Biochimica et Biophysica Acta, 1089(1991)257-258

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© 1991 ElsevierScience PublishersB.V. 0167-4781/91/$03.50 ADONIS 0167478191001570 BBAEXP 90234

Short Sequence-Paper

cDNA cloning of porcine interleukin 2 by polymerase chain reaction Jane C. Goodall 2, David C. Emery I, Michael Bailey 3, Leonard S. Enghsh 2 and Len Hall I Department of Biochemistry, Unit,ersity of Bristol, Bristol (U.K), 2 Department of Science, Bristol Polytechnic, Bristol (U.K) and 3 Department of Veterinary Medicine, Uni~'ersityof Bristol, Bristol fU.K. )

(Received8 March 1991)

Key words: lnterleukin2; Lymphokine;DNA-sequence;Polymerascchain reaction Porcine interleukin 2 (IL-2) cDNA was cloned by polymerase chain reaction (PCR), using primers derived from the corresponding bovine sequtnce. The resulting porcine DNA sequence encodes a 154 residue 11-2 primary translation product. Comparison of the mature, secreted form of porcine 11-2 with those of other species was carried out in an attempt to identify differences that might contribute to the observed differing species speciflcities.

Interleukin 2 (IL-2) is a lymphokine produced by activated T lymphocytes which plays a pivotal role in the growth and regulation of immune cells [1-5]. The cDNAs of human [6,7]' murine [8] and bovine [9,10] 11-2 have been cloned, sequenced and expressed. Work has been carried out which clearly demonstrates the ability of IL-2 from cells of various species, to support the growth of xenogeneic T lymphocytes. Human IL-2 was found to support the growth of human, porcine, murine and ovine cells, whereas porcine 11-2 acted on all but human cells. Ovine 11-2, on the other hand, only supported the growth of ovine [11] and bovine cells [12]. It was therefore of interest to determine the sequences of both ovine and porcine IL-2, to identify differences that may contribute to these differing species specificities. With this in mind, we have recently cloned and sequenced the entire coding region of ovine IL-2 cDNA [13], and now report the cloning and sequencing of porcine IL-2 cDNA tFig. 1). In both cases, total RNA was isolated from concanavalin A

The sequence data in this paper have t:een submitted to the EMBL Data Bank under the accessionnumber X56750. Abbreviations:PCR, polymerasechain reaction; IL-2, interleukin 2. Correspondence:L Hall, Department of Biochemist~,Universityof Bristol Bristol BS8 ILT, U.IL

stimulated lymphocytes and used to direct the synthesis of cDNA with reverse transcriptase. IL-2 cDNA was then specifically amplified by PCR using Du Pont ~el.,'dnase and primers based on the bovine sequence; clcr..e6 :nto pUC13 and subjected to automated DNA sequencing using a Du Pont Genesis 2000 fluorescent sequencer. The DNA sequence was completely determined on both strands with appropriate overlapping sequence runs. In addition, two totally independent cDNA clones from different experiments were sequenced, to ensure that there were no PCR induced mutations. The deduced amino acid sequence of mature porcine IL-2 was found to exhibit 72% sequence identity with human 11-2 and 72% identity with ovine 11-2 (Fig. 1). Examination of the contact regions of 11-2 (previously defined by monoclonal antibodies, deletional analysis and site-directed mutagenesis [1416]), that interact with the p55 11-2 receptor chain (amino acid residues 33-56) and the p75 11-2 receptor chain (residues 11-20), lends some support for the wider species specificity of porcine 11-2 over that of ovine !1-2 (Fig. 2). Presumably, these contact sites in the human sequence allow the binding of human IL-2 to both p55 and p75 receptor chains on the cells of all species tested to date, thereby promoting growth. Since porcine IL-2 shows a higher degree of similarity to human IL-2 than ovine IL-2, in these receptor contact regions, it might therefore be expected to possess a wider species specificity than ovine 11-2, as observed.

