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Wang, Jixin; Adelson, David Louis; Yilmaz, Ahmet; Sze, Sing-Hoi; Jin, Yuan; Zhu, James. Genomic organization, annotation, and ligand-receptor inferences of chicken chemokines and chemokine receptor based on comparative genomics, BMC Genomics, 2005; 6 (45): www1-www17.

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nd 2 May 2011

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BMC Genomics BioMed Central

Research article Open Access Genomic organization, annotation, and ligand-receptor inferences of chicken chemokines and chemokine receptor genes based on comparative genomics Jixin Wang1, David L Adelson2, Ahmet Yilmaz1, Sing-Hoi Sze3, Yuan Jin3 and James J Zhu*1

Address: 1Department of Poultry Science, Texas A & M University, College Station, TX 77843, USA, 2Department of Animal Science, Texas A & M University, College Station, TX 77843, USA and 3Department of Computer Science, Texas A & M University, College Station, TX 77843, USA Email: Jixin Wang - [email protected]; David L Adelson - [email protected]; Ahmet Yilmaz - [email protected]; Sing- Hoi Sze - [email protected]; Yuan Jin - [email protected]; James J Zhu* - [email protected] * Corresponding author

Published: 24 March 2005 Received: 12 November 2004 Accepted: 24 March 2005 BMC Genomics 2005, 6:45 doi:10.1186/1471-2164-6-45 This article is available from: http://www.biomedcentral.com/1471-2164/6/45 © 2005 Wang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract Background: Chemokines and their receptors play important roles in host defense, organogenesis, hematopoiesis, and neuronal communication. Forty-two chemokines and 19 cognate receptors have been found in the . Prior to this report, only 11 chicken chemokines and 7 receptors had been reported. The objectives of this study were to systematically identify chicken chemokines and their cognate receptor genes in the chicken genome and to annotate these genes and ligand-receptor binding by a comparative genomics approach. Results: Twenty-three chemokine and 14 chemokine receptor genes were identified in the chicken genome. All of the chicken chemokines contained a conserved CC, CXC, CX3C, or XC motif, whereas all the chemokine receptors had seven conserved transmembrane helices, four extracellular domains with a conserved cysteine, and a conserved DRYLAIV sequence in the second intracellular domain. The number of coding exons in these genes and the syntenies are highly conserved between human, mouse, and chicken although the amino acid sequence homologies are generally low between mammalian and chicken chemokines. Chicken genes were named with the systematic nomenclature used in humans and mice based on phylogeny, synteny, and . Conclusion: The independent nomenclature of chicken chemokines and chemokine receptors suggests that the chicken may have ligand-receptor pairings similar to mammals. All identified chicken chemokines and their cognate receptors were identified in the chicken genome except CCR9, whose ligand was not identified in this study. The organization of these genes suggests that there were a substantial number of these genes present before divergence between aves and mammals and more duplications of CC, CXC, CCR, and CXCR subfamilies in mammals than in aves after the divergence.

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Background species. These receptors have characteristic seven alpha- Chemokines are a family of small chemoattrative peptides helix transmembrane domains with a length between that were originally recognized to be involved in host 340–370 amino acids and have up to 80% amino acid defense as regulators of leukocyte trafficking, but more identity [1]. They also share an acidic amino terminus, a recently have also been shown to have roles in organogen- conserved sequence in the second intracellular loop, and esis, hematopoiesis, and neuronal communication [1]. one cysteine in each extracellular domain [10]. Most Their cognate receptors belong to the Class A subfamily of receptors can bind several chemokines of a single class G- coupled receptor superfamily [2]. Chemokines with high affinity [11]. Like chemokines, most chemokine are believed to have originated from gene duplications receptors are also clustered in a few chromosomal regions, and these genes underwent selection during recent evolu- such as human 2 and 3 [2]. Most amino tionary time [3]. All chemokines have a characteristic acid sequences of chemokine receptors are encoded in cysteine motif. Similarly, chemokine receptors may also one exon. be derived from a common ancestor through gene dupli- cations. All chemokine receptors share high homology At present, 42 chemokine genes have been identified in with the prototypical family member, rhodopsin [2]. human (24 CC, 15 CXC, 1 CX3C, and 2 XC) and 36 (21 CC, 13 CXC, 1 CX3C, and 1 XC) in mouse, whereas there Chemokines are highly basic , 70 to 125 amino are 11 receptors for CCLs, 6 for CXCLs, 1 for CX3CL, and acids long. Sequence identity among chemokines is usu- 1 for XCL in human and mouse. Only 11 chicken chem- ally low; however, all share a typical overall tertiary struc- okines including 4 CXC, 6 CC, and 1 XC and seven ture, which consists of at least four cysteines that form two chicken chemokine receptors including 2 CXCR and 5 disulfide bonds. Chemokines are divided into two major CCR have been reported in the literature [12-23]. Chicken (CC and CXC) and two minor (XC and CX3C) subfamilies chemokines share low sequence identity with mammals based on their four conserved cysteines. The first two [24]. Therefore, it was very difficult to assign chicken cysteines in the two major subfamilies are either adjacent chemokines to a specific mammalian counterpart based (CC) or separated by one amino acid (CXC). The first two on sequence data alone. Because of limited sequence sim- cysteines in the CX3C chemokines are separated by three ilarity, most of the reported chicken chemokines were not amino acids, whereas the XC chemokines contain only named in accordance with the systematic nomenclature of two of the cysteines [4]. The CC chemokines can be fur- mammalian chemokines. The newly available chicken ther divided into two subcategories, MCPs (monocyte draft genome sequence and a large number ESTs allow chemoattractant proteins) and MIPs (macrophage inflam- systematic identification and annotation of chicken matory proteins) based on their structural similarities [5]. chemokine and cognate receptor genes. The objectives of The members of these two CC subcategories specifically this study were to systematically identify chemokine and attract mononuclear cells but not neutrophils. The CXC chemokine receptor genes in the chicken genome, to chemokines can also be divided into two subfamilies, one name these genes according to existing systematic nomen- with an ELR (a conserved Glu-Leu-Arg preceding the first clature, and to make ligand-receptor binding inferences cysteine) motif, which is angiogenic and attracts neu- based on comparative sequence analysis. The systematic trophils, and the other without the ELR motif, whose nomenclature for these chicken genes was based on the members do not attract neutrophils [6]. phylogenetic trees and syntenies of chicken, human, and mouse genes, and ligand-receptor binding inferences were Chemokine genes are characterized by their chromosomal according to [37] and [38]. locations and similar gene structure. Most human CC and CXC genes are organized in gene clusters in mammalian Results genomes, such as human Chromosomes 4 and 17, and Chicken chemokines and chemokine receptors mouse Chromosomes 5 and 11 [7]. The genes encoding In addition to the 11 previously reported, 12 new chicken the CC subfamily contain three exons, whereas the CXC chemokine were identified. These include 7 new CC chemokine genes contain four exons [8,9]. The XC sub- chemokines named CCL1L1 (BX935885), CCL3L1 family of chemokines contains two members in human (CF258095), CCL/MCP-L2 (CK610423), CCL/MCP-L3 but only one in mouse. CX3CL1 is the only known mem- (CK610627), CCL17 (BI067703), CCL19 (BX929857), ber of the CX3C subfamily in human, mouse, rat, and and CCL21 (CR522995), 4 new chicken CXC chemokines monkey. There are extensive conserved syntenies in the named CXCL13a (BX262175), CXCL13b (BX264625), chromosomal regions containing chemokine genes and CXCL13c (CR352598), CXCL15 (BX929947), and 1 between human and mouse. CX3CL1 chemokine (assembled from CR389767, BI066258, BM426140, and our sequence: AY730688). Unlike chemokines, chemokine receptors share a higher Eleven reported chicken genes were also named accord- degree of sequence identity within a species and between ingly as CCL1L2 (L34552), CCL5 (ah294, AY037859),

