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Genome conservation between the bovine and human interleukin-8 receptor complex: Improper annotation of bovine interleukin-8 receptor b identified
Veterinary Immunology and Immunopathology 114 (2006) 335–340 www.elsevier.com/locate/vetimm
Short communication
Genome conservation between the bovine and human interleukin-8 receptor complex: Improper annotation of bovine interleukin-8 receptor b identified Gina M. Pighetti *, Magdalena Rambeaud Department of Animal Science, 114 McCord Hall, 2640 Morgan Circle, The University of Tennessee, Knoxville, TN 37996, United States Received 1 May 2006; received in revised form 28 July 2006; accepted 14 August 2006
Abstract Interleukin (IL)-8 and its receptors, CXCR1 and CXCR2, are key regulators of inflammation. However, knowledge of these receptors at the genomic level is limiting or absent in cattle. Therefore, our objective was to identify bovine orthologs of human CXCR1 and CXCR2. Alignment of bovine CXCR2 reference mRNA to the bovine genome revealed two regions of similarity on BTA2 approximately 20 kb apart and on opposite strands. Comparison with the human genome suggested the more centromeric region to be CXCR2 and the more telomeric region to be CXCR1 which contradicts the current annotation of the bovine CXCR2 reference mRNA. This observation was verified by sequencing RT-PCR products of specific regions within each predicted IL-8 receptor and comparing with human sequences using ClustalW. Further examination of coding and non-coding regions within the IL-8 receptor genome complex revealed that both bovine and canine CXCR1 and CXCR2 genes had more conserved sequences in common with the human genes than either mouse or rat, and may offer more suitable animal models for certain applications. This molecular information provides a stepping stone for greater understanding of the role each IL-8 receptor plays in inflammation and will enhance our ability to develop strategies against inflammatory based diseases. # 2006 Elsevier B.V. All rights reserved. Keywords: Bovine; Genome; Inflammation; Interleukin-8 receptors
1. Introduction Elimination of invading organisms often requires an effective inflammatory response: pathogen recognition, inflammatory mediator release, leukocyte recruitment, bacteria removal, and leukocyte removal. A key mediator of this process is the chemokine interleu-
Abbreviations: ENA-78, epithelial neutrophil activating protein 78; GRO, growth regulated oncogene; Interleukin-8, IL-8; MIP, Macrophage inflammatory protein * Corresponding author. Tel.: +1 865 974 7225; fax: +1 865 974 3394. E-mail address: [email protected] (G.M. Pighetti). 0165-2427/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2006.08.008
kin-8 (IL-8). This chemokine, mostly recognized for its ability to induce neutrophil migration, also increases cytokine production, enhances phagocytosis and reactive oxygen species generation, as well as regulating cell survival (Kettritz et al., 1998; Mitchell et al., 2003; Jozsef et al., 2006). Other structurally related chemokines such as epithelial neutrophil activating protein 78 (ENA-78), growth regulated oncogene (GRO)-a, b, g, and macrophage inflammatory protein (MIP)-1 have similar functional activities (Green et al., 1996; Li et al., 2002a,b). These chemokines mediate their effects through two primary receptors, IL-8 receptor (IL8R)-a or CXCR1 and IL-8R-b or CXCR2. CXCR1 binds IL-8 with high affinity, whereas CXCR2 is more
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promiscuous and binds other related chemokines such as those mentioned previously (Ahuja et al., 1996). Indirect evidence suggests that bovine neutrophils express both CXCR1 and CXCR2, as they display functional activity towards IL-8, ENA-78 and GRO-a (Caswell et al., 1999; Li et al., 2002a,b). Li et al., 2002a also reported CXCR1 and CXCR2 expression in bovine neutrophils by labeling with antibodies to human CXCR1 and CXCR2. However, up to this time, only one of the IL-8 receptor mRNA sequences has been publicly available, the provisional reference sequence for CXCR2 (Genbank Accession number NM_174360.2). This suggests that either only one type of IL-8 receptor exists in cattle and generates a ‘hybrid’ protein similar to both CXCR1 and CXCR2 or more likely, that the alternative IL-8 receptor transcript has not yet been identified. Due to the critical role IL-8 and its receptors share in the disease process, a more thorough understanding of gene structure, as well as the mechanisms that activate IL-8 receptor transcription and translation is needed. Therefore, as a necessary step in this direction, the primary objective of this study was to identify the presence or absence of both CXCR1 and CXCR2 orthologs in cattle. 2. Materials and methods 2.1. Sequence alignment and visualization The bovine CXCR2 mRNA provisional reference sequence (NM_174360.2) was aligned with the March 2005 build of the bovine genome using BLAT located at the UCSC browser (http://genome.ucsc.edu/) (Kent, 2002). A comparison of this region of the bovine genome with human and other species was conducted using a combination of local and global alignment that determines the degree of nucleotide conservation between genomes of different species (Couronne et al., 2003; Frazer et al., 2004a,b). This was accomplished using the GenomeVista server (http:// pipeline.lbl.gov/cgi-bin/GenomeVista). The builds available on the GenomeVista server for the human, canine, mouse, and rat genomes were May 2004, July 2004, May 2004, and June 2003, respectively. The available bovine build on this server was from March 2004 and had considerable gaps in the region of interest, therefore, a phase 2 working draft of high throughput genome sequence for clone CH240-194K2 (Genbank Accession number AC151135.5) was utilized instead. Once aligned, conserved regions were identified by evaluating the percent nucleotides identical within a 100 nucleotide window and 70% conservation. The
