GENOMICS 47, 163–170 (1998)ARTICLE NO. GE975058

Cloning and Characterization of a New Type of Mouse Chemokine

Devora L. Rossi,* Gary Hardiman,† Neal G. Copeland,‡ Debra J. Gilbert,‡Nancy Jenkins,‡ Albert Zlotnik,* and J. Fernando Bazan†,1

*Department of Immunobiology and †Department of Molecular Biology, DNAX Research Institute,Palo Alto, California 94304-1104; and ‡Mammalian Genetics Laboratory,

ABL-Basic Research Program, NCI-Frederick Cancer Researchand Development Center, Frederick, Maryland 21702

Received June 6, 1997; accepted October 7, 1997

defined by a signature cysteine motif: the a or CXCWe report here the identification and characteriza- chemokines, the b or CC chemokines, and the g or C

tion of the mouse homologue of a human CX3C chemo- chemokine lymphotactin (Kelner et al., 1994; Kennedykine described by F. Bazan et al. (1997, Nature 385, et al., 1995). These chemokine families chemoattract640–644). Termed fractalkine, this molecule consti- specific cell populations, and their cognate genes maptutes a fourth or d chemokine structural type that to unique chromosomal locations. The CXC chemokinedisplays a novel CX3C sequence fingerprint. Distinct group induces the migration of neutrophils and lym-from the a, b, or g chemokine families, the polypep- phocytes, while the CC group recruits monocytes, Ttide chain of CX3C predicts a 373-amino-acid type I cells, basophils, and eosinophils. Moreover, CXC che-transmembrane glycoprotein with the chemokine do- mokines cluster on mouse chromosome 5 (human chro-main resting on top of an extended mucin-like stalk. mosome 4), CC chemokines map to mouse chromosomeComparison of the mouse and human protein chains 11 (human chromosome 17), and the C chemokineshows a high degree of conservation in all the globu-

lymphotactin gene resides on mouse and human chro-lar segments with the exception of the stalk portion.mosome 1 (Kennedy et al., 1995). This distinctive fam-The striking identity of an amino acid stretch encom-ily clustering of chemokine genes has a few recent ex-passing a putative juxtamembrane cleavage site sug-ceptions: human MIP-3b has been mapped to chromo-gests the evolutionary conservation of both mem-some 9 (Rossi et al., 1997) and human LARC (alsobrane-bound and processed CX3C forms. Northernnamed human MIP-3a) to chromosome 2 (Hieshima etanalysis reveals the presence of mouse CX3C mRNA inal., 1997).heart, brain, lung, kidney, skeletal muscle, and testis

We have recently published (Bazan et al., 1997) thetissues. The mouse CX3C gene was further localizedidentification, structure, and in vitro analysis of humanto the central region of chromosome 8 by interspecific

backcross mapping; a related locus was detected on CX3C (or fractalkine). Interestingly, the human CX3Cchromosome 11. The novel location of this gene from gene was found to reside on human chromosome 16. Inother chemokine gene clusters adds to the notion this study we describe the isolation and sequence ofthat CX3C is a fundamentally new class of chemokine. mouse CX3C cDNA, characterize the mRNA distribu-q 1998 Academic Press tion, and define its (syntenic) chromosomal location.

In view of the fundamental role of chemokines ininflammatory diseases and the necessity to under-

INTRODUCTION stand their biological function by the use of mouse invivo models, it was critical to isolate and characterize

Chemokines are small proinflammatory peptides, 6– mouse CX3C.14 kDa in size, that direct leukocyte trafficking in im-mune surveillance. Nevertheless, other biological func-

MATERIALS AND METHODStions have been attributed to chemokines includingcostimulation of cell proliferation, lymphokine produc-

Cloning of mouse CX3C. Human brain expressed sequence tagstion (Taub et al., 1996), degranulation, cellular activa- (ESTs) that encode mouse CX3C were identified in the dbEST data-tion, adhesion, enzyme release, oxidative burst, and base at the National Center for Biotechnology Information by per-

forming BLAST searches (Altschul et al., 1990) using the chickentumor cell cytolysis (Taub et al., 1994, 1995; Lloyd etlymphotactin cDNA (Accession No. AF006742) as the query se-al., 1996).quence. Three of these ESTs were obtained from the I.M.A.G.E. Con-Chemotactic cytokines have been traditionally grouped sortium (Lennon et al., 1996) via Research Genetics (Huntsville, AL)

into three structural and functional families that are and sequenced. One of these cDNAs (Accession No. R75309) encodedthe chemokine domain, whereas the others corresponded to 3 * un-translated sequence (Accession Nos. W74988 and W16003). To clone1 To whom correspondence should be addressed. Telephone: (415)