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cCCL1L1 MAKAAGAFCILLLLTALCCQSLAQRAP--AVPDKCCFN--FHTRRIKMDNIV-- 48 cCCL1L2 MKVFSLVMVTLLLAAVWTESSGKSFR-SSY-SSCCYKNMFIQKEINTSLIR-- 49 cCCL3L1 MKVSVAALAVLLIA-ICYQTSAAPVG-SDPPTSCCFT--YISRQLPFSFVA-- 47 cCCL4L1 MKSSTAAIAVLIVAALCYQVSSTPLA-VGSNGRCCYK--FLNRALPSSKVM-- 48 cCCL5 MMTAVAVSLSILLVAALFPQASSSPFG-ADT-TVCCFN--YSVRKLPQNHVK-- 48 cCCL/MCP-L3 MPTSRSTMKGSAAALAALLLLALCSSAVAQLLDSDGLPTTCCLS--YVQRPVPRNLIA-- 56 cCCL/MCP-L1 MKGSAAALAALLLLALCSSAVAHL---DGLPTTCCFS--YVQRPVPRNLIA-- 46 cCCL/MCP-L2 MKGSAAALAALLLLALCSSAVAQL---DGLPSTCCLS--YVQRPVPRSLIA-- 46 cCCL16 MK-LSAVVLALLIASFCSRASSAPVG-PDV-PTCCTT--YITHKIPRNLIQ-- 46 cCCL17 MLSTKLVLLLLLLLSIFQYSSAAPYA----PSECCYE--HTKFALRLEALK-- 45 cCCL19 MQRLHVLCLSLLVLRCVLHVYAGNN------VLDCCLR--TSEKPIPWRIVQDY 46 cCCL20 MP-GLSTKSLILASLLGLLLLLLCSTSQAQS------NQDCCLS--YSKVRLPRKVIKGF 51 cCCL21 MALRILLPLLLLAAALLLHQAEGVDNP----ASDCCLK--TSQKAISIKWVKSY 48 ** cCCL1L1 ACYATSPQCPHRAVVFKVKNGKEICTPADRMWVKRYQQRFQVS------SYSIPS 97 cCCL1L2 RYRETPPNCSRRAIIVELKKGKKFCVDPAEGWFQQYLQGKKL------SNTST 96 cCCL3L1 MYEYTGSRCPYHGVIFTTFEGKKCCANPEEKWVQDILNVEKH------TDGSK 95 cCCL4L1 DYYETNSQCPHAGVVFITRKGREVCANPENDWVQDYMNKMEL------N 90 cCCL5 DYFYTSSKCPQAAVVFITRKGRQVCANPDARWVKEYINFLEL------Q 91 cCCL/MCP-L3 SAYITSSKCRLPAVILVTKKGKEICVNPEESWVQKRLELLQN------QEN 101 cCCL/MCP-L1 SAYITSSKCRLPAVILVTKKGREICVNPEESWVQKRLELLQK------QEN 91 cCCL/MCP-L2 SAYITSSKCRLPAVILVTKKGKEICVNPEESWVQKRLELLQK------QEN 91 cCCL16 RHYSTSTSCSKPAIIFITKKEREVCANPSDPWVQRYLQSVKR------D 89 cCCL17 SFYETSHDCLLQAIVFVTKNGTKVCSKPNAPWVKKAVKYLQK------KNNPQAV 94 cCCL19 RMQLVQDGCDIPATVFITAKGKRLCAPPQAPWVLRLREKLDT----SSARKVPNQGN 99 cCCL20 TEQLSGEVCDIDAIIFHTVRGLKACVNPKEDWVKKHLLFLSQ------KLKRMSM 100 cCCL21 SIQGPESGCVLRAVVFTTKKNKKICSSPTDPIVQKLIKSLDSKRKSTPQRKSKRQKRKQV 108 * *

AlignmentFigure 1 of amino acid sequences of chicken chemokine CC subfamilies Alignment of amino acid sequences of chicken chemokine CC subfamily. Alignment gaps are indicated by dashes. Sequences with identical amino acid in at least 50% of chicken chemokines are highlighted in gray and conserved cysteine resi- dues in dark gray.

CCL4L1 (MIP-1β, AJ243034), CCL/MCP-L1 (ah221, cysteines. Chicken CXCL8a, CXCL8b, and newly identi- AY037860/ BX933162), CCL16 (k203, Y18692), CCL20 fied chemokine CXCL15 contain the ELR (Glu-Leu-Arg) (ah189, AY037861), CXCL8a (cCAF, M16199), CXCL8b motif. Only one chicken CX3C chemokine was found (K60, Y14971), CXCL12 (SDF-1, BX936268), CXCL14 (Figure 3). The number of amino acid residues between (JSC, AF285876), and XCL1 (lymphotactin, AF006742). conserved cysteines in all chemokines is highly conserved In summary, there are 13 CCL, 8 CXCL, 1 CX3CL, and 1 between chicken and human (Table 1). XCL genes identified in the chicken genome. The informa- tion used for the nomenclature is shown in the compara- Chicken chemokines have limited amino acid sequence tive genomic maps and phylogenetic trees. similarity compared to their human counterparts. Gener- ally, chicken CXC chemokines share 27 to 60% amino Chicken chemokine amino acid sequence alignment acid identity with their human homologs except for shows that all chicken CC chemokines have four con- CXCL12, which share 73% identity with human CXCL12. served cysteines with two adjacent cysteines at the N-ter- The length of chicken chemokine CXCL polypeptides minus (Figure 1), whereas all chicken CXC chemokines ranges from 95 to 107 amino acids. Compared to their have the conserved four cysteines with the first two human homologs, chicken CXCL chemokine amino acid cysteines separated by one amino acid (Figure 2). Both sequences are shorter except for chicken CXCL8a, chicken CCLs and CXCLs show higher degrees of CXCL8b, and CXCL12, which are 4, 5, 12 amino acids sequence similarity to each other in the signal peptide longer than their respective human homologs. In contrast, sequences and sequence regions containing the last two the sequence identities between human and chicken CCL

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cCXCL8b MMGKAVAAVMALLLIS-MAGAKGMAQARSAIELRCQCIETHSKFIHPKFIQN 51 cCXCL8a MNGKLGAVLALLLVS-AALSQGRTLVKMGNELRCQCISTHSKFIHPKSIQD 50 cCXCL12 MDLRALALLAFALAVISLSEEK----PVSLTYRCPCRFFESN-VARANIKH 46 cCXCL13a MRALQAALALGLLLSSLLPGDGLSLESLLTNKRCKCVKVTAQIISLGLILA 51 cCXCL13b MRVLARGGTGAAVLPWMVLLLLLVMMSMSQAAILEVNG-NLNCRCVKTTSDYISPKRYDS 59 cCXCL13c M-AVRAALLLGLLLVVLCPGDAAILEANG-NLNCRCAKTTTAFIPLRKYES 49 cCXCL15 MSPRL--LLPLLLAATAALCWG---APPAGELRCRCVRAVAERIPPRHLVQ 46 cCXCL14 MKLLTAALLLLVIAMCLASAEG------VKCKCSRKGPK-IRFSNVRK 41 * * cCXCL8b VNLTPSGPHCKNVEVIATL--K--DGREVCLDPTAPWVKLIIKAILDKADTNNKTAS 104 cCXCL8a VKLTPSGPHCKNVEIIATL--K--DGREVCLDPTAPWVQLIVKALMAKAQLNSDAPL 103 cCXCL12 LKILST-PNCS-LQIVARL--K-SNSKQVCIDPKLKWIQEYLEKALNKPRHRTHKKKQQK 101 cCXCL13a IDVMPPGIHCRRKEIILTL--K--RNKKVCVAPEAPWIQLLIHKLTQTDVSKKEAAAVAR 107 cCXCL13b IELRPVGSTCRRIEIIIKL--K--SSAKVCVNPDAPWVKKLLKRIAG 102 cCXCL13c VEVRPVGSSCRRLEVLIKL--K--TLERICVDPNTPWVRKLLQDLPKLKKKAAPQ 100 cCXCL15 VELVPEGPHCAAPEVIATT--K--QGHTLCLSPSVPWVKLLVARFLNSAAQRS 95 cCXCL14 LEIKPRYPFCVEEMIIVTLWTKVRGEQQHCLNPKRQNTVRLLKWYRVWKEKGRVYEE 98 ** AlignmentFigure 2 of amino acid sequences of chicken chemokine CXC subfamily Alignment of amino acid sequences of chicken chemokine CXC subfamily. Alignment gaps are indicated by dashes. Sequences with identical amino acid in at least 50% of chicken chemokines are highlighted in gray and conserved cysteine resi- dues in dark gray. The conserved ELR motifs are underlined.

chemokines are generally lower than those for CXCL ENSGALT00000019505.1), CCR6 (CV039916, chemokines, ranging from 25 to 56%. Chicken chemok- BU451770, and CK987456), CCR7 (predicted sequence: ine CCL polypeptides have 89 to 108 amino acids. chr27_random_59.1), CCR8a (AJ720982), CXCR2 Chicken CCL1L2, CCL5, and CCL17 have the same amino (BX258468), CXCR5 (AJ450829), CX3CR1 (CF252942, acid length as their human counterparts. Chicken CCL4L1 BU204148, and AJ443633). In contrast to chicken chem- is shorter than corresponding human CCL4, whereas okines, chicken chemokine receptors share significant chicken CCL1L1, CCL3L1, CCL19, and CCL20 are longer amino acid identity with their human receptor counter- than the corresponding human CCLs. These differences in parts. The percents of amino acid identity between length between human and chicken chemokines are chicken and human chemokine receptors range from 48 mostly in the N- and C- termini. to 81%. The lengths of these chicken receptors range from 335 to 382 amino acids. The complete sequence of Chicken CX3CL1 encodes 441 amino acids, longer than chicken CXCR2 is unknown due to a sequence gap in the all mammalian CX3CL1 examined. It shares 20, 22, 20, chicken genome sequence. The CXCR2 EST and a partial and 22% amino acid identity with human, mouse, rat, genome sequence contain the last 170 amino acids of the and monkey CX3CL1, respectively. Sixty-six amino acid C-terminus. residues in chicken CX3CL1 are identical to residues in mammals, but the sequence identity between mammals Fourty-four amino acid residues were highly conservated is, as expected, much higher than that between chickens (>85% homologies) among all chicken chemokine recep- and mammals (Figure 3). There are more identical amino tors (Figure 4). These receptors all have seven transmem- acids between chicken and mammals at both ends of the brane helices and three extracellular loops. Of the seven sequences. transmembrane helices, helix 1 and 7 show higher degrees of sequence similarity than the other helices. The similar- Unlike the chemokines, all the chicken chemokine recep- ity between the extracellular domains of the chicken tor genes were aligned with non-chicken chemokine receptors is lower, but all have a conserved cysteine resi- receptor reference genes in the chicken genome browser. due. In contrast, the intracellular domains (except at the There was at least one chicken EST sequence aligned to C-terminus) generally have higher degrees of sequence each receptor gene except for CCR4. In addition to 7 similarity than the extracellular domains. The second reported receptors, 7 new chicken chemokine receptors intracellular domains contain a highly conserved DRY- were identified and named as CCR4 (predicted sequences: LAIV sequence.