resulting alignment with areas of conservation was visualized using VISTA (Couronne et al., 2003; Frazer et al., 2004a,b). The relationship among partial bovine IL-8R mRNA sequences generated through RT-PCR, human CXCR1 mRNA (NM_000634), and human CXCR2 (NM_001557) were assessed via ClustalW (http://www.ebi.ac.uk/clustalw/). 2.2. Generation of CXCR1 and CXCR2 cDNA from total RNA Total RNAwas isolated from bovine neutrophils using TRIzol as outlined by the manufacturer (Invitrogen, Carlsbad, CA). Neutrophils were isolated from peripheral blood of lactating Holstein cows using hypotonic lysis as described previously (Rambeaud and Pighetti, 2005). The drawing of blood was approved by the University of Tennessee Institutional Animal Care and Use Committee. Isolated RNA (2 mg) was reverse transcribed using AMV-RT according to the manufacturers instructions (Promega, Madison, WI). The resulting cDNA was then amplified by PCR using primers specific for each predicted bovine IL-8R sequence. The CXCR1 forward (50 -ggaggggtttgaggatgagt) and reverse (50 -gccaggttcagcaggtagac) primers amplify a 228 bp fragment specific for the IL-8R reference mRNA (87– 314 bp) and the predicted CXCR1 genome sequence (110,082–110,309 of AC151135.5). The CXCR2 forward (50 -gcgatgaagattttggcaat) and reverse (50 -gccaggttcagcaggtagac) primers amplify a 226 bp fragment specific for the predicted CXCR2 genome sequence (84374–84599 of AC151135.5). Samples were amplified over 30 cycles: 30 s at 94 8C, 30 s at 60 8C, and 60 s at 72 8C. The resulting PCR products were purified with a commercial kit (ZymoResearch, Orange, CA) and sent to the university’s core sequencing facility. 3. Results and discussion To date, only one provisional reference sequence has been published for bovine IL-8R and has been annotated as CXCR2 (NM_174360.2). Alignment of this reference sequence with the bovine genome (March 2005 build) using BLAT from the UCSC browser reveals significant identity with two regions of the bovine genome (Table 1). The most comprehensive alignment with the greatest nucleotide identity (98.5%) is located on the opposite strand closer to the q telomere of the chromosome (64,546,485–64,548,092). Nucleotide identity between mRNA and the genome sequence reached 100% once an approximate 50 base gap in the March 2005 build of BTA2 was filled by aligning it with
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Table 1 Bovine CXCR2 mRNA reference sequence alignment to separate regions on BTA2 and a working draft of the bovine genome using BLAT CXCR2 mRNA (NM_174360)
BTA2 region (March 2005 build)
Working draft bovine genome (AC151135.5)
Strand
Nucleotide identity
39–1744 188–1710
64,546,485–64,548,092 64,523,226–64,524,748
110,082–110,309 84,374–84,599
+
100% 95%
a working draft sequence (AC151135.5). In contrast, the second alignment located on the same strand but closer to the centromere (64,523,226–64,524,748) represents a smaller portion of the reference mRNA sequence and has only 95% nucleotide identity, suggesting this second region may contain a related IL-8 receptor, similar to what has been observed in other species. The identification of two regions on the bovine genome similar to IL-8R suggests that cows have both CXCR1 and CXCR2 genes, as has been reported for protein based studies in cattle (Li et al., 2002a,b) but not mRNA. Therefore, to gain a better understanding of the bovine IL-8R gene complex, this region of the bovine genome was compared to human, canine, mouse, and rat genomes using GenomeVista which uses a combination of local and global alignment to assess conservation of genomic regions and aid in identifying orthologous genes (Couronne et al., 2003). To accomplish this, human chromosome 2 was narrowed to contain only CXCR1 and CXCR2 genes (218,815,989–218,858,272) and alignments made with the bovine draft genome sequence (AC151135.5) (Fig. 1). Overall conservation of exon, intron, and intergenic regions between bovine
and human genomes was 29% within the IL-8R gene complex which spans approximately 43,000 bases. When only the genomic region containing the approximate 1000 base coding exon for each receptor was evaluated, 81–83% nucleotide conservation was observed between human CXCR1 (218,854,446– 218,855,439) and bases 109,332–110,331 of the draft bovine genome sequence, as well as between human CXCR2 (218,825,031–218,826,111) and bases 84,346– 85,408 of the draft bovine genome sequence. Based on annotation of the human genome, this evidence strongly suggests that the IL-8R related gene located closer to the centromere of BTA2 on initial analysis is an ortholog of CXCR2, whereas the IL-8R related gene located closer to the q telomere on the opposite strand is an ortholog of CXCR1. These findings also indicate the current annotation of the provisional reference sequence for bovine CXCR2 mRNA (NM_174360.2) is incorrect as this mRNA sequence is identical to the IL-8R related gene closer to the telomere or apparent human CXCR1 ortholog. In order to address concerns regarding annotation of the bovine IL-8R mRNA reference sequence, primers
Fig. 1. Genome conservation plot of IL-8 receptor gene complex. Nucleotide conservation between the base genome (human) and bovine, canine, rat, and mouse was assessed using GenomeVista and visualized in a VISTA plot. Nucleotide conservation was calculated in 100 bp windows and peaks represent 50–100% conservation within the VISTA plot. Regions within rectangles with dashed lines are non-coding exons and rectangles with solid lines are coding exons.