496-1115. Fax: (415) 496-1200. E-mail: bazan@dnax.org. the full-length mouse CX3C cDNA, oligonucleotides were synthesized

1630888-7543/98 $25.00

Copyright q 1998 by Academic PressAll rights of reproduction in any form reserved.

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as follows: 5*-TNACTACTAGGAGCTGCGAC-3 * and 5*-AATTCC- shown). Unfortunately, many attempts in screeningTGCACTCCAGCCAT-3 *, respectively, which correspond to sequence this library failed to produce a positive clone.derived from R75309, and 5*-CACAGACATTGGTAATGATGC-3 *, New BLASTn searches in the dbEST database withwhich corresponds to sequence derived from W74988 and W16003.

the 3 * untranslated region of a human CX3C ESTRT-PCR analysis was performed on mouse brain, kidney, and lungRNA using various oligonucleotide combinations. (H14940) (Bazan et al., 1997) retrieved two new mouse

Reaction conditions for reverse transcription were: 1 mM concen- ESTs (Accession Nos. W74988 and W16003) that likelytrations of each dNTP, 0.5 mM spermidine, 100 pmol random hexam- formed part of the 3 * untranslated region of the mouseers, 10 ml RT buffer (Boehringer Mannheim), 120 units RNase inhibi-

CX3C cDNA. The respective I.M.A.G.E. clones were ac-tor (Boehringer Mannheim), 100 u AMV RTase (Boehringer Mann-quired from Research Genetics (Huntsville, AL) andheim) in a final volume of 100 ml at 427C for 1 h. Standard Perkin–

Elmer PCR conditions were used: 10 mm Tris–HCl, pH 8.3, 50 mM sequenced. Using the previous 5* primers and a newKCl, 1.5 mM MgCl2, 0.001% gelatin, 0.2 mM dNTPs, 0.5 U AmpliTaq 3 * primer designed based on the new 3 * sequence, weDNA polymerase (Perkin–Elmer), 60 pmol each primer, 500 ng total performed several RT-PCRs with RNA from mousecDNA, for 30 cycles (947C for 1 min, 557C for 2 min, 727C for 2.5

brain, kidney, and lung. Although PCR products of var-min), followed by 15 min at 727C. Although PCR products of variousious sizes were recovered, a 3-kb product was presentsizes were recovered, a sole 3-kb product was amplified from brain

RNA. This 3-kb PCR product was gel purified and cloned into PCR only in the brain samples. The prominent brain expres-II (Invitrogen). Automated sequencing (both strands) was performed sion of the CX3C mRNA was expected since many ofon an ABI DNA sequencer to determine the complete 3065-nucleotide the human and mouse ESTs obtained from sequencesequence of the CX3C cDNA.

searches were brain-derived. The gel-purified PCRNorthern blot analysis. The mouse multiple tissue Northern blotproduct was sequenced and subcloned into the pCRIIwas purchased from Clontech Laboratories (Palo Alto, CA). A 1-kb

mouse CX3C cDNA probe was radiolabeled using the Redivue label- vector (TA Cloning kit, Invitrogen). The 3065-nt mouseing kit (Amersham). Prehybridization and hybridization were carried CX3C cDNA contains an open reading frame of 1188out at 657C in 0.5 M Na2HPO4, 7% SDS, 0.5 M EDTA, pH 8.0. Strin- bp that encodes a 395-amino-acid (aa) protein (Fig. 1).gency washes were performed at 657C twice in 21 SSC, 0.1% SDSfor 40 min and once in 0.11 SSC, 0.1% SDS (high stringency) for 20min. The membrane was exposed to X-ray film (Kodak) with intensi- Human and Mouse Protein Sequence Comparisonfying screens at 0707C until the optimum exposures were achieved.

