Human interleukin-1 beta gene

Gene, 52 (1987) 95-101 Elsevier GEN 01918

Human interleukin-1 betargene (Cytokine; recombinant DNA; molecular cloning; exon homologies; computer analysis; phage 1; genomic library)

Giuliano Bensi, Giovanni Raugei, Emanuela Palla, Valeria Carinci, Daniela Tornese Buonamassa and Marialuisa Melli Sclavo Research Center, 53100 Siena (ItaIy) Received 3 November 1986 Accepted 11 November 1986

SUMMARY

We report the nucleotide sequence of the human chromosomal gene which encodes the interleukin-1 Bprotein (IL-l /I). The gene spans a region of 7.5 kb and the coding part is divided into seven exons. Comparison with the homologous mouse gene reveals that the structural organization is conserved through evolution. In addition to this, human and murine IL- 1 /I genes show extensive sequence homology within the intervening sequences.

INTRODUCTION

IL-1 is a cytokine produced by activated macrophages which mediates several physiological responses to infections and injuries, including stimulation of thymocyte proliferation, B-lymphocyte maturation and proliferation, induction of acutephase protein synthesis by hepatocytes and induction of fever (reviewed by Dinarello, 1984). In addition to macrophages, fibroblast keratinocytes, comeal cell astrocytes, and EBV-transformed B-lymphocytes are also capable of producing IL-l

Correspondenceto: Dr. G. Bensi, Sclavo Research Center, Via Fiorentina 1, 53100 Siena (Italy) Tel. 0577-293481. Abbreviations: RNA; EBV, kilobase or otide(s); ORF, 13-acetate.

bp, base pair(s); cDNA, DNA complementary to Epstein-Barr virus; IL-l, interleukin-1; kb, 1000 bp; LPS, lipopolysaccharide; nt, nucleopen reading frame; TPA, 12-O-tetradecanoyl-

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1987 Elsevier

Science Publishers

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(Dinarello, 1984; Oppenheim et al., 1984). Monocyte cell lines produce IL-1 after treatment with agents such as TPA and LPS (Mizel et al., 1983; Oppenheim et al., 1986). In the last two years the cloning of human and murine IL-l cDNAs (Lomedico et al., 1984; Auron et al., 1984; March et al., 1985) has demonstrated the existence of at least two structurally distinct proteins, c1and 8, showing a limited homology (26 %) at the amino acid level which is in contrast with the reported functional similarity (Oppenheim et al., 1986). Recently the complete sequences of the human IL-1 a and murine IL-1 p genes have been reported (Furutani et al., 1986; Telford et al., 1986). The similarity of the structural organization of the two genes has suggested the existence of a common ancestor. Here we present the complete nucleotide sequence of a human gene coding for the IL-1 /? protein. A comparison of the murine and human sequences shows conservation both in exons and introns. Division)

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ACTTAACCTCTTGAGCTTCAGAGAGGGATAATCTTTTTATTTTATTTTATTTTATTTTGTTTTGTTTTGTTTTGTTTTGTTTTATGAGACAGAGTCTCAC

2801

TCTGTTGCCCAGGCTGGAGTGCAGTGGTACAATCTTGGCTTACTGCATCCTCCACCTC~TGAGTT~AAGCGATTCTCCTTCCT~AGTCT~~TGAATAG~~

2901

AGGATTACAGGTGCACCCCACCACACCCAGCTAATTTTT~TATTTTTAGTAGAGAAGGGGTTTCGCCAlGTTGGCCAGGCTGGTTTTGAAGTCCTGAC&T

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TTCAAGCCCTTTCCTCCTCTCTGA6CTTTCTAGTCTCTCT6ATGTCAAAG~ATGGTTCCTGGCAGGACCACCTCACCAGGCTCCCTCC~TC6CTCTCTCCGC EXON 4 . GCTCClTCCAGGACCTGGACCTCTSCCCTCTGGATGGCGGCAT~CA6CTACGAATCTCCGACEACCACTACAG~AAGGGCTTCAGGCAGGCCGCGTC ysSerPheGlnAspLeuAspLeuCysProLeuAspGlyGlyIleGlnLeuArgIleSerAspHisHisTyrSerLysGlyPheArgGlnAlaAla~s AGTTGTTGTGGCCATGGACAAGCTGAGGAAGATGCTGGTTCCCTGCECACAGACCTTCCAGGAGAATGACCTGAGCACCTTCTTTCCCTTCATCTTTGAA rValValValAleMetAspLysLeuArgLysMetLeuValProCysPraGlnThrPheGlnGluAsnAspLeu~erThrPhePheProPheIlePheGlu 3401