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were designed to amplify small coding regions (228 bases) specific to each predicted CXCR1 and CXCR2 gene. The resulting cDNA products were sequenced and similarity between the human and bovine IL-8R mRNAs assessed through ClustalW alignment (Fig. 2). The score between predicted bovine and human sequences was 74 for CXCR1 and 80 for CXCR2. Examination of the converse relationship, predicted bovine CXCR1 with human CXCR2 revealed a considerably lower score of 53, and resembles the score of 58 between predicted bovine CXCR1 and CXCR2 partial cDNAs. A similar, but less dramatic drop in score (71) was observed when the converse relationship of predicted bovine CXCR2 and human CXCR1 was examined. Greater scores for the predicted orthologs relative to the currently defined annotation lend further support for the incorrect annotation of the bovine IL-8R mRNA reference sequence. This finding has direct impact on prior research from our lab as we originally reported a series of single nucleotide polymorphisms as being in the IL-8Rb or ‘CXCR2’ gene based upon the reference sequence (Youngerman et al., 2004a,b; Rambeaud and Pighetti, 2005; Ram-
beaud et al., 2006). However, based upon current information, the polymorphisms are actually located in IL-8Ra or CXCR1. Missannotation of the IL-8R gene is not unexpected. The human CXCR1 and CXCR2 mRNAs share 89% identity within the coding region when evaluated using BLAST (data not shown). The high degree of similarity between the coding region of these genes, as well as their close proximity, complicates gene prediction (Lewis et al., 2000). The potential for improper annotation of genes also increases with the use of computer-generated annotations (Miller et al., 2004). Two basic methods are used for computer-generated gene prediction: extrinsic and ab initio (recently reviewed by Mathe et al., 2002). The first method utilizes sequence similarity and alignment to mRNA, EST, and gene sequences within and across species to develop gene predictions and is exemplified by the techniques utilized in this paper. In contrast, the second method uses Markov models developed from ‘training’ datasets to recognize specific patterns in the genome sequence. Although these methods utilize different techniques, both rely on current databases or training
Fig. 2. Clustal alignment of sequences. (A) Bovine CXCR1 partial mRNA with human CXCR1 mRNA, (B) bovine CXCR2 partial mRNA sequence with human CXCR2 mRNA sequence, and (C) predicted bovine CXCR1 and CXCR2 sequences. Nucleotides in bold with an asterisk underneath are conserved between the two species.