Interspecific mouse backcross mapping. Interspecific backcross The mouse protein sequence shows a high degree ofprogeny were generated by mating (C57BL/6J 1 Mus spretus) F1 similarity to the human CX3C protein (Fig. 2). Fivefemales and C57BL/6J males as described (Copeland and Jenkins, discrete protein segments first identified in the human1991). A total of 205 N2 mice were used to map the CX3C loci. DNA

protein sequence have close counterparts in the mouseisolation, restriction enzyme digestion, agarose gel electrophoresis,CX3C chain.and Southern blot analysis were performed essentially as described

(Jenkins et al., 1982). Fragments of 12.5 and 5.8 kb were detected The predicted signal peptide comprises 24 aa (024in BglII-digested C57BL/6J (B) DNA, and fragments of 7.4 and 6.6 to 01 aa); the next 76 amino acids (1–76 aa) form akb were detected in BglII-digested M. spretus (S) DNA. The 6.6-kb globular chemokine module as determined by directedBglII-digested M. spretus-specific fragment mapped to chromosome

sequence comparisons with C, CC, and CXC chemokine8 and defined CX3C; the 7.4-kb BglII fragment mapped to chromo-sequences (Clore and Gronenborn, 1995; Wells et al.,some 11 and defined CX3C-rsl. A description of the probes and RFLPs

for the loci linked to CX3C including LyI l, Gnao, and Cbfb has been 1996). The polypeptide chains of other chemokines typ-reported previously (Kuo et al., 1991; Wilkie et al., 1992; Bae et al., ically end shortly after the final a-helix of the chemo-1994). The probes linked to CX3C-rsl include Adralb, Il3, and Myhsfl kine fold. In this case, the CX3C chemokine domain is(McKenzie et al., 1993). Recombination distances were calculated

followed by 239 residues, rich in Gly, Pro, Ser, and Thr,as described (Green, 1981) using the computer program SPRETUSMADNESS. Gene order was determined by minimizing the number that Bazan et al. (1997) defined as an extended stalk-of recombination events required to explain the allele distribution like structure (77–315 aa). Shortly after the stalk, thepatterns. mouse CX3C displays a hydrophobic stretch of 19 resi-

dues that forms a transmembrane segment (316–334aa). The 37 C-terminal amino acids (335–371) form aRESULTScytoplasmic domain.

The mouse and human stalk segments bear struc-Computational Identification and cDNA Isolationtural and compositional resemblance to mucin and syn-decan-type transmembrane proteoglycans (Bernfield etSeveral human ESTs and one mouse EST with che-

mokine-like DNA sequence were identified in the Gen- al., 1992). Glycanation motifs for the attachment ofheparan sulfate or chondroitin sulfate to proteoglycanBank database through a BLASTn search using

chicken lymphotactin cDNA sequences (Rossi et al., Ser aa are notably not present. Although, as shown inFig. 2 the region of lowest identity between the human1996). To obtain a cDNA fragment to be used as a probe

in cDNA library screenings, specific primers were de- and the mouse proteins rests in this stalk region, themouse sequence retains a number of Ser and Thr resi-signed based on the mouse EST sequence (Accession

No. R75309). A nested PCR was set using mouse heart dues that are strongly predicted to be O-glycosylated(Gendler and Spicer, 1995). A dibasic motif, Thr-Arg-cDNA as a template (Clontech). As a result we obtained

a 133-bp cDNA fragment that was used as a probe in Arg-Gln (312–315 aa), located on the juxtamembranestalk forms a potential proteolytic cleavage site that isSouthern blot analysis of several mouse cDNA librar-

ies. A clear positive signal was detected in a mouse highly conserved in both human and mouse proteins(Fig. 2).cDNA library made from a stroma cell line (data not

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FIG. 1. Nucleotide and deduced amino acid sequence of mouse CX3C (Fractalkine). The 3065-nt cDNA contains an 1188-bp open readingframe that encodes a 395-amino-acid protein. The suggested signal peptide is marked by a double underline and numbered 024 to 01; the N-terminal chemokine module (residues 1–76 of the mature protein) is denoted in boldface text, and the boundaries are indicated by arrows. Thesignature cysteine residues are numbered 1 to 4, and the unique 3-aa insertion is in italics. The transmembrane section (residues 317–334) isdoubly underlined; the intracellular domain extends from aa 335 to 371. The dibasic cleavage motif Thr-Arg-Arg-Gln is in boldface text andunderlined. An ATTTA motif implicated in rapid mRNA turnover is detected in the 3* untranslated region at nt 1552–1556 (underlined).

