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Novel sequence of the porcine IGHA gene
Molecular Immunology 47 (2009) 147–148
Contents lists available at ScienceDirect
Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
Letter to the Editor Novel sequence of the porcine IGHA gene
a r t i c l e
i n f o
Keywords: Immunoglobulin A (IgA) IGHA Hinge Variation Splice acceptor site Pig
a b s t r a c t Two alleles of porcine IGHA have been reported previously. These have been detected by transcriptional length analysis and restriction fragment length polymorphism analysis. However, these methods may not be able to detect all polymorphism in porcine IGHA as they rely on the presence of either length variation or polymorphism in a restriction endonuclease recognition site respectively. Here we report a novel sequence occurring in the hinge region of the porcine IGHA gene that was detected using PCR-SSCP analysis. The novel sequence had two nucleotides that are missing at the splice-acceptor site relative to the previously reported IGHA-A sequence. Further identification of allelic variation in porcine IGHA may require an alternative typing system to be developed. © 2009 Elsevier Ltd. All rights reserved.
Immunoglobulin A (IgA) is the major class of antibody associated with mucosal immunity in mammals. Its primary role is as a first line of defence against invasion by inhaled and ingested pathogens at the mucosal surfaces (Wagner et al., 2003; Woof and Kerr, 2004). In pigs, two allelic variants in the heavy constant region of the IgA gene (IGHA) have been reported previously. These alleles vary in the hinge-coding region and this causes not only changes in nucleotide sequence, but also alters the length of the transcribed mRNA (Brown et al., 1995; Navarro et al., 2000a). Two typing methods have been reported for identification of these allelic variants. The first typing system described (Brown et al., 1995; Navarro et al., 2000a) was based on IgA transcriptional length analysis. A second method was developed to detect polymorphism in the genomic DNA, using PCR followed by restriction fragment length polymorphism (PCR-RFLP) analysis and which was designed to detect the presence or absence of a Dde I recognition site (Brown et al., 1995; Navarro et al., 2000b). Besides the polymorphism mentioned above, we wanted to know whether further polymorphism could be found in the porcine IGHA gene. Here we report the detection of additional IGHA polymorphism in pig using PCR-single-strand conformational polymorphism (PCR-SSCP) analysis. One hundred and thirty-one Large White × Landrace × Duroc crossbred pigs were investigated. Genomic DNA was extracted from blood samples collected on FTA cards (Whatman, Middlesex, UK) using a rapid two-step procedure (Zhou et al., 2006). A fragment of the porcine IGHA gene containing intron 1 and the hinge-coding region (exon 2) was amplified using PCR primers designed based on a published porcine IGHA sequence (GenBank accession no. U12594). These primers were 5 -tccagccagtccgtgaac3 and 5 -attggagcccaggagcag-3 . PCR amplicons were subject to SSCP analysis on 16 × 18 cm, 14% acrylamide:bisacrylamide (37.5:1 acrylamide/bis-acrylamide) gels. Electrophoresis was performed using Protein II xi cells (Bio-Rad, Hercules, CA, USA), at 260 V for 18 h 0161-5890/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2009.08.029
