Chromosomal assignment and genomic structure of Il15

GENOMICS

25, ‘701-706

(1995)

Chromosomal Assignment and Genomic Structure of //I5 DIRK M. ANDERSON,**’ LISABETHJOHNSON,* MOIRA B. GLACCUM,* NEAL G. CopELArw,t DEBRA J. GILBERT,t NANCY A. JENKINS,t VIRGINIA VALENTINE,* MARK N. KIRSTEIN,* DAVID N. SHAPIRO,+ STEPHAN W. MORRIS,+ KENNETH GRABSTEIN,* AND DAVID COSMAN* *Immunex Research and Development Corporation, Seattle, Washington 987 01; tMamma/ian Genetics Laboratory ABL-Basic Research Program, NC/-Frederick Cancer Research and Development Center, Frederick, Maryland 2 1702; and *St. Jude Children’s Research Hospital, Memphis, Tennessee 38101 Received September 9, 1994; revised November 11, 1994

stimulating factor (GM-CSF) share a common receptor ,O chain subunit (Kitamura et al., 1991; Tavernier et al., 1991), and their genes are linked on human chromosome 5 (Van Leeuwen et al., 1989). To further investigate the relatedness between IL-2 and IL-15, we have mapped the chromosomal location of both murine I115 and human IL15 and determined the genomic structure of murine 1115.

Interleukin-15 (IL-X) is a novel cytokine whose effects on T-cell activation and proliferation are similar to those of interleukind (IL-2), presumably because IL-15 utilizes the /3 and y chains of the IL-2 receptor. Murine IL-16 cDNA and genomic clones were isolated and characterized. The murine IZ15gene was found to consist of eight exons spanning at least 34 kb and was localized to the central region of mouse chromosome 8 by interspecific backcross analysis. Intron positions in a partial human IL15 genomic clone were identical with positions of corresponding introns in the murine gene. The human IL15 gene was mapped to human chromosome 4q31 by fluorescence in situ hybridization. 0 1995 Academic Press, Inc.

MATERIALSAND METHODS

INTRODUCTION

Interleukin-15 (IL-X) is a recently characterized cytokine that exhibits many of the same biological properties as IL-2, including the activation and proliferation of T cells and specific interaction with the /? and y subunits of the IL-2 receptor (Giri et al., 1994; Grabstein et al., 1994). These two hematopoietic growth factors share many properties and have similar predicted three-dimensional structures, but their expression patterns are distinct. Although IL-2 is primarily a product of activated T cells, IL-15 mRNA is detectable in a variety of cell and tissue types, including fibroblasts, epithelial cells, and monocytes, but not primary T cells (Grabstein et al., 1994). Redundancy of function is a common feature of those cytokines whose receptors are members of the hematopoietin receptor superfamily (for reviews, see Cosman, 1993 and Nicola, 1989). For example, leukemia inhibitory factor and oncostatin M have overlapping functions, share receptor components, and are closely linked on human chromosome 22 (Gearing et al., 1992; Jeffery et al., 1993). Similarly, IL-3, IL-5, and granulocyte-macrophage colony 1To whom correspondence should be addressed at Immunex Corporation, 51 University Street, Seattle, WA 98101.