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FIG. 2. Mouse–human CX3C protein sequence comparison. A high degree of sequence identity is detected at the signal peptide, chemokinedomain, transmembrane region, and intracytoplasmic tail. However, the identity index decreases at the stalk segment. SP, signal peptide;Ck, chemokine module; TM, transmembrane region; ICD, intracytoplasmic domain. A diagram of the membrane-bound CX3C structure isdepicted above the sequence comparison plot.

D1.1 (data not shown). However, CX3C was not ob-CX3C Transcripts Were Detected in Mouse Heart,served in any of the other cDNA libraries examined,Brain, Lung, Kidney, Skeletal Muscle, and Testiswhich included Th1 and Th2 polarized cells andwith the Highest Level of Expression in Brainclones; resting or activated pre-T cells; B cells; den-

To examine the expression pattern of the mouse dritic cells; and monocytic and macrophage cell lines.CX3C gene, an mRNA blot containing approximately 2mg of poly(A)/ per lane from mouse tissues (Clontech)was hybridized to the mouse CX3C cDNA probe as de-scribed under Materials and Methods (Fig. 3). This re-vealed that CX3C was expressed in adult mouse heart,brain, lung, kidney, skeletal muscle, and testis. Al-though several transcripts were observed in these tis-sues, a 4-kb mRNA species was the predominant form.Levels of this mRNA were highest in brain, with ele-vated levels also seen in lung and kidney. The addi-tional transcripts detected were approximately 7 and1.8 kb in size. Bazan et al. (1997) have found a similar FIG. 3. Tissue distribution of CX3C transcripts in adult mousepattern of expression in human tissues. tissues. The multiple tissue blot was hybridized to mouse CX3C as

outlined in the text. The first three lanes of the blot (heart, brain,As human CX3C mRNA was not observed in leuko-spleen) were exposed to X-ray film for 16 h. The other lanes receivedcytes, we tested a panel of mouse cDNA libraries froma longer exposure (4 days) to enable the visualization of the signalsdifferent leukocyte populations by Southern blot in testis and skeletal muscle. CX3C mRNA species 1.8, 4, and 7 kb

analysis. We detected low levels of CX3C expression in size were detected. Molecular weight RNA sizes in kilobases areindicated by arrows.in CH12, a B-cell line, and in a resting Th1 cell clone,

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FIG. 4. (a) Mouse chromosomal locations of CX3C and CX3C-rsl. The segregation patterns of CX3C and CX3C-rsl and flanking genes in175 and 124 backcross animals that were typed for all loci are shown at the top of the figure. For individual pairs of loci, more animalswere typed (see text). Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/6J1 M. spretus) F1 parent. The black boxes represent the presence of a C57BL/6J allele, and the white boxes represent the presence of a M.spretus allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. Partial chromosome 8and 11 linkage maps showing the location of CX3C and CX3C-rsl in relation to linked genes are shown at the bottom of the figure.Recombination distances between loci in centimorgans are shown to the left of the chromosome, and the positions of the loci, where known,are shown to the right. References for the human map positions of loci cited in this study can be obtained from GDB (Genome Data Base),a computerized database of human linkage information maintained by The William H. Welch Medical Library of the John Hopkins University(Baltimore, MD). (b) Evolution of the chemokine superfamily. An unrooted phylogenetic tree was constructed using the neighbor-joiningalgorithm from an alignment derived by Clustal W (Thompson et al., 1994) and displayed using TreeView. Each of the four chemokine genefamilies map to distinct human and mouse chromosomes. A representative collection of chemokine sequences were used to construct thetree (Accession Nos. U15607, U23772, M25897, X78686, Z11686, U92565, U84487, S71513, U26426, M21121, X12510, and 127080).