at 25 ◦ C in 0.5× TBE buffer and gels were silver-stained according to the method of Byun et al. (2009). Pig DNA samples representing the different PCR-SSCP patterns obtained were selected for amplification using Pwo Super Yield DNA polymerase (Roche Applied Science, Mannheim, Germany), according to the conditions described above. The PCR amplicons were subsequently ligated into the PCR 4 Blunt-TOPO vector (Invitrogen, Carlsbad, CA, USA) and a 2 l aliquot of the ligation mixture was used to transform competent Escherichia coli cells (One Shot INV␣F’ Invitrogen), following the manufacturer’s instructions. Twelve insert positive colonies for each transformation were picked and cultured at 37 ◦ C overnight in Terrific broth (Invitrogen), in a shaking rotary incubator (225 rpm). All clones were screened using a clonal PCR-SSCP approach as described in Zhou and Hickford (2008) and only those clones for which the SSCP patterns matched those of the corresponding genomic DNA were selected for DNA sequencing. To sequence and analyse the plasmid DNA, selected clones were extracted using a QIAprep Spin Miniprep kit (Qiagen) and sequenced in both directions using the M13 forward and reverse primers. Identical raw sequences obtained from at least three clones were subjected to further sequence analysis. Sequence analyses revealed three unique SSCP patterns representing three different nucleotide sequences. These sequences were named IGHA-A, IGHA-B and IGHA-C (Fig. 1A). Alleles IGHA-A and IGHA-B were identical to the previously reported porcine IgAa and IgAb alleles (GenBank accession nos. U12594 and S71099, respectively). A novel sequence was observed for IGHA-C. The three sequences were found to have variation in the hinge region, one (IGHA-A) potentially producing a longer hinge and the other two (IGHA-B and IGHA-C) with shorter hinges. The two short-hinged sequences had variation in their nucleotide sequence at the putative splice-acceptor site of IGHA-A. The IGHA-B allele had a G to A substitution at position c.302-1, while the IGHA-C sequence exhibited a novel two nucleotide deletion at positions
148
Letter to the Editor / Molecular Immunology 47 (2009) 147–148
result of adaptation in the IgA hinge sequence, possibly related to host–parasite interactions and/or positive selection against pathogens as has been suggested previously (Sumiyama et al., 2002; Navarro et al., 2000b). The reported sequence variation may provide greater flexibility to the IgA molecule (Brown et al., 1995), and hence further study is required to ascertain its likely effects. Acknowledgements This research was financially supported by the Gene-Marker Laboratory at Lincoln University, New Zealand, and Khon Kaen University, Thailand. The authors would like to thank Q. Fang, S.O. Byun, J. Han, J. Hu, and A. Hogan for technical assistance. Fig. 1. Polymorphism of the porcine IGHA gene. (A) Three unique PCR-SSCP patterns, corresponding to three allele sequences A, B and C, were observed for the IGHA amplicons from homozygous and heterozygous pigs. (B) Sequence comparison of the porcine IGHA alleles. Amino acids are represented in one-letter code and shown in bold. Nucleotides identical to the top sequence are represented by dashes. Nucleotides in the putative exon 2 are shaded. The putative splice-site junctions are indicated by a vertical arrow. The Dde I recognition sequence ‘C↓TNAG’ is shown in box, which is only present in allele A and missing in alleles B and C. Dots have been introduced to improve alignments. The last nucleotide of exon 1 contributes the first nucleotide of the initial codon of the hinge region and the corresponding amino acids are shown in parentheses.
c.302-2 and c.302-3. This variation in the splice-acceptor sequence would potentially lead to splicing occurring at an alternate AG site as reported previously for IgAb (Brown et al., 1995). This is at position c.314-1 and c.314-2 of GenBank sequence S71099 and is 12 nucleotides downstream from the normal splice-acceptor site in IGHA-A. This would lead to a hinge that is four amino acids shorter than that encoded by the IGHA-A sequence, and suggests that both the IGHA-B and IGHA-C sequence would have similar length hinge regions. The previously reported typing methods (Brown et al., 1995; Navarro et al., 2000a,b) would not have detected the IGHA-C variant described here. The restriction