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Chicken MRVASLQIPFALRVLC-LAAMAGGQPRAPLKCSKWCISFHRAIDQRQIKSYRETEPQCTK 59 Human MAPISLSWLLRLATFCHLTVLLAGQHHGVTKCNITCSKMTSKIPVALLIHYQQNQASCGK 60 Mouse MAPSPLAWLLRLAAFFHLCTLLPGQHLGMTKCEIMCDKMTSRIPVALLIRYQLNQESCGK 60 Rat MAPSQLAWLLRLAAFFHLCTLLAGQHLGMTKCNITCHKMTSPIPVTLLIHYQLNQESCGK 60 Monkey MAPISLSWLLHLATLCHLTVLLAGQHHGVTKCNITCSKMTSKIPVALLIHYQQNQESCGK 60 * * * Chicken KAIIFTTKRNREICANPYEPWVEKIVKKLDQEKASAASPLPRADTSPAAAVPKEPGIFQK 119 Human RAIILETRQHRLFCADPKEQWVKDAMQHLDR------QAAALTRNGGTFEK 105 Mouse RAIVLETTQHRRFCADPKEKWVQDAMKHLDH------QAAALTKNGGKFEK 105 Rat RAIILETRQHRHFCADPKEKWVQDAMKHLDH------QTAALTRNGGKFEK 105 Monkey RAIVLETRQHRLFCADPKEQWVKDAMQHLDR------QAAALTRNGGTFEK 105 * Chicken HTGLQVPPSPPATAATAASERAPTPAASTEATSKPSPAMQNATHFSAGPSAVTSGVATHS 179 Human QIGEVKPRTTPAAGGMDESVVLEPE-ATGESSSLEPTPSSQEAQRALGTSPELPTGVTG- 163 Mouse RVDNVTPGITLATRGLSPSALTKPESATLEDLALELTTISQEARGTMGTSQEPPAAVTG- 164 Rat RVDNVTPRITSTTRGLSPTALAKPESATVEDLTLEPTAISQEARRPMGTSQEPPAAVTG- 164 Monkey QVGLVKPRTTLAARGMEESAVPEPE-ATGESSSLKPTPSSREAQTALGTSPEQSTGVTG- 163

Chicken EVVSEANRESLTSAHSTADAVDMALGQRTSYPTAPARDSDSKEEPAGYATSAAGDVRGTT 239 Human ---SSGTRLPPTPKAQDGGPVGTELFRVPPVSTAATWQSSAPHQPG---PSLWAEAKTSE 217 Mouse ---SSLSTSEAQDAGLTAKPQSIGSFEAADISTT-VWPSPAVYQSG---SSSWAEEKATE 217 Rat ---SFPSTSKAQDAGLAAKPQSTGISEVAAVSTT-IWPSSAVYQSG---SSLWAEEKATE 217 Monkey ---SSGTGLPLTPKAQDGGPVGTELFRGPPVSTAAAWQSSAPHQPG---PGLWAEGKTSE 217

Chicken STSTSDPASIS------KGLDHPSLPTNVPLDTISARGSTSGTALRSSALPSTPHITEVG 293 Human APSTQDP------STQASTASSPAPEENAPSEGQRVWGQGQSPRPENSLEREEMG 266 Mouse SPSTTAP------SPQVSTTSPSTPEENVGSEGQPPWVQGQDLSPEKSLGSEEIN 266 Rat SPPTIAL------STQVSTTS--SPKQNVGSEGQPPWVQEQDSTPEKSPGPEETN 264 Monkey APSTQDPSTQASSNPRASSTQASTTSSPAPEENTPSEGQPVWGQGQSPRPENSLEREEMG 277

Chicken MVPSTPQASPSPTQNPTTAIDEGPYVHANKNFSSSAFGTGTLDHLLPSGKQGPLDMLVFT 353 Human PVPAHTDAFQ------DWGPGSMAHVSVVPVSSEGTPSREPVASGSWTPKAEEPIH 316 Mouse --PVHTDNFQ------ERGPGNTVHPSVAPISSEETPSPELVASGSQAPKIEEPIH 314 Rat --PVHTDIFQ------DRGPGSTVHPSVAPTSSEKTPSPELVASGSQAPKVEEPIH 312 Monkey PVPAHTDAFQ------DWGPGSMAHVSVVPVSSEGTPSREPVVSGSWTPKAEEPIH 327

Chicken SQIFSDQARAQATGSPSHPPALSSLSGSQMYLVIPVALIGVLIACGVAARWAYVKFEIRP 413 Human ATMDPQRLGVLITPVP------DAQAATRRQAVGLLAFLGLLFCLGVAM-FTYQSLQGCP 369 Mouse ATADPQKLSVLITPVP------DTQAATRRQAVGLLAFLGLLFCLGVAM-FAYQSLQGCP 367 Rat ATADPQKLSVFITPVP------DSQAATRRQAVGLLAFLGLLFCLGVAM-FAYQSLQGCP 365 Monkey ATMDPQRLGVLITPVP------DSQAATRRQAVGLLAFLGLLFCLGVAM-FAYQSLQGCP 380

Chicken ETTSREMVEALLYLKEGHRDNVYPMEVI 441 Human RKMAGEMAEGLRYIPRSCGSNSYVLVPV 397 Mouse RKMAGEMVEGLRYVPRSCGSNSYVLVPV 395 Rat RKMAGEMVEGLRYVPRSCGSNSYVLVPV 393 Monkey RKMAGEMVEGLRYIPRSCGSNSYVLVPV 408

FigureAlignment 3 of amino acid sequences of chicken, human, mouse, rat and monkey chemokine CX3CL1 Alignment of amino acid sequences of chicken, human, mouse, rat and monkey chemokine CX3CL1. Alignment gaps are indicated by dashes. Sequences identical in all species are highlighted in gray. The asterisk represents the conserved cysteine residues.

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Table 1: Chicken and corresponding human chemokine cysteine motifs

Families Chemokines Chicken motif Human motif

CC CCL1L1 CCX25CX15CCCX22CX15C CCL1L2 CCX24CX15CCCX22CX15C CCL3L1, CCL4L1, CCL5 CCX22CX15CCCX22CX15C CCL16 CCX22CX15CCCX21CX15C CCL17 CCX22CX15CCCX22CX15C CCL19 CCX25CX15CCCX24CX15C CCL20 CCX24CX15CCCX24CX15C CCL21 CCX25CX15CCCX24CX17C CCL/MCP-L1, -L2, -L3 CCX22CX15CCCX22CX15C CXC CXCL8a, CXCL8b CXCX24CX15C CXCX24CX15C CXCL13a, CXCL13b, CXCL13c CXCX24CX15C CXCX24CX15C CXCL12 CXCX22CX15C CXCX22CX15C CXCL14 CXCX23CX19C CXCX23CX20C 1 CXCL15 CXCX24CX15CN/A XC XCL1 CX36CCX36C CX3CCX3CL1 CX3CX21CX15CCX3CX21CX15C

1 CXCL15 is not found in humans.

Chromosomal locations and syntenies 15, 18, 23, 24, and 26, CXCL1, 2, 3, 4, 5, 6, 7, 9, 10, and Comparisons of the chromosomal segments containing 11 in chemokine clusters that share syntenies with chemokines in the human, mouse, and chicken indicate chicken clusters on 4, and 19 were not that the organization of the chemokine genes was gener- found in the chicken genome, indicating gene duplica- ally conserved between chickens and mammals (Figure 5 tions in mammals. and 6). Chicken CC and CXC chemokines are located on Chromosomes 1, 4, 6, 9, 13, 19, and Z. Like human and Chemokine receptor genes were also highly conserved mouse, there are two large clusters in the chicken genome, between chicken, human, and mouse, and were similarly located on Chromosome 19 and containing 9 CCL genes. clustered. The largest cluster of chicken chemokine recep- Two CCL1-like (CCL1L1 and 2) and three chicken MCP- tors was found on Chromosome 2, where 5 receptor genes like (CCL/MCP-L1, L2, and L3) genes related to human (CCR2, CCR5, CCR8L1, CCR9, and XCR1) were identi- and mouse MCPs, such as CCL2, 7, 8, 11, and 13, are in fied. Another cluster on Chromosome 2 contains CCR4, one cluster (Figure 5A), and CCL5, CCL16, CCL3L1, and CCR8, and CX3CR1 genes. Chicken CXCR1 and CXCR2 CCL4L1 genes in another cluster (Figure 5B). Another are also clustered as in mammals, but the chromosomal CCL cluster is located on Chromosome Z containing two segment is unknown. The remaining CCR and CXCR genes, CCL19 and CCL21 (Figure 5C). Two CXCL gene genes are individually located on Chromosomes 3 clusters are located on and contained 6 (CCR6), 27 (CCR7), 7 (CXCR4), and 24 (CXCR5). Several genes, two CXCL8 (CXCL8a and b) and one CXCL15 human chemokine receptors, such as CCR1, CCR3, genes in one cluster (Figure 6A) and three CXCL13 CCR10, CXCR3, and CXCR6 were not found in the (CXCL13a, b, and c) genes in another (Figure 6B). chicken genome, though the syntenies associated with Chicken shares the syntenies with mouse and human in these receptors are present in the chicken genome. all these regions. There is one composite cluster contain- ing one CX3CL1 and one CCL17 genes (Figure 6E). Syn- Gene structure teny was conserved in chicken on one side of this cluster. According to the chicken genome sequence, chicken Chicken CCL20, CXCL12, CXCL14, and XCL1 are individ- chemokine genes share typical three-exon CC and four- ually located on Chromosomes 9 (Figure 5D), 6 (Figure exon CXC gene structures with mammals except for 6C), 13 (Figure 6D), and 1 (Figure 6F), respectively, and CXCL13a and CXCL13b, which have only three exons. the syntenies were highly conserved between chicken, Chicken chemokine genes are shorter than the corre- mouse, and human in these four locations. Mammalian sponding human genes due to shorter introns in chickens. CCL25, CCL28, and CXCL16 were not found in the The gene structure of chemokine receptors was also con- chicken genome, although the syntenies associated with served between chicken and mammals. The EST CCL25 and CCL28 were also conserved in chickens. A sequences indicate that chicken chemokine receptor genes number of human chemokines including CCL2, 7, 8, 11, could have up to 5 exons, though the complete sequences