This reflects the absence of mouse CX3C mRNA in eral enzymes and analyzed by Southern blot hybridiza-tion for informative restriction fragment polymor-spleen.phisms (RFLPs) using a human CX3C cDNA probe. As

The Mouse CX3C Gene Is Located in the Distal a result, two fragments were detected in BglII-digestedRegion of Chromosome 8 M. spretus DNA, 7.4 kb in size (defined as CX3C-rsl)

and 6.6 kb in size, respectively (defined as CX3C). TheFor the purpose of mapping the mouse CX3C gene,C57BL/6J and M. spretus DNA were digested with sev- mapping results indicated that CX3C is located in the

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distal region of mouse chromosome 8 linked to LyI l, chemokines, adhere to charged ECM, raising their localsurface concentration and facilitating their binding toGnao, and Cbfb. Although 175 mice were analyzed for

every marker and are shown in the segregation analy- cellular receptors (Schlessinger et al., 1995; Tanaka etal., 1993). CX3C may constitute a parsimonious solu-sis (Fig. 4a), up to 188 mice were typed for some pairs

of markers. Each locus was analyzed in pairwise combi- tion to this practice.Interestingly, a particular feature is conserved innations for recombination frequencies using the addi-

tional data. The ratios of the total number of mice ex- both human and mouse CX3C proteins: the dibasic mo-tif Thr-Arg-Arg-Gln, which may constitute the targethibiting recombinant chromosomes to the total number

of mice analyzed for each pair of loci and the most likely of proteases. This motif resembles a dibasic cleavagesite found in a similar location within syndecans (Bern-gene order are centromere–LyI l–14/188–CX3C–0/

186–Gnao–7/179–Cbfb. The recombination frequen- field et al., 1992). This suggests the existence of twomouse CX3C forms: a soluble or secreted protein and acies expressed as genetic distances in centimorgans {

the standard error are –LyI l–7.5 { 1.9 [CX3C, Gnao]– membrane-bound protein. The coexistence of two pro-tein forms, secreted and membrane-bound, has never3.9 { 1.5–Cbfb. No recombinants were detected be-

tween CX3C and Gnao in 186 animals typed in common, been described for chemokines before. Releasing pro-tein from the cell membrane (without requiring a wait-suggesting that the two genes are within 1.6 cM of each

other (upper 95% confidence limit). ing period for the protein to be synthesized) is a novelmechanism of raising the chemokine local concentra-The CX3C-rsl locus mapped to the central region of

mouse chromosome 11 linked to Adralb, Il3, and tion in a rapidly regulated manner.In terms of mRNA expression, we detected threeMyhsfl. In this case 124 mice were analyzed for every

marker and are shown in the segregation analysis (Fig. main transcripts for mouse CX3C (7, 4, and 1.8 kb).These results are consistent with the multiple human4a), and up to 129 mice were typed for some pairs of

markers. Again, each locus was analyzed in pairwise CX3C transcripts observed in human tissue mRNAblots (Bazan et al., 1997). These different mRNAs maycombinations for recombination frequencies using the

additional data. The ratios of the total number of mice derive from alternative splicing events, the use of dif-ferent polyadenylation sites, or transcription initiatingexhibiting recombinant chromosomes to the total num-

ber of mice analyzed for each pair of loci and the most at different starting points. However, several unsuc-cessful attempts were made to obtain alternative splic-likely gene order are centromere–Adralb–8/123–Il3–

0/127–CX3C-rsl–8/129–Myhsfl. The recombination ing variants of human CX3C from numerous humancDNA libraries (W. Wang, pers. comm., Palo Alto,frequencies are Adralb–6.5 { 2.2–[Il3, CX3C-rsl]–6.2

{ 1.1–Myhsfl. No recombinants were detected between 1997). Another possible explanation is that the addi-tional mRNAs may originate from transcription of an-Il3 and CX3C-rsl in 127 animals typed in common, sug-

gesting that the two loci are within 2.4 cM of each other other CX3C family member. This hypothesis is in accor-dance with the results obtained from the chromosomal(upper 95% confidence limit).mapping experiments. A positive signal was detected