fragment length analysis described by Navarro et al. (2000b) detects the presence of a ‘C↓TCAG’ sequence at the splice-acceptor site of IGHA-A, which is cleaved by Dde I (recognising the sequence ‘C↓TNAG’), and which is not present in either IGHA-B or IGHA-C (Fig. 1B). Differentiation of IGHAB and IGHA-C would therefore not be possible using this PCR-RFLP approach. Likewise, the typing method based on IgA transcriptional analysis as described by Brown et al. (1995) and Navarro et al. (2000a) would probably not detect the length variation in IGHAB and IGHA-C as they only differ by two nucleotides in length in the non-coding DNA. In conclusion, we suggest that the pig has three allelic forms of the IGHA gene and the variation occurs in both gene length and sequence. The increased variation detected here might be a
References Brown, W.R., Kacskovics, I., Amendt, B.A., Blackmore, M.R., Shinde, R., Butler, J.E., 1995. The hinge deletion allelic variant of porcine IgA results from a mutation at the splice acceptor site in the first C˛ intron. J. Immunol. 154, 3836–3842. Byun, S.O., Fang, Q., Zhou, H., Hickford, J.G.H., 2009. An effective method for silverstaining DNA in large numbers of polyacrylamide gels. Anal. Biochem. 385, 174–175. Navarro, P., Christensen, R.K., Weber, P., Rothschild, M., Ekhardt, G., Butler, J.E., 2000a. Porcine IgA allotypes are not equally transcribed or expressed in heterozygous swine. Mol. Immunol. 37, 653–664. Navarro, P., Christensen, R.K., Ekhardt, G., Bosworth, B., Lunney, J.K., Rothschild, M., Lemke, J., Butler, J.E., 2000b. Genetic differences in the frequency of the hinge variants of porcine IgA is breed dependent Vet. Immunol. Immunopathol. 73, 287–295. Sumiyama, K., Saitou, N., Ueda, S., 2002. Adaptive evolution of the IgA hinge region in primates. Mol. Biol. Evol. 19, 1093–1099. Wagner, B., Greiser-Wilke, I., Antczak, D.F., 2003. Characterization of the horse (Equus caballus) IGHA gene. Immunogenetics 55, 552–560. Woof, J.M., Kerr, M.A., 2004. IgA function - variations on a theme. Immunology 113, 175–177. Zhou, H., Hickford, J.G.H., Fang, Q., 2006. A two-step procedure for extracting genomic DNA from dried blood spots on filter paper for polymerase chain reaction amplification. Anal. Biochem. 354, 159–161. Zhou, H., Hickford, J.G.H., 2008. Clonal polymerase chain reaction-single-strand conformational polymorphism analysis: an effective approach for identifying cloned sequences. Anal. Biochem. 378, 111–112.
Sajee Kunhareang Huitong Zhou Jon G.H. Hickford ∗ Gene-Marker Laboratory, Agriculture and Life Sciences Division, PO Box 84, Lincoln University, Lincoln, New Zealand ∗ Corresponding
author. Tel.: +64 3 3252811; fax: +64 3 3253851. E-mail address: [email protected] (J.G.H. Hickford) 25 August 2009 Available online 24 September 2009
Contents lists available at ScienceDirect
Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
Letter to the Editor Novel sequence of the porcine IGHA gene
a r t i c l e
i n f o
Keywords: Immunoglobulin A (IgA) IGHA Hinge Variation Splice acceptor site Pig
a b s t r a c t Two alleles of porcine IGHA have been reported previously. These have been detected by transcriptional length analysis and restriction fragment length polymorphism analysis. However, these methods may not be able to detect all polymorphism in porcine IGHA as they rely on the presence of either length variation or polymorphism in a restriction endonuclease recognition site respectively. Here we report a novel sequence occurring in the hinge region of the porcine IGHA gene that was detected using PCR-SSCP analysis. The novel sequence had two nucleotides that are missing at the splice-acceptor site relative to the previously reported IGHA-A sequence. Further identification of allelic variation in porcine IGHA may require an alternative typing system to be developed. © 2009 Elsevier Ltd. All rights reserved.
