the ACACA gene promoter, and was therefore selected because genetic variability in promoters can affect gene expression. DNA analysis was performed through denaturing high-performance liquid chromatography (DHPLC) and subsequently by direct sequencing. The analysis of DNA fragments by DHPLC using the WAVE® Nucleic Acid Fragment Analysis System is a versatile and powerful tool allowing the resolution of DNA fragments on the basis of differential retention of doublestranded versus single-stranded DNA (Hecker, 2001). At a given temperature, the difference in the melting between homo- and heteroduplex is revealed by differences in retention times. DHPLC (Oefner and Under hill, 1995) detects heteroduplexes and is used to both identify new polymorphisms as well as genotype new samples. Purpose of the present work was to search for SNPs in the 5 UTR region of the ACACA gene in some Italian local breeds of sheep, using the DHPLC, while verifying the power of this system for mutation discovery, compared to traditional sequencing.
2. Materials and methods An amplicon of 388 nucleotides (nt) was obtained by PCR amplification of the DNA of 76 sheep. The
amplified region included the DNA fragment from positions 4263 to 4651 of the locus OAR292285 (Travers et al., 2001, accession number AJ292285) and is located in the 5 UTR of the ACACA-␣ gene, starting 100 nt before the beginning of exon 1, and ending 42 nt after the end of exon 1, so to include both the whole exon 1 and the TATA signal. The sheep belonged to the following breeds: two local endangered breeds, Gentile di Puglia (26 animals) and Sopravissana (32 animals), and one Italian popular and widespread milk breed, the Sarda (15 animals). Three further animals of the Romanoff sheep breed were also added to the analysed sample, simply because they were available at the laboratory. Primers used for the amplification and PCR conditions were as follows: • Forward primer: gtg gca aac gtt gtc ttt ct; • Reverse primer: cgt atg ggc ttc act gac tg; • Thirty-five PCR cycles at annealing temperature of 60 ◦ C for 45 s were performed. The detection of sequence variation was performed on the DHPLC Transgenomic WAVE® system. DNA profiles were compared at the partial denaturation temperature of 59.3 ◦ C. The detected polymorphisms were confirmed through direct sequencing of each sample on the Perkin-Elmer ABI Prism 310 DNA sequencer. Allele frequencies at each detected SNP were calculated by direct counting, and Chi-square analysis of the differences in allele frequency between breed pairs was performed.
3. Results Three SNPs were identified in the analysed animals (Fig. 1): position 4413: G or A; position 4485: G or C; position 4507: T or C. Out of the possible theoretical 27 genotypes, only nine were detected in the analysed sample (Table 1), in detail, three different homozygous genotypes, two genotypes with one heterozygous variant; three genotypes with two heterozygous variants, and one genotype where the three variants were all heterozygous. DHPLC does not allow the detection of different homozygous genotypes, which all show only one peak profile; however, the six genotypes containing either
B. Moioli et al. / Small Ruminant Research 59 (2005) 49–53
51
Table 1 Detected genotypes in the analysed sample Genome position
Fig. 1. DNA sequence of locus OAR292285 from position 4263 to 4651, with indication of SNPs position and limits of exon I through bigger lettering point.
one or two heterozygous alleles were detectable and distinguishable (Fig. 2). Mixture of two different homozygous samples, and subsequent DHPLC analysis, allows to discriminate between the different homozygous genotypes (Fig. 3). All alleles were detected in each of the four breeds. Allele frequency at each locus by breed are presented
Number of animals
4413
4485
4507
GG AA GG GG GA GA GA GG GA
CC GG GG GC CC GG GC GC GC
TT CC TT TT TT TC TT TC TC
32 3 6 14 2 7 2 4 6
in Table 2. Significant differences in allele frequency (P < 0.01) were found between Gentile di Puglia and Sopravissana at all positions, and between Sopravissana and Sarda at position 4485 and 4507.
4. Discussion The three identified SNPs are the same that were found by comparing the two published sequences (Travers et al., 2001; Barber and Travers, 1998), the source of which were crossbred British sheep. In
Fig. 2. Representative DHPLC chromatograms showing resolution of the different genotypes: G1 = GG CC TT; G 2 = AA GG CC; G3 = GG GG TT; G4 = GG GC TT; G5 = GA CC TT; G6 = GA GG TC; G7 = GA GC TT; G8 = GG GC TC; G9 = GA GC TC.