GAP, TAGTTAGCCAAGAGCA66CAGTAGATCTCCACTTGTGTC~TCTTGGAAGTCATCAAGCCCCAGCCAACTCAATTCCCCCAGAGCCAAAGCCCTTT 3GluG

3501

AAAG6TAGAAGGCCCAGCGGGGAGACAAAACAAAGAAGGCTGGAAAC~AAAGCAATCATCTCTTTAGTGGAAACTATTCTTAAAGAAGAT~TT6AT66CT

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ACTGACATTTGCAACTCCCTCACTCTTTCTCAGGGGCCTTT~ACTTACATTGTCACCAGAGGTTCGTAACCTCCCTGTGGGCTAGTGTTAT~ACCATCA~

3701

CATTTTACCTAAGTAGCTCTGTTGCTCGGCCACAGTGAGCAGTAATAGACCTGAAGCTGGAACCCATGTCTAATAGTGTCAGGTC~ATGTTCTTAGCCAC

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CCCACTCCCAGCTTCATCCCTACTGGTSTTGTCATCAGACT~TGACCGTATATGCTCAGTGTCCTCCAAGAAATCAAATTTTGCCGCCTCGCCTCACGAG EXON 5 . GCCTGCCCTTCTGATTTTATACCTAAACAACATGTGCTCCACATTTCA AACCTATCTTCTTCGACACATGGGATAACGAGGCTTATGTGCACGATGCAC 1uProIl~PhePheAspThrTrpAspAsnGluAlaTyrValHisAspAlaP

3901

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CTGTACGATCACTGAACTGCACGCTCCGGGACTCACAGCAAAAAAGCTTGGTGATGTCTGGTCCATATGAACTGAAAGCTCTCCAC~TCCAGG6ACA~~A roValArgSerLeuAsnCysThrLeuArgAspSerGlnGlnLysSerLeuVal~etSer~lyProTyrGluLeuLysAlaLeuHisLeuGlnGlyGlnAs

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TTAACAGGAGTGTGACCCTGAGAAATTAGACGGTCAAGCACTCCCAGGACCATGTCCACCCAAGTCTCTT~GGEATAGTGCAATGTCAATTCTTCCACAA

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TATCCCCTCATTTGATGGACATGGCCTAACTGCCTGT~~GTT~TCT~TTC~T~TTGTTGA~~~TGAAACAAGAGTGCTG~AGCGATAAT~TGTCCATCC~

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CTCCCCAGTCTTCCCCCCTTGCCCCAACAGTCCGTCCCACCCAATGCAGGTGGTTCTTGTAGGGAAATTTTACCGCCCAGCAGGAACTTATATCTCTCCG

4601

CTGTAACGGGCAAAAGTTTCAAGTGCGGTGAACCCATCA~TAGCTGTGGTGATCT6CCTGGCATCGTGCCACAGTAGCCAAAGCCTCTGCACAGGAGTGT

4701

G6GCAACTAAGGCTGCTGACTTTGAAGGACA6CCTCACTCAGG6GGAAGCTATTTGCTCTCA~CCAGGCCAAGAAAATCCTGTTTCTTTGGAATCGGGTA

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GTAAGAGTGATCCCAGGGCCTCCAATTGACACTGiGAGCTCAGC~TCTCCT

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CTCCCAGTTTCTTCCCATGGGCTACTCTCTGTTCCTGAAACAGTTCTGGTGCCTGATTTCTGGCAGAAGTACAGCTTCACCTCTTTCCTTTCCTTCCACA