Immunoglobulin A (IgA) is the major class of antibody associated with mucosal immunity in mammals. Its primary role is as a first line of defence against invasion by inhaled and ingested pathogens at the mucosal surfaces (Wagner et al., 2003; Woof and Kerr, 2004). In pigs, two allelic variants in the heavy constant region of the IgA gene (IGHA) have been reported previously. These alleles vary in the hinge-coding region and this causes not only changes in nucleotide sequence, but also alters the length of the transcribed mRNA (Brown et al., 1995; Navarro et al., 2000a). Two typing methods have been reported for identification of these allelic variants. The first typing system described (Brown et al., 1995; Navarro et al., 2000a) was based on IgA transcriptional length analysis. A second method was developed to detect polymorphism in the genomic DNA, using PCR followed by restriction fragment length polymorphism (PCR-RFLP) analysis and which was designed to detect the presence or absence of a Dde I recognition site (Brown et al., 1995; Navarro et al., 2000b). Besides the polymorphism mentioned above, we wanted to know whether further polymorphism could be found in the porcine IGHA gene. Here we report the detection of additional IGHA polymorphism in pig using PCR-single-strand conformational polymorphism (PCR-SSCP) analysis. One hundred and thirty-one Large White × Landrace × Duroc crossbred pigs were investigated. Genomic DNA was extracted from blood samples collected on FTA cards (Whatman, Middlesex, UK) using a rapid two-step procedure (Zhou et al., 2006). A fragment of the porcine IGHA gene containing intron 1 and the hinge-coding region (exon 2) was amplified using PCR primers designed based on a published porcine IGHA sequence (GenBank accession no. U12594). These primers were 5 -tccagccagtccgtgaac3 and 5 -attggagcccaggagcag-3 . PCR amplicons were subject to SSCP analysis on 16 × 18 cm, 14% acrylamide:bisacrylamide (37.5:1 acrylamide/bis-acrylamide) gels. Electrophoresis was performed using Protein II xi cells (Bio-Rad, Hercules, CA, USA), at 260 V for 18 h 0161-5890/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2009.08.029
at 25 ◦ C in 0.5× TBE buffer and gels were silver-stained according to the method of Byun et al. (2009). Pig DNA samples representing the different PCR-SSCP patterns obtained were selected for amplification using Pwo Super Yield DNA polymerase (Roche Applied Science, Mannheim, Germany), according to the conditions described above. The PCR amplicons were subsequently ligated into the PCR 4 Blunt-TOPO vector (Invitrogen, Carlsbad, CA, USA) and a 2 l aliquot of the ligation mixture was used to transform competent Escherichia coli cells (One Shot INV␣F’ Invitrogen), following the manufacturer’s instructions. Twelve insert positive colonies for each transformation were picked and cultured at 37 ◦ C overnight in Terrific broth (Invitrogen), in a shaking rotary incubator (225 rpm). All clones were screened using a clonal PCR-SSCP approach as described in Zhou and Hickford (2008) and only those clones for which the SSCP patterns matched those of the corresponding genomic DNA were selected for DNA sequencing. To sequence and analyse the plasmid DNA, selected clones were extracted using a QIAprep Spin Miniprep kit (Qiagen) and sequenced in both directions using the M13 forward and reverse primers. Identical raw sequences obtained from at least three clones were subjected to further sequence analysis. Sequence analyses revealed three unique SSCP patterns representing three different nucleotide sequences. These sequences were named IGHA-A, IGHA-B and IGHA-C (Fig. 1A). Alleles IGHA-A and IGHA-B were identical to the previously reported porcine IgAa and IgAb alleles (GenBank accession nos. U12594 and S71099, respectively). A novel sequence was observed for IGHA-C. The three sequences were found to have variation in the hinge region, one (IGHA-A) potentially producing a longer hinge and the other two (IGHA-B and IGHA-C) with shorter hinges. The two short-hinged sequences had variation in their nucleotide sequence at the putative splice-acceptor site of IGHA-A. The IGHA-B allele had a G to A substitution at position c.302-1, while the IGHA-C sequence exhibited a novel two nucleotide deletion at positions
148
Letter to the Editor / Molecular Immunology 47 (2009) 147–148
result of adaptation in the IgA hinge sequence, possibly related to host–parasite interactions and/or positive selection against pathogens as has been suggested previously (Sumiyama et al., 2002; Navarro et al., 2000b). The reported sequence variation may provide greater flexibility to the IgA molecule (Brown et al., 1995), and hence further study is required to ascertain its likely effects. Acknowledgements This research was financially supported by the Gene-Marker Laboratory at Lincoln University, New Zealand, and Khon Kaen University, Thailand. The authors would like to thank Q. Fang, S.O. Byun, J. Han, J. Hu, and A. Hogan for technical assistance. Fig. 1. Polymorphism of the porcine IGHA gene. (A) Three unique PCR-SSCP patterns, corresponding to three allele sequences A, B and C, were observed for the IGHA amplicons from homozygous and heterozygous pigs. (B) Sequence comparison of the porcine IGHA alleles. Amino acids are represented in one-letter code and shown in bold. Nucleotides identical to the top sequence are represented by dashes. Nucleotides in the putative exon 2 are shaded. The putative splice-site junctions are indicated by a vertical arrow. The Dde I recognition sequence ‘C↓TNAG’ is shown in box, which is only present in allele A and missing in alleles B and C. Dots have been introduced to improve alignments. The last nucleotide of exon 1 contributes the first nucleotide of the initial codon of the hinge region and the corresponding amino acids are shown in parentheses.