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sets of known information. A lack of information as well as incorrect annotation directly impairs the effectiveness of the annotation process. Therefore, when incorrect annotations are found, they should be reported and were the focus of a recent editorial (Perkel, 2006). The ability of automated methods to accurately predict coding sequences, exons, and gene structure was initiated in 2005 as a community project EGASP (ENCODE Genome Assessment Project) (Guigo and Reese, 2005). Preliminary results indicate sensitivity and specificity of exon predictions were greatest for expression-derived methods (0.8) and least for ab initio (0.5) methods. The final results are expected to be published soon and should provide a comprehensive assessment of current annotation methods and serve as a basis for development and refinement of gene prediction methodologies. The observed similarity of the bovine to the human genome in the region of the IL-8R complex suggests cattle may be useful as an alternative animal model for human studies. To examine this possibility, the level of nucleotide conservation at the genome level was evaluated between the human IL-8R complex and that of bovine, canine, mouse and rat species (Fig. 1). The overall conservation of nucleotide sequence in this approximately 42,000 bp region of the human genome was greater in canine (37%) and bovine (29%) relative to both mouse (9%) and rat (9%). Because the degree of nucleotide conservation for the CXCR1 and CXCR2 coding exons in the 42,000 bp region was similar for all species (6–7%), the primary difference in overall conservation was observed in non-coding exons, introns, and intergenic regions. These non-protein coding regions of the genome contain regulatory sequences such as promoters, enhancers, silencers, and non-coding RNA which influence transcription and translation. Greater conservation of nucleotides in nonprotein coding regions increases the probability of key regulatory regions being conserved. For instance, comparison of the SIM2 interval on human chromosome 21 (365 kDa) with six different species, revealed that horse, cow, pig, dog, and cat genomes had more sequences in common with humans than mice (Frazer et al., 2004a,b). Subsequent evaluation of several conserved sequences present in species other than mice demonstrated these sequences were capable of influencing SIM2 promoter activity. This supports the hypothesis that moderate nucleotide conservation between the bovine and human IL-8 receptor gene complex increases the probability of more functional regulatory elements being conserved. Therefore, the cow may be a more suitable animal model for certain
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types of studies evaluating CXCR1 and CXCR2 gene expression. In conclusion, with the availability of the bovine genome sequence and aid of bioinformatics tools, we have been able to identify bovine orthologs of the human CXCR1 and CXCR2 genes and verify their presence through mRNA expression. This process also revealed that the current annotation of the provisional bovine CXCR2 mRNA reference sequence (NM_174360.2) is incorrect. Moreover, moderate conservation of the bovine IL-8R gene complex with the human complex indicates that cattle may provide a more suitable animal model for certain studies, as well as allow the use of human reagents to better understand the bovine system. This information provides a stepping stone for greater understanding of the role each of these receptors plays in the inflammatory response and will enhance our ability to develop preventive and therapeutic strategies against inflammatory based diseases such as mastitis and pneumonia. Acknowledgements The critical review of this manuscript by Arnold Saxton, Cheryl Kojima, Angela Pollock, and Rose Clift is appreciated greatly. The research reported within this manuscript was supported by The College of Veterinary Medicine Center of Excellence and The University of Tennessee Agricultural Experiment Station. References Ahuja, S.K., Murphy, P.M., Tiffany, H.L., 1996. The CXC chemokines growth-regulated oncogene (GRO)-alpha, -beta, -gamma, neutrophil activating peptide-2, and epithelial cell-derived neutrophil activating peptide-78 are potent agonists for the type B, but not the type A, human interleukin-8 receptor. J. Biol. Chem. 271, 20545– 20550. Caswell, J.L., Middleton, D.M., Gordon, J.R., 1999. Production and functional characterization of recombinant bovine interleukin-8 as a specific neutrophil activator and chemoattractant. Vet. Immunol. Immunopathol. 67, 327–340. Couronne, O., Poliakov, A., Bray, N., Ishkhanov, T., Ryaboy, D., Rubin, E., Pachter, L., Dubchak, I., 2003. Strategies and tools for whole-genome alignments. Genome Res. 13, 73–80. Frazer, K.A., Pachter, L., Poliakov, A., Rubin, E.M., Dubchak, I., 2004a. VISTA: computational tools for comparative genomics. Nucleic Acids Res. 32, W273–W279. Frazer, K.A., Tao, H., Osoegawa, K., de Jong, P.J., Chen, X., Doherty, M.F., Cox, D.R., 2004b. Noncoding sequences conserved in a limited number of mammals in the SIM2 interval are frequently functional. Genome Res. 14, 367–372. Green, S.P., Chuntharapai, A., Curnutte, J.T., 1996. Interleukin-8 (IL8), melanoma growth-stimulatory activity, and neutrophil-activating peptide selectively mediate priming of the neutrophil NADPH
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oxidase through the type A or type B IL-8 receptor. J. Biol. Chem. 271, 25400–25405. Guigo, R., Reese, M.G., 2005. EGASP: collaboration through competition to find human genes. Nat. Methods 2, 575–577. Jozsef, L., Khreiss, T., El Kebir, D., Filep, J.G., 2006. Activation of TLR-9 induces IL-8 secretion through peroxynitrite signaling in human neutrophils. J. Immunol. 176, 1195–1202. Kent, W.J., 2002. BLAT—The BLAST-like alignment tool. Genome Res. 12, 656–664. Kettritz, R., Gaido, M.L., Haller, H., Luft, F.C., Jennette, C.J., Falk, R.J., 1998. Interleukin-8 delays spontaneous and tumor necrosis factor-alpha- mediated apoptosis of human neutrophils. Kidney Int. 53, 84–91. Lewis, S., Ashburner, M., Reese, M.G., 2000. Annotating eukaryote genomes. Curr. Opin. Struct. Biol. 10, 349–354. Li, F., Zhang, X., Gordon, J.R., 2002a. CXCL8(3-73)K11R/G31P antagonizes ligand binding to the neutrophil CXCR1 and CXCR2 receptors and cellular responses to CXCL8/IL-8. Biochem. Biophys. Res. Commun. 293, 939–944. Li, F., Zhang, X., Mizzi, C., Gordon, J.R., 2002b. CXCL8((3– 73))K11R/G31P antagonizes the neutrophil chemoattractants present in pasteurellosis and mastitis lesions and abrogates neutrophil influx into intradermal endotoxin challenge sites in vivo. Vet. Immunol. Immunopathol. 90, 65–77.