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TTGATCAAGTTGTTCCGCTCCTGTGGATGGGCACATTGCCAGCCAGTGACACAATGGCTTCCTTCCTTCCTTCCTTCAGCATTTAAAATGTAGACCCTCT

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TTCATTCTCCGTTCCTACTGCTATGAGGCTCTGAGAAACCTCAGGCCTTTGAGGGGAAACCCTAAATCAACAAAATGACCCTGCTATTGTCTGTGAGAA~

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TCAAGTTATCCTGTGTCTT;GGCCAAGGA;CCTCACTGTG~ATG~AT~CTA~TC~TGGGGCCTA~GGGTTGGGG~ EXON 6 GACCETGCACTGCTGTETCCCTAACCACAAGACCCCCTfGACAAAATAG blValPheSerMetSerPheValGln6lyGluGluSerAsnAspLysileF

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CTGTGGCCTTGGGCCTCAAGGAAAAGAATCTGTACCTGTCCTGCGTGTTGAAAGATGATAAGCCCACTCTACAGCTGGAG roValAlateuGlyLeuLys6luLysA~nLeuTyrLeuSerCysValLsuLysAspAspLysProThrLeuGlnLeuGlu

TAA~T6AATGCTATGGAAT

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GAAGCCCTTCTCAGCCTCCTGCTACCACTTATTCCCAGACAACCACCTTCTCCCCGCCCCCATCCCTAGGAAAAGCTGGGAACAGGTCTATTTGACAATT

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TTGCATTAATGTAAATAAATTTAACATAATTTTTAACTGCGTGCAACCTTCAATCCTGCTGCAGAAAATTAAATCATTTTGCCGATGTTATTATGTCCTA

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CCATAGTTACAACCCCAAC;GATTATATATTGTTAGGGCTGCTCTCATT;GATAGACACCTTGGGAAATAGATGACTTAAAGGGTCCCATTATCACGTCC

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ACTCCACTCCCAAAATCACCACCACTATCACCTCCAGCTTTCTCAGCAAAAGCTTCATTTCCAAGTT6ATGTCATTCTAGGACCATAA6GAAAAATACAA

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TAAAAAGCCCCTGGAAAETAG6TACTTCAAGAA6CTCTAGCTTAATTTTCACCCCCCAAAAAAAAAAAATTCTCACCTACATTATGCTCCTCA6CATTTG

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GCACTAA6TT~AGAAAAGAAGAGGGCTCTTTTAAATAAATTCACACAGAAAGTTGGGCCCAGTTACAACTCAGGAGTCTGGCTCCTGATCATGTGACCT

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EXON GCTCGTCAGTTTCCTTTCTGGCCAACCCAAAGAACATCTTTCCCATAGCATCTTTGTCCCTTGCCCCACAAAAATTCTTCTTTCTCTTTCGT6CAGAGT6 SerV 7 TAGATCCCA;AAATTACCCAAAGAAGAAGAT~GAAAAGC6, alAspProLysAsnTyrProLysLysLysHetGluLysArgPheValPheAsnLysIle6luIlsAsnAsnLysLeuGluPheGluSerAla6lnPhePr

6301

CAACTGGTACATCAGCACCTCTCAAGCAGAAACATGCCCCGTCTTCCTGGGAGGGACCAAAGEC66CCASGATATAACT6ACTTCACCATGCAATTTGTG oAsnTrpTyrIleSerThrSerGlnAlaGluAsnMetProValPheLeu6lyGlyThrLysGlyGlyGlnAspIleThrAspPhsThrMst6lnPhsVal

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TCTTCCTAA;GAGAGCTGTACCCA6A~AGTCCTGTOCTGA~TAC~~CT~ SerSerEnd

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TAGCCTG6ACTTTCCTGTTGTCTACACCAATGCCCAACT6CCT6CCTTA6G6TA6TGCTAAGA66ATCTCCT6TCCATC~6CC~~ACA6TCAGCTCTCT,

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CCTTTCAGGGCCAATCCCCAGCCCTTTTGTTGAGCCAGGCCTCTCTCACCTCTCCTACTCACTTAAA6CCCGCCTGACA6AAACCACGGCCACATTTGGT 'I