DISCUSSION in mouse chromosome 11, which does not correspondto the authentic mouse gene localization (see below).In this study we report the mouse CX3C protein se-

quence and compare it to its human ortholog. Along The presence of ESTs from 13.5 to 14.5 pc mouseembryo in the dbEST database demonstrates thatwith the similar hydrophobic stretches that compose

the signal peptide and transmembrane regions, the CX3C is expressed during early development in themouse. Although it is reasonable to hypothesize thatchemokine and cytoplasmic domains of mouse and hu-

man CX3C proteins are highly conserved (Fig. 2). While chemokines may be key factors in cell migration duringorgan development, their specific role in this processthe sequence identity decays significantly in the stalk

regions, the chain length, amino acid composition, and remains unclear.Although mouse CX3C transcripts are widely distrib-O-glycosylated character are closely maintained; in ad-

dition, a putative processing site is conserved. Interest- uted among several tissues, the most abundantly ex-pressed was the 4-kb brain RNA. The high level ofingly, the presence of a chemokine module fused to a

highly glycosylated scaffold may unify two functions, CX3C expression in this tissue suggests a role for CX3Cas a mediator of inflammation in the CNS. Expressioncell recruitment and adhesion, in one molecule. These

two roles are usually played by two different kind of of other chemokines (MCP-1, IP-10, and MIP-1a) hasbeen reported in astrocytes (Berman et al., 1996; Gla-molecules: the chemokines and the cell adhesion mole-

cules. During an inflammatory process, the expression binski et al., 1996) and microglial cells (Meda et al., 1996).Furthermore, Berman et al. (1996) have detected mono-of both types of molecules is upregulated (Vaddi and

Newton, 1994; Wang and Feuerstein, 1995) to ensure cyte chemoattractant protein-1 (MCP-1) expression atthe onset of inflammation, in the CNS of rats with ex-rapid access of leukocytes to the inflammation site.

In addition, the general resemblance of the CX3C perimental autoimmune encephalomyelitis (EAE), andobserved a direct correlation with disease activity.stalk to structural proteins of the extracellular matrix

(ECM) may indicate an ability to interact with other These findings suggest that brain-derived chemokinesmay support the infiltration of circulating inflamma-ECM components such as integrins or selectins (Shi-

mizu and Shaw, 1993). Many cytokines, including tory cells during CNS inflammatory diseases, such as

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tin by Pan, Y., Lloyd, C., Zhou, H., Dolich, S., Deeds, J., Gonzalo,multiple sclerosis or Alzheimer’s disease, in which in-J. A., Vath, J., Gosselin, M., Ma, J., Dussault, B., Woolf, E., Alperin,flammatory processes are implicated in the pathogene-G., Culpepper, J., Gutierrez-Ramos, J. C., and Gearing, D. (1997).sis of the disease (Breitner, 1996; Patterson, 1995). Neurotactin, a membrane-anchored chemokine upregulated in brain

The chromosomal mapping results showed two possi- inflammation. Nature 387: 611–617.ble locations for the mouse CX3C gene: chromosome 8or chromosome 11. The central region of mouse chromo-

REFERENCESsome 8 shares regions of homology with human chro-mosomes 19p and 16q (summarized in Fig. 4a). In par- Altschul, S., Gish, W., Miller, W., Myers, E., and Lipman, D. (1990).ticular, Gnao has been mapped to 16q13. The tight Basic local alignment search tool. J. Mol. Biol. 215: 403–410.linkage between CX3C and Gnao in mouse suggests Bae, S., Ogawa, E., Maruyama, M., Oka, H., Satake, M., Shigesada,that CX3C will map to 16q, as well. Recently, CX3C has K., Jenkins, N., Gilbert, D., Copeland, N., and Ito, Y. (1994).

PEBP2 alpha B/mouse AML1 consists of multiple isoforms thatbeen mapped to human chromosome 16 (Bazan et al.,possess differential transactivation potentials. Mol. Cell. Biol. 14:1997). Given the syntenic relationship between mouse3242–3252.and human chromosomes, we postulate that the mouse

Bazan, F., Bacon, K., Hardiman, G., Wang, W., Soo, K., Rossi, D.,chromosome 8 locus represents the authentic CX3C Greaves, D., Zlotnik, A., and Schall, T. (1997). A new class of mem-gene. The Cx3C-rsl locus maps to a region of mouse brane-bound chemokine with a CX3C motif. Nature 385: 640–644.chromosome 11 that is syntenic with human chromo- Berman, J., Guida, M., Warren, J., Amat, J., and Brosnan, C. (1996).somes 5q and 17 (summarized in Fig. 4). This related Localization of monocyte chemoattractant peptide-1 expression in

the central nervous system in experimental autoimmune encepha-sequence, which may represent a pseudogene or an-lomyelitis and trauma in the rat. J. Immunol. 156: 3017.other member of the CX3C gene family, is likely to map

Bernfield, M., Koyenyesi, R., Kato, M., Hinkes, M., Spring, J., Gallo,to human 5q given its tight linkage to IL3 in mouse.R., and Lose, E. (1992). Biology of the syndecans: A family of trans-We have compared our interspecific map of chromo-membrane heparan sulfate proteoglycans. Annu. Rev. Cell Biol. 8:some 8 to a composite mouse linkage map that reports 365–393.