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cCCR9 MTSLDYYRNDSAGLSVIGNPINDTELMCDRR 31 cCCR6 MSTTVFGTTEFFDTDYAS------LISTVCSKS 27 cCCR7 MQTGKRAARWDGCHVFCAGNNVTDDYDANTTI--DYNMFEMMCEKK 44 cCCR8b MEQRKKPTGRHTRALYLWFFPSQESKMNPTDLFLSTTEYDYGY--DENTAPCNEG 59 cCCR8a MDGDLRSLLAGGKQEDLLADFHFSPT--VNSSASYDNMY-YPELATDCEFE 48 cCCR2 MEN-YTDLGDMDVTTTFDYGD-----TAPCMGT 27 cCCR5 MEN-YTDLGDMP-TTTFDYGD-----TAPCMGT 26 cCCR4 MSSSSTESLEADTTTFYDFIDNYNDAPQPCSKE 33 cCX3CR1 MTEAPPEVTTEYVFYES-----ALACDES 24 cXCR1 MDEEQYPSGWDDNYSFEYVLNE-----SNVCEMG 29 cCXCR1 MGTFYADELLDILYNYTSDYCNYSLVLPDID-----VSSSPC-RN 39 cCXCR2 ------cCXCR5 MGPVSYSSETYDLSLVELSGYYEAENTTPSLEGYFCFNPSSSVVGN 46 cCXCR4 MDGSMDGLDLSSGILIEFADNGSEEIGSADYGDYGEPCFQH 41 hCCR1 METPNTTEDYDTTTEFDYGD-----ATPCQKV 27 * cCCR9 -QVWQFARAFLPMFFWLIFFVGTVGNALVVLIYCKYRFRRSMMDRYLLHLAVADLLLLFT 90 cCCR6 -EVRSFTKVFLPVAYSLICIVGLVGNIFVVMTFALYERTKSMTDVYLFNMAIADILFVLT 86 cCCR7 -EVRDFRAAFLPAMYSLICFTGLLGNGLVMLTYIYFKRLKTMTDIYLLNLALADILFLLT 103 cCCR8b NSFPRFKSLFLPILYCLVFVFCLLGNSLVLWILLTRKRLMTMTDICLLNLAASDLLFIVP 118 cCCR8a -SIPAFASSFFPVLYSILFVIGLMGNALVVWVLTAFKKIRAMTDVYLLNLAISDLVFVFS 107 cCCR2 -EEKHFAANFLPPLYSLVVIFGFIGNILVVLILVKYKKLKSMTDIYLLNLAISDLLFIFS 86 cCCR5 -EEKHFAANFLPPLYSLVVIFGFIGNILVVLILVKYKKLKSMTDIYLLNLAISDLLFVFS 85 cCCR4 N-FKRFAASFFPVLYTLVFLIGLIGNTLVIVVLFKYKRLKSMTDVYLLNLAISDLLFVLS 92 cCX3CR1 -DIQAFGKIFLPLFYIAVFALGLAGNVMVVLAIVKEGSKKSITDIYLMNLAVSDLLFVIS 83 cXCR1 N-YFIFYTHFTTVLYTLAFLLSLLGNTLVLWILFKYENLTSLTNIFIMNLCISDLVFSCM 88 cCXCR1 -EGSVANKYLVAFIYCLAFLLSMVGNGLVVLVVTSGHINRSVTDVYLLNLAVDDLLFALS 98 cCXCR2 ------cCXCR5 -QRDPFRKVFIPLAYLLMFVLGTVGNALVLVILERFKRSRTTTENFLFHLTLANLALLLT 105 cCXCR4 -ENADFNRIFLPTIYSIIFLTGIIGNGLVIIVMGYQKKQRSMTDKYRLHLSVADLLFVIT 100 hCCR1 -NERAFGAQLLPPLYSLVFVIGLVGNILVVLVLVQYKRLKNMTSIYLLNLAISDLLFLFT 86 ED1 Helix 1 ID1 Helix 2

cCCR9 LPFWATA-ASSGWIFRNFMCKVVNSMYKINFYGCILFLTCISFDRYLTIVQATKAKSSKQ 149 cCCR6 LPLWAVNYAADKWIFGNFICKMAKGIYAINFSCGMLLLAFISVDRYIAIVQATKSFKLRA 146 cCCR7 LPFWATS-AATFWCFGEFACKAVYCICKMSFFSGMLLLLSISIDRYFAIVQAASAHRFRP 162 cCCR8b LPFQAYY-ASDQWVFGNALCKIMGGIYYTGFYSSIFFITLMSIDRYIAIVHAVYA--MKI 175 cCCR8a LPFLAQYSLVSQWTFGNAMCKIVSSAYFIGFYSSAFFITIMSIDRYLAIVHSVYA--LKV 165 cCCR2 LPFWAYY-AAHDWIFGDALCRILSGVYLLGFYSGIFFIILLTVDRYLAIVHAVFA--LKA 143 cCCR5 LPFWAYY-AAHDWIFGDALCRILSGVYLLGFYSGIFFIILLTVDRYLAIVHAVFA--LKA 142 cCCR4 LPFWSYF-MIDQWVFGTPWCKIISWIYLVGFYSGIFFIMLMSIDRYLAIVRAVFS--MKA 149 cCX3CR1 LPFWASN-TVRGWTLGTIPCKVVSSLYYIGFFGGMFFITVISIDRYLAIVRATYS--MRS 140 cXCR1 LPFWAVD-QTFGWIFGEFLCKAVNAIFSIGYYSGVFFLTLMTILRYLSVVSPLST--LRS 145 cCXCR1 LPLWAVYWAHE-WVFGTVMCKAILVLQESNFYSGILLLACISVDRYLAIVYGTRA--ATE 155 cCXCR2 ------cCXCR5 FPFSVVE-SLAGWVFGTFLCKILSAVHKINFYCSSMLLGCIAVDRYLAIVYAIHT--YRK 162 cCXCR4 LPFWSVD-AAISWYFGNVLCKAVHVIYTVNLYSSVLILAFISLDRYLAIVHATNS--QRP 157 hCCR1 LPFWIDYKLKDDWVFGDAMCKILSGFYYTGLYSEIFFIILLTIDRYLAIVHAVFA--LRA 144 Helix 2 ED2 * Helix 3 ID2

cCCR9 RRILRSKVVCFAVWLASVSLCLPEIM-YSQSKQIGAVTV-CKMTYP----PNIGMAFRVAV 204 cCCR6 RTLAYSKLICLAVWASAILISSSSFLYSESYGFATNETQICDHRFDKTSDSIVLKSLL--- 204 cCCR7 RMIFISKVTCILIWLLAFVLSIPELV-HSGVNNYDSHPR-CSIIASDLQTFSTGIKVS--- 218 cCCR8b RTASCGTMISLVLWLVAGLASVPNIVF-NQQLEIEQSVQ-CVPVYPPG-----NNIWKVTT 229 cCCR8a RTTKHGIIASLALWAVAILASVPGLVF-FREVDEDNRTQ-CIPHYPGS-----GNSWKVFS 219 cCCR2 RTVTYGILTSIVTWAVALFASVPGIVF-HKTQQEHTRYT-CSAHYPQEQ----RDEWKQFL 198 cCCR5 RTVTYGILTSIVTWAVALFASVPGIVF-HKTQQENTQCT-CSFHYPSDA----LINWQHSY 197 cCCR4 RTAFHGLIASLTVWLVALLASVPELVF-RESFVEQNYTT-CKLRYPSN-----YLTWKLFY 203 cCX3CR1 RTIKHSLLITCGVWATAVLVSVPHFVF-SQMFEN----D-CIPVLPQEL----MNIWPVFC 191 cXCR1 QTQYCGSLVCLLVWTCSILIVVPEMIHTTVVETLEEVST-CDYDDWK------WKKVD 196 cCXCR1 KRH-WVKFVCVGIWVFSVLLSLPVLLFREAFVSDRNGTV-CYERIG----NENTTKWRVVL 210 cCXCR2 ------RNGTVCYEGIGNE---NTTK-WRVVL 22 cCXCR5 RRARSIHLTCTAIWLSSLLLTLPDLIFMEVWTDESNRSI-CYFPEAG---IHGNNVWLAT- 218 cCXCR4 RKLLAEKIVYVGVWLPAVLLTVPDIIF-ASTSEVEGRYL-CDRMYPHDN------WLISF 209 hCCR1 RTVTFGVITSIIIWALAILASMPGLYFSKTQWEFTHHT--CSLHFPHES----LREWKLFQ 199 Helix 4 * ED3