Mathe, C., Sagot, M.F., Schiex, T., Rouze, P., 2002. Current methods of gene prediction, their strengths and weaknesses. Nucleic Acids Res. 30, 4103–4117. Miller, W., Makova, K.D., Nekrutenko, A., Hardison, R.C., 2004. Comparative genomics. Annu. Rev. Genomics Hum. Genet. 5, 15– 56. Mitchell, G.B., Albright, B.N., Casewell, J.L., 2003. Effect of interleukin-8 and granulocyte colony stimulating factor on priming and activation of bovine neutrophils. Infect. Immun. 71, 1643– 1649. Perkel, J.M., 2006. Why you should be annotating. The Scientist 20, 71. Rambeaud, M., Pighetti, G.M., 2005. Impaired neutrophil migration associated with specific bovine CXCR2 genotypes. Infect. Immun. 73, 4955–4959. Rambeaud, M., Clift, R., Pighetti, G.M., 2006. Association of a bovine CXCR2 gene polymorphism with neutrophil survival and killing ability. Vet. Immunol. Immunopathol. (epub ahead of print). Youngerman, S.M., Saxton, A.M., Oliver, S.P., Pighetti, G.M., 2004a. Association of CXCR2 polymorphisms with subclinical and clinical mastitis in dairy cattle. J. Dairy Sci. 87, 2442–2448. Youngerman, S.M., Saxton, A.M., Pighetti, G.M., 2004b. Novel single nucleotide polymorphisms and haplotypes within the bovine CXCR2 gene. Immunogenetics 56, 355–359.
Short communication
Genome conservation between the bovine and human interleukin-8 receptor complex: Improper annotation of bovine interleukin-8 receptor b identified Gina M. Pighetti *, Magdalena Rambeaud Department of Animal Science, 114 McCord Hall, 2640 Morgan Circle, The University of Tennessee, Knoxville, TN 37996, United States Received 1 May 2006; received in revised form 28 July 2006; accepted 14 August 2006
Abstract Interleukin (IL)-8 and its receptors, CXCR1 and CXCR2, are key regulators of inflammation. However, knowledge of these receptors at the genomic level is limiting or absent in cattle. Therefore, our objective was to identify bovine orthologs of human CXCR1 and CXCR2. Alignment of bovine CXCR2 reference mRNA to the bovine genome revealed two regions of similarity on BTA2 approximately 20 kb apart and on opposite strands. Comparison with the human genome suggested the more centromeric region to be CXCR2 and the more telomeric region to be CXCR1 which contradicts the current annotation of the bovine CXCR2 reference mRNA. This observation was verified by sequencing RT-PCR products of specific regions within each predicted IL-8 receptor and comparing with human sequences using ClustalW. Further examination of coding and non-coding regions within the IL-8 receptor genome complex revealed that both bovine and canine CXCR1 and CXCR2 genes had more conserved sequences in common with the human genes than either mouse or rat, and may offer more suitable animal models for certain applications. This molecular information provides a stepping stone for greater understanding of the role each IL-8 receptor plays in inflammation and will enhance our ability to develop strategies against inflammatory based diseases. # 2006 Elsevier B.V. All rights reserved. Keywords: Bovine; Genome; Inflammation; Interleukin-8 receptors
1. Introduction Elimination of invading organisms often requires an effective inflammatory response: pathogen recognition, inflammatory mediator release, leukocyte recruitment, bacteria removal, and leukocyte removal. A key mediator of this process is the chemokine interleu-
Abbreviations: ENA-78, epithelial neutrophil activating protein 78; GRO, growth regulated oncogene; Interleukin-8, IL-8; MIP, Macrophage inflammatory protein * Corresponding author. Tel.: +1 865 974 7225; fax: +1 865 974 3394. E-mail address: [email protected] (G.M. Pighetti). 0165-2427/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2006.08.008
kin-8 (IL-8). This chemokine, mostly recognized for its ability to induce neutrophil migration, also increases cytokine production, enhances phagocytosis and reactive oxygen species generation, as well as regulating cell survival (Kettritz et al., 1998; Mitchell et al., 2003; Jozsef et al., 2006). Other structurally related chemokines such as epithelial neutrophil activating protein 78 (ENA-78), growth regulated oncogene (GRO)-a, b, g, and macrophage inflammatory protein (MIP)-1 have similar functional activities (Green et al., 1996; Li et al., 2002a,b). These chemokines mediate their effects through two primary receptors, IL-8 receptor (IL8R)-a or CXCR1 and IL-8R-b or CXCR2. CXCR1 binds IL-8 with high affinity, whereas CXCR2 is more