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TCTAAGAAACCCTCTGTCATTCGCTCCCACATTCTGATGAGCAACC6CTTCCCTATTTATTTATTTATTTGTTT6TTT6~TTTATTCAT~6GTCTAATT~

6801

ATTCAAAGGGGGCAAGAAGTAGCAGTGTCTGTAAAGAGTTTGGACTGGTGTGCTCTCTTTAAATCAA

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GTCCTTTAATTAAGACTGAAAATATATAAGCTCAGATTATTTAAAT6GGAATATTTATAAATGAGCAAATATCATACTGTTCAATGGTT~TGAAATAAA~

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AAGAAAAAAAAAGG6TCTTTCCTGATCATCATTGTCTT6TCTTGGATTTGACACT6AACAGTAAA6ACAAACAG6GCT6TGAGAGTTCTTGGGGGA

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CTAAAGCCCACCTCCTCATTGCTGAGTGC;GCAAAGTCACCCATCCCCT~TATTTCT~T~

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GTTCAACAG;AGGATATTC;GTGCACATC;GGAACAGGA;CT~~TA~TAACA~CTACCA6TG~TTTATCTAT~

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AATGCACCAAACATCTGTTGAGCAAGCGCTATGTACGAGGGTCCCTCCTCA6ATAGGAGAGGCAGCTA

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6TTATAAGCAGAAACAAGGTAACATGACA;GTAGAGTAA~ATAAAGAAC~A

Fig. 1. Complete nucleotide sequence of the human IL-l Bgene. Position + 1 represents the proposed transcription start point. The putative TATA box and the sequences of exons are boxed. Putative CAAT boxes and the Ah repetitive sequence are underlined. The sequence in exon 2 indicated with a broken line is complementary to the synthetic oligodeoxynucleotide used in the primer extension experiment (RESULTS AND DISCUSSION, section a).

MATERIALS AND METHODS

an automatic DNA synthesizer (Applied Biosysterns, Model 380A).

(a) Plasmids and phage vectors (c) Restriction mapping, nucleotide sequencing and The Ml3 derivatives mp18 and mp19 (Messing, 1983), tg130 and tg131 (Kieny etal., 1983), pEMBL18 and pEMBL19 (Dente et al., 1983) were used as plasmid vectors for subcloning. (b) Enzymes, chemicals and oligodeoxynucleotides

Restriction endonucleases, T4 DNA ligase and DNA polymerase I (large fragment) were purchased from Boehringer. The 32P-labelled compounds were from NEN. AMV reverse transcriptase was from P.H. Steheling & Cie AG. Exonucleases III and VII were from BRL. In some experiments, specific sequencing primers were prepared from the determined sequence and used to analyze the adjacent regions. These primers (15mer) were synthesized by

primer elongation

Restriction maps of the A recombinant phages were obtained with the method described by Rackwitz et al. (1984). Sequence analysis was carried out using the dideoxy chain termination method (Sanger et al., 1977). Primer elongation was carried out as previously described (Bensi et al., 1985). (d) Computer analysis

Computer analysis was performed using the programs prepared by the University of Wisconsin Genetics Computer Group (Devereux et al., 1984).