cCCR9 LVLKVTIGFFLPLLVMVIC-YTLIIHTLLQAKRCQKHKSLKIITMIITAFLLSQFPYNIVL 264 cCCR6 LCLQVGFGFFIPFVFMIFC-YAFIVKSLQQAQNSKRNKAINVIVLIVVVFLVCQVPYNTVL 264 cCCR7 ---QMVFGFLVPLVVMSVC-YLIIIKTLLQARNFEKNKAIKVIIAVVIVFVVFQLPYNGVM 275 cCCR8b QFAANILGLLIPFSILIHC-YAQILRNLRKCKNQN------LFWTPFNVVL 289 cCCR8a NSEVNILGWLFPVSILIFC-YHNILRNLQRCHTQNKYKAMKLVFIVVIVFFLFWTPINIML 279 cCCR2 ALKMNILGLVIPMIIMI-CSYTQIIKTLLQCRNEKKNKAVRLIFIIMIVYFFFWAPYNICI 258 cCCR5 ILKMNILGLIIPMIIMIFC-YSQILRTLFGCRNEKKQKAVRLIFVIMIFYFIFWTPFHVAS 257 cCCR4 TLEINILGLLLPLIVMAFC-YSMIIKTLLHCRNEKKNKAVRMIFAVMIVFFFFWTPYNIVI 263 cCX3CR1 NVELNTAGFFIPVCIICYC-YCGIIKTLLYCKNQKKARAIKLTLAVVIVFFLFWTPYNVLI 251 cXCR1 IYQRNIL-FLISFGIIIFC-YINILIILLRTRSRRKHRTVKLILVIVVAFFLSWAPYNILS 255 cCXCR1 RVLRRPSALPCPSWSCFTA-TEVTVHTLLQTKNVQKQRAMKVILAVVLVFLVCWLPYNITL 270 cCXCR2 RVLPQTLGFALPLLVMLFC-YGVTVHTLLQTKNVQKQRAMKVILAVVLVFLVCWLPYNITL 82 cCXCR5 RFLYHSVGFFMPLLVMCYC-YMAIVRTLCQSQRLQRQKAVRVAILVTGVFLLCWSPYHIVI 278 cCXCR4 RFQHILVGLVLPGLIILTC-YCIIISKLSHSKGHQKRKALKTTVILILTFFACWLPYYIGI 269 hCCR1 ALKLNLFGLVLPLLVMIIC-YTGIIKILLRRPNEKKSKAVRLIFVIMIIFFLFWTPYNLTI 259 Helix 5 * ID3 Helix 6

cCCR9 LIKTINMYTGAVYSCQTINGLDIGLQVTQSIAFLHSCLNPFLYVFAGERFRMALARMVQS 324 cCCR6 LMAVANMGK-TEKSCDSDNIMAYAKYTTETIAFLHCCLNPVLYAFIGVKFRSYFVKIMKD 323 cCCR7 LAKTISVFN-NTSSCDESKKLDMADDVTYTLACFRCCLNPFLYAFIGVKFRNDLFKLLKE 334 cCCR8b FLDSLQSLL-IIDNCQASSQITLALQLTETISFIHCCLNPVIYAFAGVTFKAHLKRLLQP 348 cCCR8a LLDSLRSLH-IIDDCQNSQRLDLALELAETLSLVHCCLNPIIYAFVGEKFKKYLCEAFGK 338 cCCR2 LLRDFQDSF-SITSCEISGQLQKATQVTETISMIHCCINPVIYAFAGEKFRKYLRSFFR- 316 cCCR5 FVHTFQTSF-FSPDCDSQSRLEKTIQVTETISMVHCCINPVIYAFVGEKFRKYLHMFFR- 315 cCCR4 LLQLLEATG-VIRNCQASRNLDYASQITESLGLFHCCLNPVIYFFMGEKFKKYLKMLFKN 322 cCX3CR1 FLETLRHYE-LFISCNQIKSLDYAMHLTETIAFSHCCLNPLIYAFAGEKFRKYLHRVCFK 310 cXCR1 FLITFPP-----PTCQYEKDTLLAFHISRKIAFSHCCLNPVLYVFAGVKFKSHLFRLCGQ 310 cCXCR1 VSDTLMRTRAITETCERRKHIDTALSITQVLGFSTVASTPSSTASSGRSFATASSRSWHS 330 cCXCR2 VSDTLMRTRAITETCERRKHIDTALSITQVLGFAHSCINPIIYAFIGQKFRNSFLKILAQ 142 cCXCR5 FLNTLTKLEAFAKDCLLEDHLDTAIMVTEAIGFTHCCLNPIIYAFIGVKFRNDFFRILHE 338 cCXCR4 SIDTFILLGVIRHRCSLDTIVHKWISITEALAFFHCCLNPILYAFLGAKFKTSAQNALTS 329 hCCR1 LISVFQDFLFT-HECEQSRHLDLAVQVTEVIAYTHCCVNPVIYAFVGERFRKYLRQLFHR 318 ED4 * Helix 7 * ID4

cCCR9 TGRYWLGGQDQCSSLGDSQEHSSNWSFAMLGRRRVRNSLTLSTNLASSVVPASCQVFV 382 cCCR6 LWCRRYKKYNKR----SSRINSDIYLSRQTSEILTDNASSFTI 362 cCCR7 LGCLSQQRLWQL----SSCRES----KRFSFAMETETTTTFSP 369 cCCR8b CARILWSPTRG-----SGVT--QSSLVLSQISGCSDS---AGVL 382 cCCR8a YAHFLLICKGH-----SAFNRRNTDRRTSMYTASSQSSFVGSVL 377 cCCR2 KQIASHFSKYC-----PVFYADTVERASSTYTQSTGEQEVSAAL 355 cCCR5 KHVATHLCKKC-----PSLYREKLERVSSTFTASTAEHDISTGL 354 cCCR4 WQLPGDICKWC-----GLHITYHTESTGSFHTQSTGDQEAL 358 cCX3CR1 YCPCLCFCGPCNHYDVRPSVSYAESMVNSNITLNTSDQDGTVFL 354 cXCR1 YLPCCDEVSRIG------SQAKFHYEDASIY 335 cCXCR1 VASSARMLWHATAAPPTLSPLATPPPPSEPHCSPRPSACSPGTPRTPAS 379 cCXCR2 RGFISKD------AVARYGRTSYTSTSGNTS--TTL 170 cCXCR5 LGCISQET------LQEILEVTRKGCGIESDNTTSISTF 371 cCXCR4 VSRGSSLKILSK------SKRGGHSSVSTESESSSFHSS 362 hCCR1 RVAVHLVKWLPFLSVD-----RLERVSST-SPSTGEHELSAGF 355

AlignmentFigure 4 of amino acid sequences of chicken chemokine receptors with human CCR1 Alignment of amino acid sequences of chicken chemokine receptors with human CCR1. Alignment gaps are indi- cated by dashes. Sequences with identical amino acid in at least 50% or 85% of the chicken chemokines are highlighted in gray and dark gray, respectively. Asterisks represent the conserved cysteine residues. ED and ID denote extracellular and intracel- lular domains, respectively. Seven transmembrane spanning domains of chicken chemokine receptors were predicted using the SMART program and these consensus domains are indicated with a box. The N-terminal sequence of chicken CXCR2 is cur- rently unknown.

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Mouse chr11:80040k Human chr17:27795k Chicken chr19:4586k Mouse chr11:83040k Human chr17:31115k Chicken chr19:7997k PSMD11 AATF PSMD11 CCT6B TAF15 TAF15 SPACA3 SUPT4H1 AK006357 PSPH CCL5 CCL5 ACCN1 ACCN1 RAD52B BZRAP1 NF2 CCL9 CCL2 CCL16 LPO CCL2 CCL/MCP-L1 CCL7 CCL6 CCL14 EPX CCL7 CCL/MCP-L2 CCL15 CCL3 CCL5 (AH294) CCL11 CCL11 CCL/MCP-L3 CCL23 CCL16 (K203) CCL12 CCL8 CCL4 CCL18 CCL1L1 CCL3 CCL13 TCF2 CCL4L1( MIP-1β) CCL8 CCL4 CCL1L2 CCL3L1 CCL1 CCL1 AATF TBC1D3 LOC124842 HIP1 CCT6B CCT6B AATF Mouse chr11:84117k Human chr17:32490k WBSCR20A LIG3 LIG3 LIG3 RAD51L3 RAD51L3 RAD51L3 Mouse chr5:132730k Human chr7:74684k HIP1 HIP1 CCL26 CCL24 TAF15 Mouse chr11:82505k Human chr17:30472k Chicken chr19:4198k CCL24 RHBDL7 A RHBDL7 POR POR POR Mouse chr4:41805k Human chr9:34603k Chicken chrZ_random:7421k Mouse chr11:133190k Human chr7:75269k Chicken chr19:4072k DCTN3 DCTN3 DCTN3 B OPRS1 OPRS1 ARID3A GALT GALT GALT Mouse chr1:83360k Human chr2:228160k Chicken chr9:4180k IL11RA IL11RA CNTFR HRB HRB HRB CCL27 CCL27 IL11RA SLC19A3 SLC19A3 CCL19 CCL19 CCL19 SLC19A3 CCL20 CCL20 (AH189) CCL21 CCL21 CCL21 CCL20 FLJ25955 KIAA1045 UBAP2 4933429 FLJ25955 FLJ20701 DNAJB5 DNAJB5 DNAJB5 D11Rik FLJ20701 VCP VCP VCP DNER DNER DNER

FANCG FANCG FANCG Mouse chr1:85225k Human chr2:230430k Chicken chr9:3650k Mouse chr4:42256k Human chr9:35071k Chicken chrZ_random:7974k D C Mouse chr8:4222k Human chr19:7890k Chicken chr28:881k Mouse chrUn_random:20703k Human chr5:42835k Chicken chrZ:6659k

APBA3 AJ296079 SEPP1 SNAPC2 SNAPC2 MATK TMLHE ZNF131 SEPP1 CTXN CTXN ELAVL1 ZFP131 MGC42105 TIMM44 TIMM44 MGC42105 ELAVL1 E130304F04Rik HMGCS1 HMGCS1 ELAVL1 LMNB2 CCL25 CCL25 CCL28 CCL28 FBN3 LASS4 TIMM44 3110070M22RiK FLJ21657 FLJ21657 SHC1 LASS4 FBN3 AK049201 PAIP1 PAIP1 8D6A SLC10A2 MVP C920008G01Rik NNT NDUFA7 NNT TRH3 HMGCS1 Mouse chr8:5100k Human chr19:8293k Chicken chr28:57k Mouse chrUn_random:23586k Human chr5:43750k Chicken chrZ:6856k E F