52
B. Moioli et al. / Small Ruminant Research 59 (2005) 49–53
Fig. 3. Representative DHPLC chromatograms showing resolution of the mixtures of homozygous genotypes: G1 + 2 = GG CC TT + AA GG CC; G1 + 3 = GG CC TT + GG GG TT; G2 + 3 = AA GG CC + GG GG TT. Table 2 Allele frequency at each locus by breed Breed
Allele frequency (%) Genome position 4413
Gentile di Puglia Sarda Sopravissana Romanoff All breeds
Genome position 4485
Genome position 4507
A
G
C
G
C
T
23.08a 16.67 6.25b 33.33 15.13
76.92a 83.33 93.75b 66.67 84.87
46.15a 50.00ac 82.81b 33.33 61.84
53.85a 50.00ac 17.19b 66.67 38.16
28.85a 16.67ac 1.56b 33.33 15.13
71.15a 83.33ac 98.44b 66.67 84.87
Different letters indicate significant differences in allele frequency between breeds at P < 0.01.
the Italian local breeds, the analysed genome region showed high variability; however, genetic variability was found within breed, because at least one individual of each analysed breeds possesses each detected allele. The significant differences in allele frequencies between the Sopravissana breed and the other two Italian breeds confirm the different genetic structure of the Italian local breeds, as reported in previous biodiversity studies (Napolitano et al., 2002). Although this study could not provide SNPs that allow discrimination between breeds, it does provide knowledge of novel SNPs that might be of value in conservation programmes. This work had also the purpose to verify the power of DHPLC for mutation discovery, in particular when multiple SNPs are found in the same amplicon. In fact, we have not found in literature any reference of multiple SNPs detection through DHPLC. In this work, the obtained profiles indicate that it is possible to discriminate between different genotypes also when more than one allele is found at heterozygous status within the same
amplicon. In order to be able to prove this opportunity, all analysed amplicons were also sequenced. In future trials, once different repeatable profiles are identified through DHPLC, only one amplicon for each type of profile will need to be fully sequenced. In order to discriminate between the homozygous genotype profiles, because each of them shows one single peak, it will be opportune to make mixtures of two homozygous samples and sequence only those that make a heteroduplex profile evident. With this system, the number of amplicons that must be sequenced can be highly reduced.
Acknowledgements The authors thank Dr. Stravros Papadimitriou of Trangenomic Ltd. for his assistance in the use of DHPLC and in interpreting DNA chromatograms. This work was supported by the Italian Ministry of Agriculture.
B. Moioli et al. / Small Ruminant Research 59 (2005) 49–53
References Barber, M., Travers, M., 1998. Elucidation of a promoter activity that directs the expression of acetyl-CoA carboxylase ␣ with an alternative N-terminus in a tissue-restricted fashion. Biochem. J. 333, 17–25. Hecker, K.H., 2001. Automated high-accuracy mutation screening with the WAVE® nucleic acid fragment analysis system. In: Proceedings of the SPIE V 4626 on Tools for Molecular Analysis and High-Throughput Screening, p. 57. Luo, X., Park, K., Lopez-Casillas, F., Kim, K.H., 1989. Structural features of the acetyl-CoA carboxylase gene: mechanisms for the generation of mRNAs with 5 end heterogeneity. Proc. Natl. Acad. Sci. U.S.A. 86, 4042–4046.
53
Napolitano, F., Parente, A., Catillo, G., Moioli, B., 2002. In: Proceedings of the 53th EAAP Annual Meeting on Genetic Diversity between Gentile di Puglia, Sopravissana and Sarda breeds, Using Microsatellite Markers, Cairo, September 1–4. Oefner, P.J., Underhill, P.A., 1995. Comparative DNA sequencing by denaturing high-performance liquid chromatography (DHPLC). Am. J. Hum. Genet. 57, 266. Travers, M., Vallance, A.J., Gourlay, H.T., Gill, C.A., Klein, I., Bottema, C.B.K., Barber, M., 2001. Promoter I of the ovine acetyl-CoA carboxylase ␣ gene: an E-box motif at 114 in the proximal promoter binds upstream stimulatory factor (USF)-1 and USF-2 and acts as an insulin-response sequence in differentiating adipocytes. Biochem. J. 359, 273– 284.