Polyadenylated mRNA (1 Zsolation of murine IL-15 cDNA clones. pg) from the murine bone marrow stromal cell line +/+ (Boswell et al., 1990) was used to direct first-strand cDNA synthesis (Superscript Preamplification System; Bethesda Research Laboratories, Gaithersburg, MD). A portion of the murine IL-15 mature coding region was amplified by PCR using human IL-E-specific primers (Grabstein et al., 1994; 5’-TAAAACAGAAGCCAACTG-3’ and B’XAAGAAGTG’ITGATGAACAT-3 ’ ), under conditions described previously (Anderson et al., 1990). The amplified product from this reaction was subcloned into pBluescript SK(-) (Stratagene, La Jolla, CA) and sequenced to confirm its identity based on comparison with the human IL-15 cDNA sequence. The subcloned fragment was then random prime labeled with [(r-32PldCTP and used to probe a +/+ cDNA library (Anderson et al., 1990) to isolate murine IL-15 cDNA clones. Additional amplifications were performed using murine IL-15-specific primers with first-strand cDNA derived from a murine fetal liver epithelial cell line to isolate additional murine IL-15 cDNA clones. Interspecific mouse backcross mapping. Interspecific backcross progeny were generated by mating (C57BI&J X Mus spretus)F, females and C57BL16J males as described (Copeland and Jenkins, 1991). A total of 205 Nz mice were used to map the 1115 locus. DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, Southern blot transfer, and hybridization were performed essentially as described (Jenkins et al., 1982). All blots were prepared with Hybond-N+ nylon membrane (Amersham, Arlington Heights, IL). The probe, a murine IL-15 cDNA fragment containing the entire coding region and - 130 bp of 5’ noncoding sequence, was labeled by nick-translation with [a-32PldCTP and washed to a final stringency of 1.0~ SSCP, 0.1% SDS, 65°C. Fragments of 5.8 and 4.2 kb were detected in BamHI-digested C57BL&J DNA, and fragments of 10.5 and 4.2 kb were detected in BamHI-digested M. spretus DNA. In addition, Z’aqI digestion produced major fragments of 7.6, 5.2, and 4.4 kb (C57BI&J) and 5.5 and 4.2 kb (M. spretus). The presence or absence of the 10.5-kb BamHI and 5.5- and 4.2-kb TaqI M. spretusspecific fragments, which cosegregated, was followed in backcross mice. The BamHI and TaqI data were combined. A description of the probes and restriction fragment length poly-

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ANDERSON

morphisms for the loci linked to 1115, including mineralocorticoid receptor (Mlr), mitochondrial uncoupling protein (Ucp), and junB oncogene (Junb), has been reported previously (Newton et al., 1994). Recombination distances were calculated as described (Green, 1981) using the computer program SPRETUS MADNESS. Gene order was determined by minimizing the number of recombination events required to explain the allele distribution patterns. Isolation of murine Zl15 genomic clones. Murine 1115 genomic clones were isolated from a murine (C57BL/6 strain) genomic phage library prepared in the Lambda FIX II vector (Stratagene). The library was initially screened with a random prime-labeled coding region probe under conditions of high stringency. Positive phage were replated and screened with end-labeled oligonucleotide probes corresponding to 5’ noncoding, coding, and 3 ’ untranslated regions of the murine IL-15 cDNA. Three overlapping candidate genomic murine 1115 phage DNAs, comprising the entire murine IL-15 coding region and 3’ untranslated region, but not the 5’ noncoding region, were identified. A second probing of the phage library was performed using a 5’ noncoding region probe (the first 200 bp of Fig. 1A) to obtain additional B’specific genomic clones. Candidate 1115 genomic clones were digested with Not1 and subcloned into pGEMl1 (Promega, Madison, WI) or pBluescript SK(-) for further analysis. DNA sequencing and mapping of intron-exon boundaries. DNA sequencing was performed using the Z’aq DyeDeoxy Terminator Cycle Sequencing kit on an automated ABI DNA sequencer Model 373A (Applied Biosystems, Foster City, CA). The positions of introns were determined by sequencing across intron-exon boundaries in the genomic clones, using murine IL-15 cDNA-specific sequencing primers. Zsolation of a human genomic IL15 clone. A full-length human IL-15 cDNA clone was random prime labeled and used to screen a human placenta genomic phage library prepared in the Lambda DASH II vector (Stratagene) under conditions of high stringency. Identity of putative IL15 clones was confirmed by partial sequencing of candidate clones using oligonucleotide primers derived from the cDNA sequence. A clone with an -13-kb insert (designated h7-1) that contains 3’ human IL15 coding sequences was used for fluorescence in situ hybridization studies. Hybrid cell lines with the prefix Somatic cell hybrid analysis. “GM” were obtained from the National Institute of General Medical Sciences’ Human Genetic Mutant Cell Repository (Coriell Institute for Medical Research, Camden, NJ); characterization and human chromosome content of these hybrids is described fully in the Repository catalog. The preparation of human x hamster somatic cell hybrid lines A2, A4, A5, and Cl has been described (Morris et al., 1991); these hybrids contain a der(3) and/or der(51 from a balanced translocation, t(3;5)(q25,l;q34), together with other human chromosomes. Southern hybridizations of hybrid DNAs were performed at 42°C for 16 h in 5~ SSC, 50% formamide, 0.5% SDS, 10% dextran sulfate with 100 pglml salmon sperm DNA, stringent washes were in 0.1~ SSC/O.l% SDS at 58°C. Fluorescence in situ hybridization. Peripheral blood lymphocytes from a normal donor were synchronized with bromodeoxyuridine and stimulated with phytohemagglutinin as a source of metaphase chromosomes. The human IL15 genomic DNA clone, X7-1, was nick-translated with digoxigenin-11-UTP and hybridized overnight at 37°C to fixed metaphase chromosomes as described (Morris et al., 19921. Simultaneous hybridization with a digoxigenin-labeled chromosome 4-specific a-satellite DNA probe (Oncor, Inc., Gaithersburg, MD, Cat. No. P5007) was performed to confirm chromosome identity. Signals were detected by incubating slides with fluorescein-conjugated sheep anti-digoxigenin antibodies (Boehringer Mannheim, Indianapolis, IN) followed by counterstaining in propidium iodide solution containing DABCO (1,4-diazabicyclo[2.2.2]octane; Sigma Chemical, St. Louis, MO). Fluorescence microscopy was performed with a Zeiss standard microscope equipped with fluorescein epifluorescence filters.