FigureGenomic 5 organization (syntenies) of human, mouse, and chicken CCLs, CX3CLs, and XCLs Genomic organization (syntenies) of human, mouse, and chicken CCLs, CX3CLs, and XCLs

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Mouse chr5:82830k Human chr4:66000k Chicken chr4:45310k Mouse chr5:91084k Human chr4:77195k Chicken chr4:50539k

CEK7 CEK7 ASAHL ASAHL SDAD1 SDAD1 SDAD1 ASAHL ANKRD17 ANKRD17 EREG CXCL9 CXCL9 AFP AFP AREG CXCL10 CXCL10 SCARB2 CXCL11 CXCL11 AFM RASSF6 HERC3 ART3 ART3 RASSF6 CXCL8 ANKRD17 NUP54 NUP54 CCNG2 CXCL6 RASSF6 SCARB2 SCARB2 CXCL5 CXCL1 CCNG2 CCNG2 CXCL13c CXCL7 CXCL8b (K60) CXCL13 CXCL13 CXCL13b CXCL13a CXCL4 CXCL4 CXCL8a (cCAF) CNOT6L CNOT6L CXCL15 CXCL7 CXCL15 MRPL1 MARPL1 CNOT6L CXCL1 FRAS1 FRAS1 CXCL5 CEK7 FRAS1 CXCL2 CXCL3 LOC395922 Mouse chr5:94100k Human chr4:79824k Chicken chr4:35220k EPGN CXCL2 FLJ10808 Mouse chr11:70043k Human chr17:4560k EREG EREG ARRB2 ARRB2 AREG AREG CXCL16 CXCL16 Synteny not found in chickens Mouse chr5:89964k Human chr4:75686k Chicken chr4:52185k TM4SF5 ZMYND15 TM4SF5 A Mouse ch11: 70125k Human chr17:4634k Mouse chr6:117014k Human chr10:90430k Chicken chr6:17350k B

LIPF Mouse chr13:54922k Human chr5:134390k Chicken chr13:14547k FRMPD2 ZNF22 ERCC6 MAPK8 PITX1 PITX1 RASSF4 ZNF22 ARHGAP22 H2AFY H2AFY PITX1 RASSF4 BC027057 DCNP1 H2AFY CXCL12 NEUROG1 CXCL12 DRG11 NEUROG1 NEUROG1 BC017939 CXCL14 (JSC) C630010N09 ERCC6 CXCL14 CXCL14 HNRPA3 AK126812 AK016198 CXCL12 (SDF-1) E230025K15 ZNF32 IL-9 IL-9 HNRPF LIPF IL-9 LOC283031 FBXL3B FBXL3B FXYD4 ANKRD22 LECT2 HNRPF TGFBI LECT2 FXYD4 TGFBI SMAD5 TGFBI Mouse chr6:118327k Human chr10:43186k Chicken chr6:18345k SMAD5 SMAD5

C Mouse chr13:55850k Human chr5:135550k Chicken chr13:13974k

Mouse chr8:93875k Human chr16:55680k Chicken chr11:504k D CPNE2 GPR56 Mouse chr1:165481k Human chr1:164838k Chicken chr1:78241k NIP30 GPR161 CPNE2 KIAA1972 DOK4 AK004618 RPL34P1 TTF2 ARL2BP ARL2BP POLR2C LOC375035 TM4SF11 TM4SF11 CPNE2 AY255596 GPR161 CCL22 CCL22 NIP30 2010005O13Rik TBX19 MGC3794 CX3CL1 CX3CL1 KIAA1972 TBX19 XCL2 TBX19 CCL17 CCL17 ARL2BP XCL1 XCL1 XCL1 POLR2C POLR2C CX3CL1 DPT DPT DPT DOK4 DOK4 CCL17 ATP1B1 ATP1B1 ATP1B1 GPR56 MGC10992 RAN10 NME7 NME7 NME7 GPR114 NUTF2 BLZF1 BLZF1 BLZF1 GPR56 Mouse chr8:94364k Human chr16:56258k Chicken chr11:897k Mouse chr1:164325k Human chr1:166079k Chicken chr1:77763k E F

FigureGenomic 6 organization (syntenies) of human, mouse, and chicken CXCLs Genomic organization (syntenies) of human, mouse, and chicken CXCLs

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were not available. However, the expressed sequences appear to be shorter between chicken and mammalian show that the amino acid sequences of identified chicken chemokine receptors than those between chicken and receptors are mostly encoded in a single exon as are most mammalian chemokines, which probably due to highly of the mammalian chemokine receptors. Chicken ESTs conserved transmembrane domains in these receptors. aligned with the chicken genome sequence indicate that Chicken CCR4, CCR6, CCR7, CCR9, CXCR2, CXCR4, these receptor mRNAs have approximately 2 kb of 5' UTR, CXCR5, CX3CR1, and XCR1 are closely related to a mam- as do those found in humans. malian ortholog based on the phylogenetic analysis. There are two distantly related (relative to the distance Phylogenetic analyses and nomenclatures between mouse and human CCR8) CCR8 genes in chick- The phylogenetic trees (Figure 7A,7B, and 8) show that ens. The one closer to human CCR8 is named as CCR8a chicken CCL5, 16, 17, 19, and 20 and all seven CXCLs are and the other as CCR8b. There are also two chicken CCRs closely related to single specific human and/or mouse closely related to human and mouse CCR2 and CCR5, but chemokines. The phylogenetic trees together with the syn- the phylogenetic analysis could not distinguish them as tenies associated with these genes (Figure 5 and 6) either CCR2 or CCR5. Because these two receptors are strongly indicate that these genes are the orthologs of located in a conserved chromosomal region on chicken, those found in mammals; therefore, they are named human, and mouse chromosomes, these two chicken accordingly. The phylogenetic results show that chickens CCRs were named as CCR2 and CCR5 based on the syn- have two CXCL8 and three CXCL13 genes (only one copy teny in which CCR2 is closer to XCR1 than CCR5. each in mammals), indicating gene duplications of these genes in aves. One chicken CXCL related to mouse In summary, 23 chemokine and 14 chemokine receptor CXCL15 but not to human CCLs is named as cCXCL15, genes were identified from the chicken genome in this which is also supported by the synteny of the chemokine study. Many chicken genes display high degrees of similar- cluster (Figure 6A). Chicken CCL21 is named according to ity with their human and mouse orthologs in terms of relatedness to the human and mouse and the highly con- gene structure, sequence homology, and synteny. Chicken served synteny (Figure 5C). According to the phylogenetic has significantly fewer CCLs, CXCLs, CCRs, and CXCRs tree in Figure 7B, two directly linked chicken CCLs are than mammals, but it has the same number of CX3C, XC, remotely related to human and mouse CCL1. The synteny and cognate receptors as mouse. The results of phyloge- associated with these genes also indicates that they may be netic analyses generally agree with the comparative chro- CCL1-like genes (Figure 5A); therefore, they are named as mosomal locations and syntenies of the genes. The CCL1L1 and CCL1L2. Three closely related chicken CCLs independent nomenclature of chicken chemokines and that are directly linked to the CCL1-like genes are related chemokine receptors suggests that the chicken may have to a group of clustered human and mouse MCP CCLs (2, ligand-receptor pairings similar to mammals. The organi- 7, 8, 11, and 13) in the phylogenetic tree. The synteny and zation of these genes suggests that there were a substantial phylogenetic tree do not provide information to a specific number of these genes present before divergence between mammalian ortholog, though these three chicken genes aves and mammals and more gene duplications of CC, are somewhat more similar to human CCL13 and mouse CXC, CCR, and CXCR subfamilies in mammals than in CCL2. The results indicate that these genes are chicken aves after the divergence. MCP-like (Figure 5A and 7B); therefore, they are named as CCL/MCP-like (CCL/MCP-L1, -L2, and -L3). A chicken Discussion CCL gene that is directly linked to CCL16 and CCL5 (Fig- We systematically searched for chicken chemokine and ure 5B) is distantly related to chicken CCL5 in the tree. chemokine receptor genes in the recently available draft This gene has been reported as MIP-1β-like chemokine chicken genome sequence. Without this information, it [13], which is CCL4 in humans and mouse. Therefore, it may have taken years to find chicken chemokines and is named as CCL4L1 in order to conform to the report. their receptors. The independent nomenclature of chicken Another CCL in this cluster that does not display related- chemokines and chemokine receptors and mammalian ness to other CCLs in the phylogenetic tree (Figure 7B) is chemokine-receptor binding information suggest that named as CCL3L1 because this chemokine displays high- most of the genes have been identified. One exception est sequence similarity to a human CCL3-like chemokine, was CCL25, the only known ligand of CCR9 in mammals, and it shares synteny with human CCL3 genes (Figure which was not found in this study though its receptor was 5B). Overall, CXCLs are more conservative among identified. Likewise, CXCL14, and CXCL15 were identi- chicken, human, and mouse than CCLs. fied in both chickens and mice, but their receptors are unknown; therefore, it is very likely that there are Chicken chemokine receptors can also be named accord- additional chicken chemokine and chemokine receptor ing to mammalian nomenclature based on phylogenetic genes in the chicken genome. analysis (Figure 7) and syntenies. The genetic distances

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58 hCCL3

50 mCCL3 hCCL4 40 70 mCCL4 20 hCCL18 cCCL4L1 cCCL5 49 96 h5CCL 20 99 m5CCL cCCL16 100 hCCL19 59 48 hCCL16 77 mCCL19 h4CCL1