c.302-2 and c.302-3. This variation in the splice-acceptor sequence would potentially lead to splicing occurring at an alternate AG site as reported previously for IgAb (Brown et al., 1995). This is at position c.314-1 and c.314-2 of GenBank sequence S71099 and is 12 nucleotides downstream from the normal splice-acceptor site in IGHA-A. This would lead to a hinge that is four amino acids shorter than that encoded by the IGHA-A sequence, and suggests that both the IGHA-B and IGHA-C sequence would have similar length hinge regions. The previously reported typing methods (Brown et al., 1995; Navarro et al., 2000a,b) would not have detected the IGHA-C variant described here. The restriction fragment length analysis described by Navarro et al. (2000b) detects the presence of a ‘C↓TCAG’ sequence at the splice-acceptor site of IGHA-A, which is cleaved by Dde I (recognising the sequence ‘C↓TNAG’), and which is not present in either IGHA-B or IGHA-C (Fig. 1B). Differentiation of IGHAB and IGHA-C would therefore not be possible using this PCR-RFLP approach. Likewise, the typing method based on IgA transcriptional analysis as described by Brown et al. (1995) and Navarro et al. (2000a) would probably not detect the length variation in IGHAB and IGHA-C as they only differ by two nucleotides in length in the non-coding DNA. In conclusion, we suggest that the pig has three allelic forms of the IGHA gene and the variation occurs in both gene length and sequence. The increased variation detected here might be a
References Brown, W.R., Kacskovics, I., Amendt, B.A., Blackmore, M.R., Shinde, R., Butler, J.E., 1995. The hinge deletion allelic variant of porcine IgA results from a mutation at the splice acceptor site in the first C˛ intron. J. Immunol. 154, 3836–3842. Byun, S.O., Fang, Q., Zhou, H., Hickford, J.G.H., 2009. An effective method for silverstaining DNA in large numbers of polyacrylamide gels. Anal. Biochem. 385, 174–175. Navarro, P., Christensen, R.K., Weber, P., Rothschild, M., Ekhardt, G., Butler, J.E., 2000a. Porcine IgA allotypes are not equally transcribed or expressed in heterozygous swine. Mol. Immunol. 37, 653–664. Navarro, P., Christensen, R.K., Ekhardt, G., Bosworth, B., Lunney, J.K., Rothschild, M., Lemke, J., Butler, J.E., 2000b. Genetic differences in the frequency of the hinge variants of porcine IgA is breed dependent Vet. Immunol. Immunopathol. 73, 287–295. Sumiyama, K., Saitou, N., Ueda, S., 2002. Adaptive evolution of the IgA hinge region in primates. Mol. Biol. Evol. 19, 1093–1099. Wagner, B., Greiser-Wilke, I., Antczak, D.F., 2003. Characterization of the horse (Equus caballus) IGHA gene. Immunogenetics 55, 552–560. Woof, J.M., Kerr, M.A., 2004. IgA function - variations on a theme. Immunology 113, 175–177. Zhou, H., Hickford, J.G.H., Fang, Q., 2006. A two-step procedure for extracting genomic DNA from dried blood spots on filter paper for polymerase chain reaction amplification. Anal. Biochem. 354, 159–161. Zhou, H., Hickford, J.G.H., 2008. Clonal polymerase chain reaction-single-strand conformational polymorphism analysis: an effective approach for identifying cloned sequences. Anal. Biochem. 378, 111–112.
Sajee Kunhareang Huitong Zhou Jon G.H. Hickford ∗ Gene-Marker Laboratory, Agriculture and Life Sciences Division, PO Box 84, Lincoln University, Lincoln, New Zealand ∗ Corresponding
author. Tel.: +64 3 3252811; fax: +64 3 3253851. E-mail address: [email protected] (J.G.H. Hickford) 25 August 2009 Available online 24 September 2009