RESULTS

Isolation amplification

of murine

IL-15

primers

based

cDNA clones. on the human

We used IL-15 se-

ET AL.

quence to amplify a portion of the coding region of murine IL-15. We then used this amplified product to screen a murine cDNA library to obtain full-length murine IL-15 cDNA clones. One clone, mIL-15-149, contained the entire predicted IL-15 coding region interrupted by a 1280-bp insert (at the position of intron 5, Fig. 1A) and flanked by 465 bp of 5’ and 300 bp of 3’ noncoding sequences. Additional murine IL-15 cDNA clones isolated from this library were sequenced and found to contain various insertions and/or deletions as determined by comparison with the human IL-15 cDNA. To ascertain that these isolated cDNA clones represented splicing intermediates, we used oligonucleotide primers designed to amplify the entire coding region of murine IL-15 in a PCR amplification using cDNA from a murine fetal liver epithelial cell line. This amplification yielded a DNA fragment that was cloned, sequenced, and determined to encode the uninterrupted murine IL-15 coding region shown in Fig. 1A. Recombinant murine IL-15 generated in a yeast expression system utilizing this cDNA was biologically active in a CTLL proliferation assay (data not shown). A comparison of the deduced amino acid sequences of murine and human IL-15 is shown in Fig. 1B. Murine and human IL-15 share 73% sequence identity at both the nucleotide and the amino acid levels. IL15gene structure analysis. Three overlapping genomic murine I115 clones were isolated from the C57BL/6 library using a coding region cDNA hybridization probe (Fig. 2, clones X14, X2, and h10.3). The 5’ end of clone X14 terminated in intron 2. Both clones obtained from a second library screen using a 5’ noncoding region cDNA probe (X13, X18) terminated at their 3 ’ ends in intron 1. No genomic clones containing exon 2 were obtained. The murine I115 locus is therefore estimated to be at least 34 kb in length. Sequence analysis of these genomic clones with murine IL-15 cDNA-specific sequencing primers identified seven introns in the murine I115 gene; their positions are shown in Fig. 1A. This gene structure differs from the four exomthree intron structure of the I12 gene; however, the positions of the last three introns of the Ill5 gene delineate exons encoding predicted helix-forming domains of IL-15 (Grabstein et al., 1994). The positions of introns in the partial human IZ15 genomic clone were found to be identical with the corresponding positions of introns 5, 6, and 7 of the murine gene (data not shown). Murine Ill5 chromosomal mapping. The mouse chromosomal location of Ill5 was determined by interspecific backcross analysis using progeny derived from matings of l(C57BU6J X M. spretus)F1 X C57BL/6Jl mice. This interspecific backcross mapping panel has been typed for over 1600 loci that are well distributed among all of the autosomes as well as the X chromosome (Copeland and Jenkins, 1991). The mapping results indicate that IZ15 is located in the central region of mouse chromosome 8 linked to Mlr, Ucp, and Junb