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of alpha and beta chemokines. FASEB J. 9: 57–62.of the composite map that lacks mouse mutations withCopeland, N., and Jenkins, N. (1991). Development and applicationsa phenotype that might be expected for an alteration

of a molecular genetic linkage map of the mouse genome. Trendsin this locus (data not shown).Genet. 7: 113–118.In Fig. 4b, we examine the divergence of the chemo-

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kine class more closely related to C and CC familiesGlabinski, A., Balasingam, V., Tani, M., Kunkel, S., Strieter, R.,than to the CXC grouping. Interestingly, each chemo- Yong, V., and Ransohoff, R. (1996). Chemokine monocyte chemoat-

kine type segregates to distinct human and mouse chro- tractant protein-1 is expressed by astrocytes after mechanical in-jury to the brain. J. Immunol. 156: 4363–4368.mosomes with some exceptions, as outlined above (e.g.,

MIP-3a/LARC, MIP-3b). Green, E. (1981). Linkage, recombination and mapping. In ‘‘Geneticsand Probability in Animal Breeding Experiments,’’ pp. 77–113,In summary, we have isolated a mouse d chemokineOxford Univ. Press, New York.with a strikingly different modular structure and a new

Hieshima, K., Imai, T., Opdenakker, G., Van Damme, J., Kusuda,gene locus. The preliminary data shown in this studyJ., Tei, H., Sakaki, Y., Takatsuki, K., Miura, R., Yoshie, O., andwill set the basis for future in vivo mouse studies. Fi- Nomiyama, H. (1997). Molecular cloning of a novel human CC

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flammatory diseases. The use of transgenic mice and Jenkins, N., Copeland, N., Taylor, B., and Lee, B. (1982). Organiza-tion, distribution, and stability of endogenous ecotropic murineinflammatory disease in vivo models will provide in-leukemia virus DNA sequences in chromosomes of Mus musculus.sight into the effects of CX3C in inflammation and CNSJ. Virol. 43: 26–36.autoimmune diseases.

Kelner, G., Kennedy, J., Bacon, K., Kleyensteuber, S., Largaespada,D., Jenkins, N., Copeland, N., Bazan, J., Moore, K., Schall, T., andZlotnik, A. (1994). Lymphotactin: A cytokine that represents a newACKNOWLEDGMENTSclass of chemokine. Science 266: 1395–1399.

Kennedy, J., Kelner, G., Kleyensteuber, S., Schall, T., Weiss, M.,We thank D. Liggett for oligonucleotide synthesis and Deborah B.Yssel, H., Schneider, P., Cocks, B., Bacon, K., and Zlotnik, A.Householder for excellent technical assistance. We are grateful to G.(1995). Molecular cloning and functional characterization of hu-Burget and M. Andonian for their help in the preparation of theman lymphotactin. J. Immunol. 155: 203–209.figures for the manuscript. We thank C. Huffine, A. Helms, and D.

Gorman for automated DNA sequencing. This research was sup- Kuo, S., Mellentin, J., Copeland, N., Gilbert, D., Jenkins, N., andported, in part, by the National Cancer Institute, DHHS, under con- Cleary, M. (1991). Structure, chromosome mapping, and expres-tract with ABL. DNAX Research Institute is supported by Schering- sion of the mouse Lyl-1 gene. Oncogene 6: 961–968.Plough. Lennon, G., Auffray, C., Polymeropoulos, M., and Soares, M. (1996).

The I.M.A.G.E. Consortium: An integrated molecular analysis ofgenomes and their expression. Genomics 33: 151–152.Note added in proof. During the review process of this article,

mouse CX3C/fractalkine was published under the name of neurotac- Lloyd, A., Oppenheim, J., Kelvin, D., and Taub, D. (1996). Chemo-

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