RESULTS AND DISCUSSION

(a) Isolation and structural analysis of the human IL-l j? gene To isolate the IL-l /? gene, a human phage 1 genomic library (Bensi et al., 1985) was screened with IL-l B cDNA previously isolated in our laboratory (unpublished data). Two overlapping IL-l p clones were isolated out of 700000 recombinants analyzed. Phage 2 hilb8 includes only the last 2.3 kb of the 3’ end of the gene, while phage 1 hilb4 contains the entire gene plus 2 kb of the 5’ flanking region. Hind111 restriction fragments of the two recombinants were mapped and subcloned into Ml3 or pEMBL vectors in both orientations. Progressive deletions were obtained by digestion with exonucleases III and VII enzymes (Yanisch-Perron et al., 1985). The sequence obtained from the overlapping clones is shown in Fig. 1. Seven exons with the expected consensus sequences at the splice junctions (Breathnach et al., 1978) can be identified by the comparison with the human cDNAs previously reported (Auron et al., 1984; March et al., 1985). The deduced IL- 1 B protein is identical to that described by March et al. (1985). The third intron contains a member of the Ah family of repetitive sequences in the opposite transcriptional direction with respect to the IL-l gene. The transcription initiation site has been determined by primer extension analysis, using a synthetic oligodeoxynucleotide spanning 11 nt of the 5’ untranslated region and 4 nt of the coding region (see Fig. 1). The resulting cDNA band (Fig. 2, lane 2) migrates in a denaturing 6 y0 polyacrylamide gel to a position corresponding to a length of 91 nt, placing the start point of transcription 87 nt upstream from the ATG codon. This result is in agreement with the observation that most eukaryotic mRNAs start with an A residue. Approximately 30 nt upstream from the start point of transcription, the sequence CATAAAA is closely related to the Goldberg-Hogness consensus box (Breathnach and Chambon, 1981), while two possible CAAT boxes (Benoist et al., 1980) are found beginning at nt positions -75 and -123 (Fig. 1).

Fig. 2. Primer extension analysis of IL-I /I mRNA carried out according to Bensi et al. (1985). The primer used is a synthetic oligodeoxynucleotide complementary to the sequence indicated with a broken line in Fig. 1. Lane 1 shows the electrophoresis on a denaturing 6% polyacrylamide gel of the primer extension product of a control reaction carried out annealing the synthetic oligodeoxynucleotide to total human liver RNA, which does not contain IL-l B mRNA. Under these conditions no cDNA band is detectable. Lane 2 shows the result of the same experiment carried out using total RNA from induced U937 monocyte cell line (Amento et al., 1985). The arrow indicates the size in nt of the cDNA synthesized. The M, marker is a sequencing ladder obtained by sequencing the corresponding genomic DNA fragment, using the same synthetic oligodeoxynucleotide as sequencing primer. IL-l B induction in U937 monocyte cell line was obtained by adding 500 ng/ml each of retinoic acid and TPA to a culture of cells grown to a density of 1 x 106/ml in RPM1 medium in the presence of 10% fetal calf serum. The stimulation was carried out for 8 h.

(b) Comparative analysis of IL-1 genes The comparison of the human and murine IL-l /? genes shows evolutionary conservation of intronexon domains, such that the length of the exons is almost identical in the two species, except for exon 7

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Fig. 3. Structure of the human and murine IL-1 j genes. A map of the human IL-l B sequence shown in Fig. 1 is compared with that of the murine IL-I /I gene sequence published by Telford et al. (1986). Black boxes represent the exons; arrowheads represent the beginning and the end of the ORFs; H, Hind111 sites.

which is 200 bp longer in the human gene at the 3’ untranslated region (Fig. 3). This is very similar to what is found comparing human IL-l a to the murine IL- 1 /I gene, where the number and the length of the exons is also highly conserved, although the sequences have completely diverged. Based on this observation Telford et al. (1986) have proposed the existence of a common ancestral gene for IL- 1 a and /3 and of functional protein domains corresponding to the exon regions. In addition to the similar structural organization, human and murine IL-l /3genes show extensive sequence homology of the exon and intron regions. This is shown by the dot matrix

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Fig. 4. Dot matrix comparison of the human and murine IL-1 j genes. The programs Compare and Dotplot of the University of Wisconsin Genetics Computer Group have been used with window 21 and stringency 15. Dots indicate segments of homology between the two genes. Exons (black boxes) and the Alu sequence (open box) are indicated; arrowheads delimit the ORFs.

sequence comparison of Fig. 4. The diagonals represent the alignment of the two sequences, beginning at the same position of the two 5’ flanking regions. The shift between diagonals is due to the presence of an Ah sequence in the third intron of the human gene and to insertion of about 200 and 400 bp in the first intron and in the 3’ flanking region of the murine gene, respectively. The considerable conservation of the intron sequences is in contrast with what is found for genes better conserved in evolution than the IL-l genes. The large intron of the rabbit figlobin gene compared to that of the homologous mouse gene, shows no sequence homology, suggesting that a divergence of 70 million years is sufficient to randomize completely the nucleotide sequence (Van Ooyen et al., 1979). This observation may imply a role of the intron sequences in the function of the IL-l fi gene. A role of introns in the production of stable RNA has been proposed (Nishioka and Leder, 1979) and sequences regulating gene expression can be located within intron regions (Gillies et al., 1983; Grosschedl and Baltimore, 1985).