1115

A

GENE

CHARACTERIZATION

703

CTTCTGTCCAGCCACTCTTCCCCAGAGTTCTCTTCTTCATTCTCT

100

GCGCCCAAAAGACTTGCAGTGCATCTCTCCTGCATC

200

&l CTGCTGTGTTTGGAAGGCTGAGTTCCACATCTAACATCT~~GCTCA~GAGGT~GG~G~TCCACCTTGA~CATGGCCCTCTGGCTCTTC~G~CTGCC 12 TCTTCATGGTCCTTGCTGGTGAGGTCCTTAAGAACACAGATGGCA 13 GCTGGAAGCCCATCGCCATAGCCAGCTCATCTCATCTTC~~TTG~GCTCTTACCTGGGCATTAAGTAATGTTTTG~C~TATAT~G~TACATC MKILKPYMRNTS 14 CATCTCGTGCTACTTGTGTTTCCTTCTAAACAGTCACTTTTGTCTTCATTTTGGGCTGTGTCAGTGTAGGTCTCCCTAAA ISCYLCFLLNSHFLTEAGIHVFILGCVSVGLPK i.5 ACAGAGGCCAACTGGATAGATGTAAGATATGACCTGGAGACT TEANWIDVRYDLEKIESLIQS IHIDTTLYTDSDF

300 400 500

600

700

1s TTCATCCCAGTTGCAAAGTTACTGCAATGAACTGCTTTCTCCTGGAATTGCAGGTTATTTTACATGAGTACAGTAATCTTAATGAAACAGTAAG HPSCKVTAMNCFLLELQVILHEYSNMTLNETVR $1 AAACGTGCTCTACCTTGCAAACAGCACTCTGTCTTCTAACCA NVLYLANSTLSSNKNVAESGCKECEELEEKTFT

800

900

GAGTTTTTGCAAAGCTTTATACGCATTGTCC~TGTT~T~CACGTCCTGACTG~TGC~GCCTCTTCCGTGTTTCTGTTATT~GGTACCTC~C EFLQSFIRIVQMFINTS

1000

CTGCTGCTCAGAGGCAGCACAGCTCCATGCATTTGAAATCTGCTGGGCAAACTAAGCTTCCTAATC

1100

TTGGAAATGAAGAGAGGAAAAGAGCTCGTCTCAGACTTATTTTTGCTTGCTTATTTTT~TTTATTGCTTCATTTGTACATATTTGT~TAT~CAG~G

1200

ATGTGGAATAAFiGTTGTATGGATATTTTATCAATTGAiWTTT-

B

1250

M0tm MKILKPYMRNTSISCYLCFLLNSHFLTEAGIHVFILGCVSVGLPKTE~IDVRYDLEKI Human

I I II I II III1 IIIIIIIII/IIIIIIIII I IiIIIIIII I II II MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTE~VISDLKKI ESLIQSIHIDTTLYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVRNVLYLS I IIII III III1 II IIIIIIIII IIIIIIIIl I II I III EDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLII~

60 60 120 120

TLSSNKNVAESGCKECEELEEKTFTEFLQSFIRIVQMFINTS162 IIII II IIIIIIIlIIIII IIIIII IIIIIIIII SLSSNGWVTESGCKECEELEEKNIKEFLQ~F~HI~QMFINTS

162

FIG. 1. DNA and deduced amino acid sequence of murine IL-K (A) Sequence of isolate mIL-15-149, with intervening sequence at intron position 5 removed. The positions of introns are indicated above the sequence by arrows. (B) Amino acid comparison of murine and human IL-15 The amino-terminus is underlined; predicted N-linked glycosylation sites are in boldface. The nucleotide sequence data reported in this _ paper _ have been deposited with the EMBL, GenBank, and DDBJ Nucleotide Sequence databases under Accession No. ui4332.