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promiscuous and binds other related chemokines such as those mentioned previously (Ahuja et al., 1996). Indirect evidence suggests that bovine neutrophils express both CXCR1 and CXCR2, as they display functional activity towards IL-8, ENA-78 and GRO-a (Caswell et al., 1999; Li et al., 2002a,b). Li et al., 2002a also reported CXCR1 and CXCR2 expression in bovine neutrophils by labeling with antibodies to human CXCR1 and CXCR2. However, up to this time, only one of the IL-8 receptor mRNA sequences has been publicly available, the provisional reference sequence for CXCR2 (Genbank Accession number NM_174360.2). This suggests that either only one type of IL-8 receptor exists in cattle and generates a ‘hybrid’ protein similar to both CXCR1 and CXCR2 or more likely, that the alternative IL-8 receptor transcript has not yet been identified. Due to the critical role IL-8 and its receptors share in the disease process, a more thorough understanding of gene structure, as well as the mechanisms that activate IL-8 receptor transcription and translation is needed. Therefore, as a necessary step in this direction, the primary objective of this study was to identify the presence or absence of both CXCR1 and CXCR2 orthologs in cattle. 2. Materials and methods 2.1. Sequence alignment and visualization The bovine CXCR2 mRNA provisional reference sequence (NM_174360.2) was aligned with the March 2005 build of the bovine genome using BLAT located at the UCSC browser (http://genome.ucsc.edu/) (Kent, 2002). A comparison of this region of the bovine genome with human and other species was conducted using a combination of local and global alignment that determines the degree of nucleotide conservation between genomes of different species (Couronne et al., 2003; Frazer et al., 2004a,b). This was accomplished using the GenomeVista server (http:// pipeline.lbl.gov/cgi-bin/GenomeVista). The builds available on the GenomeVista server for the human, canine, mouse, and rat genomes were May 2004, July 2004, May 2004, and June 2003, respectively. The available bovine build on this server was from March 2004 and had considerable gaps in the region of interest, therefore, a phase 2 working draft of high throughput genome sequence for clone CH240-194K2 (Genbank Accession number AC151135.5) was utilized instead. Once aligned, conserved regions were identified by evaluating the percent nucleotides identical within a 100 nucleotide window and 70% conservation. The
resulting alignment with areas of conservation was visualized using VISTA (Couronne et al., 2003; Frazer et al., 2004a,b). The relationship among partial bovine IL-8R mRNA sequences generated through RT-PCR, human CXCR1 mRNA (NM_000634), and human CXCR2 (NM_001557) were assessed via ClustalW (http://www.ebi.ac.uk/clustalw/). 2.2. Generation of CXCR1 and CXCR2 cDNA from total RNA Total RNAwas isolated from bovine neutrophils using TRIzol as outlined by the manufacturer (Invitrogen, Carlsbad, CA). Neutrophils were isolated from peripheral blood of lactating Holstein cows using hypotonic lysis as described previously (Rambeaud and Pighetti, 2005). The drawing of blood was approved by the University of Tennessee Institutional Animal Care and Use Committee. Isolated RNA (2 mg) was reverse transcribed using AMV-RT according to the manufacturers instructions (Promega, Madison, WI). The resulting cDNA was then amplified by PCR using primers specific for each predicted bovine IL-8R sequence. The CXCR1 forward (50 -ggaggggtttgaggatgagt) and reverse (50 -gccaggttcagcaggtagac) primers amplify a 228 bp fragment specific for the IL-8R reference mRNA (87– 314 bp) and the predicted CXCR1 genome sequence (110,082–110,309 of AC151135.5). The CXCR2 forward (50 -gcgatgaagattttggcaat) and reverse (50 -gccaggttcagcaggtagac) primers amplify a 226 bp fragment specific for the predicted CXCR2 genome sequence (84374–84599 of AC151135.5). Samples were amplified over 30 cycles: 30 s at 94 8C, 30 s at 60 8C, and 60 s at 72 8C. The resulting PCR products were purified with a commercial kit (ZymoResearch, Orange, CA) and sent to the university’s core sequencing facility. 3. Results and discussion To date, only one provisional reference sequence has been published for bovine IL-8R and has been annotated as CXCR2 (NM_174360.2). Alignment of this reference sequence with the bovine genome (March 2005 build) using BLAT from the UCSC browser reveals significant identity with two regions of the bovine genome (Table 1). The most comprehensive alignment with the greatest nucleotide identity (98.5%) is located on the opposite strand closer to the q telomere of the chromosome (64,546,485–64,548,092). Nucleotide identity between mRNA and the genome sequence reached 100% once an approximate 50 base gap in the March 2005 build of BTA2 was filled by aligning it with
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337
Table 1 Bovine CXCR2 mRNA reference sequence alignment to separate regions on BTA2 and a working draft of the bovine genome using BLAT CXCR2 mRNA (NM_174360)
BTA2 region (March 2005 build)
Working draft bovine genome (AC151135.5)
Strand
Nucleotide identity
39–1744 188–1710
64,546,485–64,548,092 64,523,226–64,524,748
110,082–110,309 84,374–84,599
+
100% 95%