13 99 hCCL15 44 cCCL19 12 h3CCL2 hCCL21 54 68 mCCL6

100 mCCL21 98 mCCL9 99 mCCL10 cCCL21 20 30 h6CCL2

98 hCCL25 99 h4CCL2 mCCL25 m4CCL2 65 cCCL/MCP-L2 26 cCCL20 99 cCCL/MCP-L1 24 cCCL/MCP-L3 99 hCCL20 mCCL2 75 mCCL20 30 22 hCCL8 99 hCCL22 10 hCCL11 94 2 mCCL11 mCCL22 hCCL13

26 cCCL17 42 mCCL8 mCCL7 80 hCCL17 5 h7CCL 92 mCCL17 24 31 hCCL2 99 hCCL28 50 mCCL12 cCCL3L1 mCCL28 80 hCCL1 99 hCCL27 mCCL1 20 cCCL1 99 mCCL27 32 cCCL1L1

0.2 0.2 A B

PhylogeneticchemokinesFigure 7 trees of the chemokine CC subfamily constructed using the amino acid sequences of chicken, human, and mouse Phylogenetic trees of the chemokine CC subfamily constructed using the amino acid sequences of chicken, human, and mouse chemokines. The numbers on the branches are bootstrap values (percentage that the simulation sup- ports the original interpretation). Human, mouse, and chicken are abbreviated as h, m, and c, respectively. The scale bar reflects the horizontal distance at which amino acid sequences differ by 20% between two sequences. A and B are the phyloge- netic trees of CCLs that are not located on Chromosome 19 or located on Chromosome 19, respectively.

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45 hCXCL1 99 hCXCL3 93 hCXCL2 mCXCL1

14 42 mCXCL2

98 hCXCL4 mCXCL4

10 34 mCXCL5

99 hCXCL5 98 hCXCL6 3 hCXCL7 49 mCXCL7 hCXCL8 45 74 cCXCL8b 94 cCXCL8a cCXCL15 44 44 mCXCL15

99 cCXCL13c cCXCL13b

69 cCXCL13a 46 48 hCXCL13 93 mCXCL13

99 hCXCL9 mCXCL9

50 100 hCXCL10 70 mCXCL10 67 hCXCL11 100 mCXCL11 cCXCL14

100 hCXCL14 100 mCXCL14 cCXCL12

100 hCXCL12 98 mCXCL12 hCXCL16 100 mCXCL16

0.2

mouseFigurePhylogenetic chemokines 8 trees of the chemokine CXC subfamily constructed using the amino acid sequences of chicken, human, and Phylogenetic tree of the chemokine CXC subfamily constructed using the amino acid sequences of chicken, human, and mouse chemokines. The numbers on the branches are bootstrap values (percentage that the simulation sup- ports the original interpretation). Human, mouse, and chicken are abbreviated as h, m, and c, respectively. The scale bar reflects the horizontal distance at which amino acid sequences differ by 20% between two sequences.

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Although most of the systematic nomenclature of the gene with partial sequence. Further study including chicken genes was unambiguous based on both phyloge- sequencing expressed sequences is needed to confirm netic trees and syntenies, the information that was used to these genes. name seven chicken CCLs as CCL1L1, CCL1L2, CCL3L1, CCL4L1, CCL/MCP-L1, CCL/MCP-L2, and CCL/MCP-L3 Conclusion and to distinguish two chicken chemokine receptors into Based on the organization, syntenies, and phylogenetic CCR2 and CCR5 is inadequate. CCR2 and CCR5 are trees of chicken, mouse, and human chemokine and closely related and tightly linked in the human, mouse, chemokine receptor genes, we conclude that there may be and chicken genomes. The phylogenetic analysis indicates a substantial number of chemokine and cognate receptor these genes were duplicated after the divergence between genes before divergence between aves and mammals. The mammals and aves. Chicken CCL/MCP-L1, -L2, and -L3 presence of a few chicken chemokine and chemokine were related to a group of clustered mouse and human receptor paralogs and orthologs of the mammalian genes MCP CCLs; therefore, specific cognate receptors must be indicated that most chicken chemokine and the receptor tested to distinguish them. In humans, the chemokines of genes shared common ancestors with the human and this MCP group and MIPs, such as CCL3, CCL4, and mouse genes. There were significantly more gene duplica- CCL5, can bind to more than one receptor, such as CCR1, tions of CC, CXC, CCR, and CXCR subfamilies in mam- 2, 3, and/or 5, but not both CCR2 and CCR5. CCR1 and mals than in aves after the divergence of mammals and CCR3 were not found in the chicken genome and proba- aves. The mammalian and chicken genome sequences and bly are not present in the species. Therefore, chicken the genes identified in this study can be used for further CCR2 and CCR5 may be two receptors that recognize investigation of the molecular evolution of these gene these two groups of CCL chemokines, such as CCR2 for families and as a model for the study of the divergence MCPs and CCR5 for MIPs. Interestingly, two CCL1 like between aves and mammals. Avian and mammalian spe- (CCL1L1 and CCL1L2) and two CCL1 receptor (CCR8a cies may share similar chemokine-receptor binding pat- and CCR8b) genes were found in the chicken genome. terns. The results of this study may be used as functional The ligand-receptor binding for these four genes can not inferences for these chicken genes before they are experi- be determined in this study. Nerveless, the names mentally tested. assigned based on comparative analysis in this study may prove useful in order to apply the functional and Methods physiological knowledge from other species to chickens. Gene identification Further lab testing must be carried out to confirm the lig- To identify syntenies, genes closely linked to human and and-receptor binding and to understand their biological mouse chemokines were identified and localized on the functions. chicken genome using the UCSC genome browser [25]. Expressed Sequence Tags (ESTs) and chicken mRNA Chicken chemokine ESTs are highly represented in the sequences in the corresponding chromosomal regions EST database. There are several ESTs aligned to each iden- were then identified and, if necessary, assembled with the tified chicken chemokine gene in the UCSC Genome CAP3 program [26,27]. These sequences were aligned browser. The sequences assembled from ESTs probably with the corresponding chicken genomic sequence and contained most, if not all, of the full-length chemokine any deletions or insertions corrected. Sequences were then mRNA sequences. Promoter sequences with a typical submitted to ORF Finder (Open Reading Frame Finder) TATAA were detected with promoter prediction software [28] and the open reading frames were used as queries in (data not shown). However, there were only a few ESTs BLASTP [29,30] searches against the non-redundant pro- that partially cover chicken chemokine receptor genes. tein database in Genbank [31]. Sequences that produced Some of these EST contain translation start sites. These significant alignments with chemokines were identified as EST sequences and reported complete coding sequences putative chicken chemokine sequences. To identify indicate that the amino acid sequences of chicken chem- chicken chemokine receptors, all sequences of putative okine receptors are mostly encoded in one exon. The pre- chicken chemokine receptors including ESTs, mRNAs, dicted amino acid sequences were of the expected length and predicted sequences were retrieved from the UCSC and aligned very well with the coding sequences of non- Genome Browser. The identified ESTs were used to deter- chicken reference genes in the UCSC genome browser. mine the translation start sites for the receptors. If the The conserved gene structure of this receptor family and translation start sites could not be determined from ESTs, high sequence similarity between chicken and mammals translation start sites were based on the most likely pre- suggest that the predicted coding sequences were very dicted sequences from non-chicken reference genes in the accurate, especially for those with ESTs containing transla- UCSC Genome Browser. tion start sites. CCR4 is the only predicted gene that does not have a matching EST and CXCR2 is the only identified

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100 hCCR10

98 mCCR10 hCXCR3 60 100 mCXCR3

100 hCXCR5 mCXCR5 100 cCXCR5 42 85 hCXCR1 96 hCXCR2 66 mCXCR2 mCXCR1 100 cCXCR1 96 cCXCR2 cXCR1 100 hXCR1 100 mXCR1

100 hCCR1

98 mCCR1 hCCR3 99 mCCR3

100 94 hCCR5 87 mCCR5 97 100 mCCR2 hCCR2 73 76 cCCR5 100 cCCR2

100 hCCR4 98 mCCR4 99 cCCR4

100 hCCR8 50 mCCR8 cCCR8a 93 97 cCCR8b

100 hCX3CR1 mCX3CR1 100 cCX3CR1

100 hCXCR4 mCXCR4 100 cCXCR4

100 hCCR6 100 mCCR6 cCCR6 91 100 hCCR7

60 mCCR7 100 cCCR7 hCXCR6 100 mCXCR6 93 100 hCCR9 mCCR9 99 cCCR9

0.1 okineFigurePhylogenetic receptors 9 trees of chemokine receptors constructed using the amino acid sequences of chicken, human, and mouse chem- Phylogenetic tree of chemokine receptors constructed using the amino acid sequences of chicken, human, and mouse chemokine receptors. The numbers on the branches are bootstrap values (percentage that the simulation supports the original interpretation). Human, mouse, and chicken chemokines are abbreviated as h, m, and c, respectively, followed by the receptor named. The scale bar reflects the horizontal distance at which amino acid sequences differ by 10% between two sequences.