(Fig. 3). Although 99 mice were analyzed for every marker and are shown in the segregation analysis (Fig. 3), as many as 153 mice were typed for some pairs of markers. Each locus was analyzed in pair-wise combinations for recombination frequencies using the additional data. The ratios of the total number of mice exhibiting recombinant chromosomes to the total number of mice analyzed for each pair of loci and the most likely gene centromere-Mlr-4/153-Ill5order are O/115-Ucp-l/ll%Junb. The recombination frequenA2

A14 no.3

118 ____ E . ..+

....

E

:

E

E

:

yi Exons

!

E

E E

I,

!

!

E

‘Lib

!

FIG. 2. Structure of murine 1115. Restriction enzyme EcoRV cleavage map of murine 1115 gene. The extent of the phage clones is shown above the map. Open bars indicate noncoding exons; solid bars indicate coding exons.

ties (expressed as genetic distances in centimorgans tthe standard error> are Mlr-2.6 + 1.3-(1115, Ucp)-0.9 + 0.8-Junb. No recombinants were detected between I115 and Ucp in 115 animals typed in common, suggesting that th8 two loci are within 2.6 CMof each other (upper 95% confidence limit). Human-rodent somatic cell hybrid analysis. Southern blot hybridizations of human genomic DNAs digested with multiple restriction enzymes using the full-length human IL-15 cDNA as a probe revealed a hybridization pattern consistent with a single-copy gene (not shown). Hybridization of this probe to panels of EcoRI- or HindIIIdigested DNAs prepared from somatic cell hybrid cell lines produced a pattern of segregation for all humanspecific restriction fragments that was consistent with localization to chromosome 4 (Fig. 4A); no hybrids discordant with localization to chromosome 4 were identified. Hybridization of the IL-15 cDNA probe to genomic DNA prepared from the hybrid GM11449 that contains the 4q25-qter segment of the chromosome only permitted gross regional localization of the gene.

704

ANDERSON

ET AL

the centromere of the hybridization signal relative to the entire length of the long arm of chromosome 4, we assigned the human IZ15 locus to band q31. DISCUSSION

We report the cloning of a murine IL-15 cDNA, the characterization of the genomic structure of murine

A 7.5 kb -

t

1115 UCP

4q28-q31

JWtb

19~13.2

3.5 kb -

FIG. 3. 1115 maps to the central region of mouse chromosome 8. 1115 was placed on mouse chromosome 8 by interspecific backcross analysis. The segregation patterns of 1115 and flanking genes in 99 backcross animals that were typed for all loci are shown at the top of the figure. Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/6J x M. spretus) F, parent. The black boxes represent the presence of a C57BI&J allele, and 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. A partial chromosome 8 linkage map showing the location of 1115 in relation to linked genes is 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 loci in human chromosomes, 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 Johns Hopkins University (Baltimore, MD).

Fluorescence in situ hybridization (FISH) analysis. To confirm and to refine more precisely our localization of1115 to human chromosome 4q25-qter, we performed FISH of metaphase chromosomes prepared from peripheral blood lymphocytes of a normal donor using a genomic I115 phage clone. A representative hybridization of human genomic 1115 phage clone h7-1 to a metaphase chromosome preparation is shown in Fig. 4B. Fluorescence signal observed with the clone was specific to the distal third of the long arm of a Group B chromosome. Simultaneous hybridization using a chromosome 4-specific a-satellite DNA probe confirmed the identity of the chromosome. Specific labeling of 70 to 74 (95%) chromosome 4 chromatids with the X7-1 clone at an identical position on the long arm was observed in representative metaphases that were selected for photography. No other nonrandom signals suggestive of alternative localizations or of cross-hybridization with related sequences were seen in any of the metaphase spreads examined. Based upon the distance from

FIG. 4. Human IL15 maps to chromosome 4q31. (A) Southern blot analysis of HindIII-digested DNAs from representative somatic cell hybrids probed with a full-length IL-15 cDNA. Hu, Ha, and Mu: human, hamster, and mouse genomic DNAs, respectively. The positive hybrid GM11687 contains intact chromosome 4 as its only human DNA, whereas hybrid GM11449 contains the chromosome 4 subfragment 4q25-qter only. The negative hybrids (GMO9934, A2, A4, A5, Cl) contain various human chromosomes other than chromosome 4. (B) Fluorescence in situ hybridization to human metaphase chromosomes with an IL15 genomic clone. The position of the IL15 locus on chromosome 4q31 is indicated (arrows). The identity of the chromosomes was verified by simultaneous hybridization with a chromosome 4-specific a-satellite DNA probe (arrowheads).