NOTE

After this paper was submitted for publication, Clark et al. (1986) published the sequence of a human IL- 1 /Igene. The coding sequences of the two genes are identical. Differences are observed within the 5’ and 3’ nontranslated regions and within the intron sequences, possibly indicating polymorphism. The existence of polymorphism within the IL-1 B gene had already been suggested by the comparison of the cDNA sequence published by Auron et al. (1984) with the one reported by March et al. (1985).

Differences between the two sequences are present both in the coding and nontranslated regions of the two cDNAs.

ACKNOWLEDGEMENTS

We thank G. Ratti and S. Ricci for supplying oligodeoxynucleotides, S. Palei for technical assistance, G. Corsi for photographic work, J. Telford for revising the manuscript and Antonella Mori for secretarial assistance.

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Dinarello, C.A.: Interleukin-1. Rev. Infect. Dis. 6 (1984) 51-95. Furutani, Y., Notake, M., Fukui, T., Ohue, M., Nomura, H., Yamada, M. and Nakamura, S.: Complete nucleotide sequence of the gene for human interleukin 1 a. Nucl. Acids Res. 14 (1986) 3167-4179. Gillies, S.D., Morrison, S.L., Oi, V.T. and Tonegawa, S.: A tissue-specific transcription enhancer element is located in the major intron of a rearranged immunoglobuhn heavy chain gene. Cell 33 (1983) 717-728. Grosschedl, R. and Baltimore, D.: Cell type specificity of immunoglobulin gene expression is regulated by at least three DNA sequence elements. Cell 41 (1985) 885-897. Hoppenheim, J.J., Kovacs, E.J., Matsushima, K. and Durum, S.K.: There is more than one interleukin 1. Immunology Today 7 (1986) 45-55. Kieny, M.P., Lathe, R. and Lecocq, J-P.: New versatile cloning and sequencing vectors based on bacteriophage M13. Gene 26 (1983) 91-99. Lomedico, P.T., Gubler, U., Hellman, C.P., Dukovich, M., Giri, J.G., Pan, Y.E., Collier, K., Semionow, R., Chua, A.O. and Mizel, S.B.: Cloning and expression of murine interleukin-1 cDNA in Escherichiu co/i. Nature 312 (1984) 458-462. March, J.C., Mosley, B., Larsen, A., Cerretti, D.P., Braedt, G., Price, P., Gillis, S., Henney, C.S., Kronheim, S.M., Grabstein, K., Conlon, P.J., Hopp, T.P. and Cosman, D.: Cloning, sequence and expression of two distinct human interleukin-1 complementary DNAs. Nature 315 (1985) 641-647. Nishioka, Y. and Leder, P.: The complete sequence of a chromosomal mouse alpha-globin gene reveals elements conserved throughout vertebrate evolution. Cell 18 (1979) 875-882. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chain terminating inhibitors. Proc. Nat]. Acad. Sci. USA 74 (1977) 5463-5467. Telford, J.L., Macchia, G., Massone, A., Carinci, V., Palla, E. and Melli M.: The murine interleukin 1 B gene: structure and evolution. Nucl. Acids Res. 14 (1986) 9955-9963. Van Ooyen, A., Van den Berg, J., Mantei, N. and Weissmann, C.: Comparison of total sequence of a cloned rabbit p-globin gene and its flanking regions with a homologous mouse sequence. Science 206 (1979) 337-344. Yanisch-Perron, C., Vieira, J. and Messing, J.: Improved Ml3 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33 (1985) 103-I 19. Communicated by H.G. Zachau.