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Sequence analyses Human and mouse chemokine receptors hCCR1 Complete amino acid sequences of currently known (NM_001295), hCCR2 (NM_000647), hCCR3 human and mouse chemokines were retrieved from Gen- (NM_001837), hCCR4 (NM_005508), hCCR5 bank. The amino acid sequences of all putative chicken (NM_000579), hCCR6 (NM_004367), hCCR7 chemokines were predicted based on the open reading (NM_001838), hCCR8 (NM_005201), hCCR9 frames of the expressed nucleotide sequences (ESTs or (NM_006641), hCCR10 (AY429103), hCXCR1 mRNAs). The amino acid sequences were grouped accord- (NM_000634), hCXCR2 (BC037961), hCXCR3 ing to CC, CXC, and CX3C motifs and aligned using the (NM_001504), hCXCR4 (AY728138), hCXCR5 ClustalW program [32,33]. The similarity of the amino (NM_032966), hCXCR6 (NM_006564), hCX3CR1 acid sequences was determined based on alignments with (NP_001328), and hXCR1 (NM_005283), mCXCR1 the most likely human or mouse orthologs. Human CCR1 (AY749637), mCXCR2 (NM_009909), mCXCR3 was included in the multiple alignments of chicken chem- (NM_009910), mCXCR4 (NM_009911), mCXCR5 okine receptors for comparison. The seven transmem- (NM_007551), mCXCR6 (NM_030712), mCCR1 brane domains were predicted using the SMART program (NM_009912), mCCR2 (NM_009915), mCCR3 [34]. (NM_009914), mCCR4 (NM_009916), mCCR5 (NM_009917), mCCR6 (NM_009835), mCCR7 For comparison, human chemokines hCCL1 (GenBank (NM_007719), mCCR8 (NM_007720), mCCR9 accession number: (NM_002981), hCCL2 (BC009716), (NM_0099130), mCCR10 (AF215982), mCX3CR1 hCCL3 (BC071834), hCCL4 (NM_002984), hCCL5 (NM_009987), and mXCR1 (NM_011798), and reported (BC008600), hCCL7 (NM_006273), hCCL8 chicken cCCR2 (CAF28776), cCCR5 (BI393893, (NM_005623), hCCL11 (BC017850), hCCL13 CAF28777), cCCR8L1(CAF28778), cCCR9 (CAF28781), (BC008621), hCCL14(BC045165), hCCL15 cCXCR1 (AAG33964), cCXCR4 (NP_989948), and (NM_032964), hCCL16 (NM_004590), hCCL17 cXCR1 (CAF28779), were also retrieved from GenBank (BC069107), hCCL18 (BC069700), hCCL19 for comparisons. (BC027968), hCCL20 (BC020698), hCCL21 (BC027918), hCCL22 (BC027952), hCCL23 Phylogenetic analyses of protein sequences of chicken, (NM_145898), hCCL24 (BC069072), hCCL25 human, and mouse chemokines and chemokine receptors (NM_005624), hCCL26 (BC069394), hCCL27 were based on the amino acid sequences using neighbor- (AJ243542), hCCL28 (AF220210), hCXCL1 (BC011976), joining with options selected for bootstrap test, pairwise hCXCL2 (BC015753), hCXCL3 (BC065743), hCXCL4 deletion and Poisson correction, using MEGA3 [35,36]. (NM_002619), hCXCL5 (BC008376), hCXCL6 For ligand-receptor inference, the first 20 amino acids (BC013744), hCXCL7 (BC028217), hCXCL8 (leading peptide) of all chemokines were removed before (BC013615), hCXCL9 (BC063122), hCXCL10 the phylogenetic analysis and chicken CCLs were divided (BC010954), hCXCL11 (BC012532), hCXCL12 into two groups, one group located on Chromosomes 4 (BC039893), hCXCL13 (BC012589), hCXCL14 and 19 and the other from other chromosomes. Synte- (BC003513), and hCXCL16 (BC017588), and nies, phylogenetic trees, and sequence homologies were hCX3CL1(NM_002996) and mouse chemokines CCL1 the combined information used for naming chicken (NM_011329), mCCL2 (NM_011333), mCCL3 chemokines and their cognate receptors according to the (NM_011337), mCCL4 (NM_013652), mCCL5 recommendations of the IUIS/WHO Subcommittee on (BC033508), mCCL6 (BC002073), mCCL7 (BC061126), Chemokine Nomenclature [37]. These chicken genes were mCCL8 (NM_021443), mCCL9 (NM_011338), mCCL10 named according to their closest predicted human or (U15209), mCCL11 (NM_011330), mCCL12 mouse orthologs if all information supports the nomen- (BC027520), mCCL17 (BC028505), mCCL19 clature. If there was more than one chicken gene similar to (BC051472), mCCL20 (BC028504 (NM_009138), a human and/or mouse gene, these gene was named as in mCCL27 (BC028511), mCCL28 (BC055864), and the human and/or mouse followed by a letter with alpha- mCX3CL1 (BC054838) were retrieved from the GenBank. bet order. If a specific human or mouse ortholog could Reported chicken chemokines K60 (Y14971), cCAF not be reliablely determined, the chicken genes were (M16199), MIP-1β (AJ243034), k203 (Y18692), AH294 named according to a closest human or mouse ortholog (AY037859), AH221 (AY037860), AH189 (AY037861), followed by an "L" and a number based on the informa- JSC (AF285876), SDF-1(BX936268), Clone 391 (L34552) tion available. This nomenclature also used the existing and lymphotactin (AF006742) are included in this study. systematic names reported in the literature to avoid Rat (BC070938) and monkey (AF449286) CX3CL1 were confusion. also retrieved for CX3CL sequence analysis. There are sev- eral human chemokine-like genes in the human genome, which were not included in this study.

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Table 2: Systematic names, chromosomal locations (kb), and putative identified cognate receptors of chicken chemokines1

Nomenclature Chromosomal location Ligand name Putative receptor Chromosomal location

CCL1L1 chr19:4,491–4,492 I-309/TCA CCR8a and/or CCR8b chr2:43,465–43,469 CCL1L2 chr19:4,493–4,495 chr2:41,804–41,811 CCL/MCP-L1 chr19:4,495–4,496 MCP-? CCL/MCP-L2 chr19:4,498–4,499 MCP-? CCR2 chr2:41,768–41,769 CCL/MCP-L3 chr19:4,507–4,508 MCP-? CCL16 chr19:258–261 HCC CCL3L1 chr19:240–242 MIP-1α CCL4L1 chr19:250–253 MIP-1β CCR5 chr2:41,784–41,786 CCL5 chr19:263–266 RANTES CCL17 chr11:768–771 TARC CCR4 chr2:43,501–43,503 CCL19 chrZ_random:7,804–7,809 MIP-3β CCR7 chr27_random:661–673 CCL21 chrZ_random:7,810–7,810 SLC CCL20 chr9:4,119–4,122 MIP-3α CCR6 chr3:38,589–38,596 Not found2 CCL25 CCR9 chr2:41,880–41,882 CXCL8b chr4:51,462–51,466 IL-8 CXCR2 chrUn:136,108–136,109 CXCL8a chr4:51,475–51,479 CXCR13 chrUn:25,460–25,462 CXCL12 chr6:18,184–18,195 SDF-1 CXCR4 chr7:31,441–31,443 CXCL13a chr4:35,453–35,455 BCA-? CXCL13b chr4:35,455–35,457 BCA-? CXCR5 chr24:5,242–5,247 CXCL13c chr4:35,457–35,459 BCA-? CXCL14 chr13:14,231–14,239 BRAK Unknown4 CXCL15 chr4:51,500–51,501 Lungkine Unknown4

CX3CL1 chr11:758–764 Fractalkine CX3CR1 chr2:43,480–43,490 XCL1 chr1:780,81–78,086 Lymphotactin XCR1 chr2:41,831–41,833

1 The systematic naming, ligand naming, and putative receptors are according to [37] and [38]. 2 CCL25 was not identified in this study. 3 The ligand and receptor binding has been experimentally tested [39] 4 The information is currently not available in humans or mice.

Polymerase chain reaction (PCR) and DNA sequencing cell-derived factor-1; MCPs, monocyte chemoattractant Chicken EST or mRNA sequences were identified for all proteins; MIPs, macrophage inflammatory proteins. chemokine genes. All sequences contained complete putative open reading frames except for CX3CL1. How- Authors' contributions ever, partial chicken CX3CL1 gene sequences (BM426140, JW collected most of the data and drafted the manuscript. BI066258, and CR389767) were identified, with a gap of DLA contributed to the interpretation of the data and final 123 nucleotides between the ESTs. Forward (TGTGA- approval of the manuscript. AY performed the DNA CATCGGGAGTCGCTAC) and reverse (AAAATC- sequencing and assisted with the preparation of the man- CCCAGCGTTTGCTACT) PCR primers were used to uscript. SHS and YJ designed computer programs to amplify across the gap using cDNA prepared from white search chicken chemokine sequences in chicken EST data- blood cells. PCR was performed as follows: An initial base. JJZ provided the conception and design of the study, denaturation step at 94°C for 2 min and 35 cycles of collected some of the data, conducted phylogenetic anal- denaturation, annealing, and extension at 94°C for 30 ysis, and revised the manuscript. sec, 59°C for 45 sec, and 72°C for 1 min., and a final extension step was carried out at 72°C for 10 min. Unin- Acknowledgements corporated nucleotides were removed from amplified The authors sincerely thank Mr. Suresh Xavier for his assistance in setting PCR products using BioMax spin-50 mini-columns (Mill- up the chicken EST database for this study. This research project was finan- ipore, Billerica, MA). BigDye terminator cycle sequencing cially supported by Life Science Task Force at Texas A&M University. This reaction kits and an ABI Prism 377XL DNA Sequencer research work should also be credited to all persons including the funding agencies who contributed to chicken genome and EST sequencing and the (Applied Biosystems) were used for DNA sequencing. genome browser. Without the chicken genetic information, this research would have taken years and substantial funds to accomplish. The authors List of abbreviations also greatly appreciate the inputs from the reviewers of this manuscript. Abbreviations: cCAF, chicken chemotactic and angiogenic factor; JSC, Jun-suppressed chemokine; SDF-1, stromal

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