1115 GENE

705

CHARACTERIZATION

1115, and the chromosomal mapping of the murine I115 and human IL15 gene loci. Comparison of murine and human IL-15 revealed 73% identity at both the nucleotide and the amino acid levels. The murine I115 gene contains eight exons and seven intervening introns and is at least 34 kb in length. Preliminary characterization of human genomic clones suggests that this structure is conserved in human IL15. The last three introns of I115 occur within the mature protein coding domain and delineate exons encoding predicted helix-forming domains, a structure that corresponds to the intronic positions of the I12 gene, as well as other members of the helical cytokine family (Fujita et al., 1983; reviewed in Bazan, 1992). The murine I115 gene maps to the central region of chromosome 8. We have compared our interspecific map of chromosome 8 with a composite mouse linkage map of many uncloned mouse mutations (compiled by M. T. Davisson, T. H. Roderick, A. L. Hillyard, and D. P. Doolittle and provided from GBASE, a computerized database maintained at The Jackson Laboratory, Bar Harbor, ME). IZ15 mapped to a region of the composite map that lacks mouse mutations with a phenotype that might be expected for an alteration in this locus (data not shown). The central region of mouse chromosome 8 shares regions of homology with human chromosomes 4q and 19q (summarized in Fig. 3). In particular, the gene encoding mitochondrial uncoupling protein (Ucp), a component of the inner membrane of adipose tissue mitochondria that acts to induce thermogenesis, is known to reside at 4q31 (Cassard et al., 1990). The tight linkage in mouse suggested that IL15 would reside on 4q as well. We have mapped the human IL15 genomic locus to human chromosome 4q31 by Southern analysis of DNAs prepared from somatic cell hybrid lines segregating human chromosomes and by metaphase fluorescence in situ hybridization experiments using a human IL15 genomic DNA clone. Human I115 joins several other genes that are located on chromosome arm 4q that encode growth factors/chemokines. For example, the region 4q13 -93 1 contains the loci encoding amphiregulin, IL-8, fibroblast growth factor (FGF)-2 and FGF-5, three distinct GRO genes, the interferon-inducible cytokine IP-10, epidermal growth factor, and IL-2 (Murray and Van Ommen, 1991). The gene encoding IL-2 (located at 4q26-q27) and the human IL15 gene at 4q31 lie in relatively close proximity, but the lack of primary sequence homology between these two T-cell growth factors indicates no significant evolutionary relatedness. However, the gene structures of IL2 and IL15 are similar, as mentioned above, suggesting a common ancestry shared by other members of the helical cytokine family. There are no commonly recurring, nonrandom abnormalities of the 4q31 segment in human neoplastic diseases. Although IL-15 mRNA is known to be expressed in a number of human tissues (including placenta, skeletal muscle, heart, lung, liver, and kidney), additional studies of the biologic functions

of IL-15 will be necessary to determine whether any human disorders may be related to abnormalities of this gene locus. ACKNOWLEDGMENTS The authors thank Debbie Barnhart for excellent technical assistance and Anthony Namen for the murine fetal liver epithelial cell line. This research was supported, in part, by the National Cancer Institute (NCI), DHHS, under Contract NOl-CO-74101, NC1 Grant CA01702 (S.W.M.), by Cancer Center CORE Grants CA21765 and CA23099 from the NCI, and by the American Lebanese Syrian Associated Charities (ALSAC). REFERENCES Anderson, D. M., Lyman, S. D., Baird, A., Wignall, J. M., Eisenman, J., Rauch, C., March, C. J., Boswell, H. S., Gimpel, S. D., Cosman, D., and Williams, D. E. (1990). Molecular cloning of mast cell growth factor, a hematopoietin that is active in both membrane bound and soluble forms. Cell 63: 235-243. Bazan, J. F. (1992). Unraveling 410-413.