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Fig. I. Nucleolide and deduced amino acid sequences o f porcine inlerleukin 2 c D N A . Differences between the porcine (upper row), ovine (middle row) and human (bottom row) IL-2 peptide ~quences are shown.

^: Proposedp75 bindingsite !1 15 20 ImmamIL-2 QLQ . . . . . . . . . . . . . . LEHLLLD I~rciae IL-2 KKQ . . . . . . . . . . . . . . LEPLLLD mm~elL-2 T A E A Q Q Q Q Q Q Q Q Q Q Q Q H L E Q L L MD ovineIL-2 MKE . . . . . . . . . . . . . . VKSLLLD bovinelL-2 MKE . . . . . . . . . . . . . . VKS L L L D B: ProposedI)55 binding~te

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Fig. 2. Comparison of proposed i n ~ u k i n 2 ~ce~or binding sites.

Future ~vork gill involve expressing the cloned ovine and porcine IL-2 cDNAs for affinity labelling studies, to further examine the species specificities of these

products. References I Morgan, D.A., Ruscett, F.W. and Gallo, R.C. (1976) Science 193, 1007-1008. 2 Robb, RJ., Munck, A. and Smith ILA. (1981) J. Exp. Med. 154, 1455-1474. 3 Smith, K.A. (1988) Science 240. 1169-1176.

4 Waldmann, T.A., Goldman, C.K., Robb, R.J., Depper, J.M. and Leonard, W.J. (1984)J. Exp. Med. 160, 1450-1466. 5 Siegel, J.P.. Sharon, M., Smith, P.L and Leonard, W.J. (1987) Science 238, 75-78. 6 Devos, R., Plaetinck, G., Cheroutre, H., Simons` G., Degrave, W., Tavernier, J , Remaut, E. and Fiers, W. (1983) Nucleic Acids Res. 11, 4307-4323. 7 Taniguchi, T., Matsui, H., Fujita, T., Takaoka, C., Kashima, N, Yoshimoto, R. and Hamuro, J. (1983) Nature 303, 305-310. 8 Kashima, N., Nishi-Takaoka, C., Fujita, T., Taki, S., Yamada, G., Hamuro, J. and Taniguchi, T. (1985) Nature 313, 402-405. 9 Cerretti, D.P.. McKeraghan, K., Larson, A., Cosman, D. and Baker, P.E. (1986) Proc. Natl. Acad. Sci. USA 83, 3223-3227. 10 Reeves, R., Spies, A.G., Nissen, M.S., Buck, C.D., Weinherg, A.D.0 Barr, PJ., Magnuson, N.S. and Magnuson, J.A. (1986) Proc. Natl. Acad. Sci. USA 83, 3228-3232. I! English, L.S., Binns` R.M. and Licence, S.T., (1985) Vet. lmmunol, lmmunupathol. 9, 59-69. 12 Denyer, M.S., Martin, S. and Wardley, R.C. (1987) Res. Vet. Sci. 42, 252-254. 13 Goodall, J.C., Emery, D.C., Perry, A.C.F., Eng;i;h, LS. and Hall, L (1990) Nucleic. Acids Res. 18, 5883. 14 Kuo, L and Robb, RJ. (1986) J. lmmunol. 137, 1538-1543. 15 Ju, G., Collins, L, Kaftka, ILL, Tsien, W.-H., Chizzonite, R., Crowl, R., Bhatt, R. and Kilian, P.L (1987) J. Biol. Chem. 262, 5723-5731. 16 Collins, L, Tsien, W.-H., Seals, C., Hakimi, J., Webber, D., Balm, P., Hoskins, J., Greene, W.C., Toome, V. and Ju, G. (1988) Pro(:. Natl. Acad. Sci. USA 85, 7709-7713.

cDNA cloning of porcine interleukin 2 by polymerase chain reaction.

Porcine interleukin 2 (IL-2) cDNA was cloned by polymerase chain reaction (PCR), using primers derived from the corresponding bovine sequence. The res...
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