a working draft sequence (AC151135.5). In contrast, the second alignment located on the same strand but closer to the centromere (64,523,226–64,524,748) represents a smaller portion of the reference mRNA sequence and has only 95% nucleotide identity, suggesting this second region may contain a related IL-8 receptor, similar to what has been observed in other species. The identification of two regions on the bovine genome similar to IL-8R suggests that cows have both CXCR1 and CXCR2 genes, as has been reported for protein based studies in cattle (Li et al., 2002a,b) but not mRNA. Therefore, to gain a better understanding of the bovine IL-8R gene complex, this region of the bovine genome was compared to human, canine, mouse, and rat genomes using GenomeVista which uses a combination of local and global alignment to assess conservation of genomic regions and aid in identifying orthologous genes (Couronne et al., 2003). To accomplish this, human chromosome 2 was narrowed to contain only CXCR1 and CXCR2 genes (218,815,989–218,858,272) and alignments made with the bovine draft genome sequence (AC151135.5) (Fig. 1). Overall conservation of exon, intron, and intergenic regions between bovine
and human genomes was 29% within the IL-8R gene complex which spans approximately 43,000 bases. When only the genomic region containing the approximate 1000 base coding exon for each receptor was evaluated, 81–83% nucleotide conservation was observed between human CXCR1 (218,854,446– 218,855,439) and bases 109,332–110,331 of the draft bovine genome sequence, as well as between human CXCR2 (218,825,031–218,826,111) and bases 84,346– 85,408 of the draft bovine genome sequence. Based on annotation of the human genome, this evidence strongly suggests that the IL-8R related gene located closer to the centromere of BTA2 on initial analysis is an ortholog of CXCR2, whereas the IL-8R related gene located closer to the q telomere on the opposite strand is an ortholog of CXCR1. These findings also indicate the current annotation of the provisional reference sequence for bovine CXCR2 mRNA (NM_174360.2) is incorrect as this mRNA sequence is identical to the IL-8R related gene closer to the telomere or apparent human CXCR1 ortholog. In order to address concerns regarding annotation of the bovine IL-8R mRNA reference sequence, primers
Fig. 1. Genome conservation plot of IL-8 receptor gene complex. Nucleotide conservation between the base genome (human) and bovine, canine, rat, and mouse was assessed using GenomeVista and visualized in a VISTA plot. Nucleotide conservation was calculated in 100 bp windows and peaks represent 50–100% conservation within the VISTA plot. Regions within rectangles with dashed lines are non-coding exons and rectangles with solid lines are coding exons.
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were designed to amplify small coding regions (228 bases) specific to each predicted CXCR1 and CXCR2 gene. The resulting cDNA products were sequenced and similarity between the human and bovine IL-8R mRNAs assessed through ClustalW alignment (Fig. 2). The score between predicted bovine and human sequences was 74 for CXCR1 and 80 for CXCR2. Examination of the converse relationship, predicted bovine CXCR1 with human CXCR2 revealed a considerably lower score of 53, and resembles the score of 58 between predicted bovine CXCR1 and CXCR2 partial cDNAs. A similar, but less dramatic drop in score (71) was observed when the converse relationship of predicted bovine CXCR2 and human CXCR1 was examined. Greater scores for the predicted orthologs relative to the currently defined annotation lend further support for the incorrect annotation of the bovine IL-8R mRNA reference sequence. This finding has direct impact on prior research from our lab as we originally reported a series of single nucleotide polymorphisms as being in the IL-8Rb or ‘CXCR2’ gene based upon the reference sequence (Youngerman et al., 2004a,b; Rambeaud and Pighetti, 2005; Ram-
beaud et al., 2006). However, based upon current information, the polymorphisms are actually located in IL-8Ra or CXCR1. Missannotation of the IL-8R gene is not unexpected. The human CXCR1 and CXCR2 mRNAs share 89% identity within the coding region when evaluated using BLAST (data not shown). The high degree of similarity between the coding region of these genes, as well as their close proximity, complicates gene prediction (Lewis et al., 2000). The potential for improper annotation of genes also increases with the use of computer-generated annotations (Miller et al., 2004). Two basic methods are used for computer-generated gene prediction: extrinsic and ab initio (recently reviewed by Mathe et al., 2002). The first method utilizes sequence similarity and alignment to mRNA, EST, and gene sequences within and across species to develop gene predictions and is exemplified by the techniques utilized in this paper. In contrast, the second method uses Markov models developed from ‘training’ datasets to recognize specific patterns in the genome sequence. Although these methods utilize different techniques, both rely on current databases or training
Fig. 2. Clustal alignment of sequences. (A) Bovine CXCR1 partial mRNA with human CXCR1 mRNA, (B) bovine CXCR2 partial mRNA sequence with human CXCR2 mRNA sequence, and (C) predicted bovine CXCR1 and CXCR2 sequences. Nucleotides in bold with an asterisk underneath are conserved between the two species.