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Boswell, H. S., Mochizuki, D. Y., Burgess, G. S., Gillis, S., Walker, E. B., Anderson, D., and Williams, D. E. (1990). A novel mast cell growth factor (MCGF-3) produced by marrow-adherent cells that synergizes with interleukin 3 and interleukin 4. Exp. Hematol. 18: 794-800. Cassard, A. M., Bouillaud, F., Mattei, M. G., Hentz, E., Raimbault, S., Thomas, M., and Ricquier, D. (1990). Human uncoupling protein gene: Structure, comparison with rat gene, and assignment to the long arm of chromosome 4. J. Cell. Biochem. 43: 255-264. Copeland, N. G., and Jenkins, N. A. (1991). Development and applications of a molecular genetic linkage map of the mouse genome. Trends Genet. 7: 113-118. Cosman, D. (1993). The hematopoietin kine 5: 95-106.

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Cyto-

Fujita, T., Takaoka, C., Matsui, H., and Taniguchi, T. (1983). Structure of the human interleukin 2 gene. Proc. Natl. Acad. Sci. USA 80: 7437-7441. Gearing, D. P., Comeau, M. R., Friend, D. J., Gimpel, S. D., Thut, C. J., McGourty, J., Brasher, K. K., King, J. A., Gillis, S., Mosley, B., Ziegler, S. F., and Cosman, D. (1992). The IL-6 signal transducer, gp130: An oncostatin M receptor and affinity converter for the LIF receptor. Science 255: 1434-1437. Giri, J. G., Ahdieh, M., Eisenman, J., Shanebeck, K., Grabstein, K., Kumaki, S., Namen, A., Park, L. S., Cosman, D., and Anderson, D. (1994). Utilization of the B and y chains of the IL-2 receptor by the novel cytokine IL-15. EMBO J. 13: 2822-2830. Grabstein, K. H., Eisenman, J., Shanebeck, K., Rauch, C., Srinivasan, S., Fung, V., Beers, C., Richardson, J., Schoenborn, M. A., Ahdieh, M., Johnson, L., Alderson, M. R., Watson, J. D., Anderson, D. M., and Giri, J. G. (1994). Cloning of a novel T cell growth factor that interacts with the p chain of the interleukin-2 receptor. Science 264: 965-968. Green, E. L. (1981). “Linkage, Recombination, 77-113, Oxford Univ. Press, New York.

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Jeffery, E., Price, V., and Gearing, D. P. (1993). Close proximity of the genes for leukemia inhibitory factor and oncostatin M. Cytokine 5: 107-111. Jenkins, N. A., Copeland, N. G., Taylor, B. A., and Lee, B. K. (1982). Organization, distribution, and stability of endogenous ecotropic murine leukemia virus DNA sequences in chromosomes of Mus musculus. J. Viral. 43: 26-36. Kitamura, T., Sato, N., Arai, K., and Miyajima, A. (1991). Expression cloning of the human IL-3 receptor cDNA reveals a shared 0 sub-

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ET AL.

Newton, G., Weremowicz, S., Morton, C. C., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., and Lawler, J. (1994). Characterization of human and mouse cartilage oligomeric matrix protein. Submitted for publication. Nicola, N. (1989). Hematopoietic growth factors and their receptors. Anna Rev. Biochem. 58: 45-77. Tavernier, J., Devos, R., Cornelis, S., Tuypens, T., Van der Heyden, J., Fiers, W., and Plaetinck, G. (1991). A human high affinity interleukin-5 receptor (ILBR) is composed of an IL5-specific (Ychain and a p chain shared with the receptor for GM-CSF. Cell 68: 11751184. Van Leeuwen, B. H., Martinson, M. E., Webb, G. C., and Young, I. G. (1989). Molecular organization of the cytokine gene cluster, involving the human IL-3, IL-4, IL-5, and GM-CSF genes, on human chromosome 5. Blood 73: 1142-1148.