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sets of known information. A lack of information as well as incorrect annotation directly impairs the effectiveness of the annotation process. Therefore, when incorrect annotations are found, they should be reported and were the focus of a recent editorial (Perkel, 2006). The ability of automated methods to accurately predict coding sequences, exons, and gene structure was initiated in 2005 as a community project EGASP (ENCODE Genome Assessment Project) (Guigo and Reese, 2005). Preliminary results indicate sensitivity and specificity of exon predictions were greatest for expression-derived methods (0.8) and least for ab initio (0.5) methods. The final results are expected to be published soon and should provide a comprehensive assessment of current annotation methods and serve as a basis for development and refinement of gene prediction methodologies. The observed similarity of the bovine to the human genome in the region of the IL-8R complex suggests cattle may be useful as an alternative animal model for human studies. To examine this possibility, the level of nucleotide conservation at the genome level was evaluated between the human IL-8R complex and that of bovine, canine, mouse and rat species (Fig. 1). The overall conservation of nucleotide sequence in this approximately 42,000 bp region of the human genome was greater in canine (37%) and bovine (29%) relative to both mouse (9%) and rat (9%). Because the degree of nucleotide conservation for the CXCR1 and CXCR2 coding exons in the 42,000 bp region was similar for all species (6–7%), the primary difference in overall conservation was observed in non-coding exons, introns, and intergenic regions. These non-protein coding regions of the genome contain regulatory sequences such as promoters, enhancers, silencers, and non-coding RNA which influence transcription and translation. Greater conservation of nucleotides in nonprotein coding regions increases the probability of key regulatory regions being conserved. For instance, comparison of the SIM2 interval on human chromosome 21 (365 kDa) with six different species, revealed that horse, cow, pig, dog, and cat genomes had more sequences in common with humans than mice (Frazer et al., 2004a,b). Subsequent evaluation of several conserved sequences present in species other than mice demonstrated these sequences were capable of influencing SIM2 promoter activity. This supports the hypothesis that moderate nucleotide conservation between the bovine and human IL-8 receptor gene complex increases the probability of more functional regulatory elements being conserved. Therefore, the cow may be a more suitable animal model for certain
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types of studies evaluating CXCR1 and CXCR2 gene expression. In conclusion, with the availability of the bovine genome sequence and aid of bioinformatics tools, we have been able to identify bovine orthologs of the human CXCR1 and CXCR2 genes and verify their presence through mRNA expression. This process also revealed that the current annotation of the provisional bovine CXCR2 mRNA reference sequence (NM_174360.2) is incorrect. Moreover, moderate conservation of the bovine IL-8R gene complex with the human complex indicates that cattle may provide a more suitable animal model for certain studies, as well as allow the use of human reagents to better understand the bovine system. This information provides a stepping stone for greater understanding of the role each of these receptors plays in the inflammatory response and will enhance our ability to develop preventive and therapeutic strategies against inflammatory based diseases such as mastitis and pneumonia. Acknowledgements The critical review of this manuscript by Arnold Saxton, Cheryl Kojima, Angela Pollock, and Rose Clift is appreciated greatly. The research reported within this manuscript was supported by The College of Veterinary Medicine Center of Excellence and The University of Tennessee Agricultural Experiment Station. References Ahuja, S.K., Murphy, P.M., Tiffany, H.L., 1996. The CXC chemokines growth-regulated oncogene (GRO)-alpha, -beta, -gamma, neutrophil activating peptide-2, and epithelial cell-derived neutrophil activating peptide-78 are potent agonists for the type B, but not the type A, human interleukin-8 receptor. J. Biol. Chem. 271, 20545– 20550. Caswell, J.L., Middleton, D.M., Gordon, J.R., 1999. Production and functional characterization of recombinant bovine interleukin-8 as a specific neutrophil activator and chemoattractant. Vet. Immunol. Immunopathol. 67, 327–340. Couronne, O., Poliakov, A., Bray, N., Ishkhanov, T., Ryaboy, D., Rubin, E., Pachter, L., Dubchak, I., 2003. Strategies and tools for whole-genome alignments. Genome Res. 13, 73–80. Frazer, K.A., Pachter, L., Poliakov, A., Rubin, E.M., Dubchak, I., 2004a. VISTA: computational tools for comparative genomics. Nucleic Acids Res. 32, W273–W279. Frazer, K.A., Tao, H., Osoegawa, K., de Jong, P.J., Chen, X., Doherty, M.F., Cox, D.R., 2004b. Noncoding sequences conserved in a limited number of mammals in the SIM2 interval are frequently functional. Genome Res. 14, 367–372. Green, S.P., Chuntharapai, A., Curnutte, J.T., 1996. Interleukin-8 (IL8), melanoma growth-stimulatory activity, and neutrophil-activating peptide selectively mediate priming of the neutrophil NADPH
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