Cloning, sequence and expression of bovine interleukin 1α and interleukin 1β complementary DNAs

0161-5890/88$3.00+ 0.00 Pergamon Press plc

molecular Immunology,Vol. 25, No. 5, PP. 429-437, 1988 Printed in Great Britain.

CLONING, SEQUENCE AND EXPRESSION OF BOVINE INTERLEUKIN 1a AND INTERLEUKIN l/3 COMPLEMENTARY DNAs CHARLES R. MALISZEWSKI,* PAUL E. BAKER, MICHAEL A. SCHOENBORN,BRIAN S. DAVID COSMAN, STEVEN GILLIS and DOUGLAS PAT CERRETTI Immunex Corporation,

DAVIS,

Seattle, WA 98101, U.S.A.

(First received 8 September 1987; accepted in revised form 25 November 1987) Abstract-Interleukin 1 (IL-l) is a cytokine which mediates a variety of immunoregulatory and inflammatory activities. Using human IL-la and IL-la probes, cDNAs for the corresponding bovine genes were isolated from an alveolar macrophage library. The open reading frames of the bovine IL-lcr and IL-ID cDNAs encode proteins of 268 and 266 amino acids, respectively, each with a predicted mol. wt of approx. 31,000. Both forms of bovine IL-I exhibit a high degree of sequence homology with IL-I gene products from other mammalian species. Based upon comparisons with human IL-1 amino acid sequences, the post-translationally processed, mature forms of bovine IL-1 would occur as 17-l 8,000 mol. wt proteins. Sequences encoding mature bovine IL-la and IL-lb were inserted into E. coli expression plasmids and biologically active proteins were synthesized as judged by the ability of the recombinant proteins to induce proliferation of bovine thymocytes. Both IL-la and IL-ID exist as single genomic copies. In addition, bovine IL-IS mRNA is approx. IO-fold more abundant than IL-la: mRNA in stimulated alveolar macrophages.

species for each form of IL-l (Lomedico et al., 1987; Hopp et al., 1986), which suggests that the retention of both molecules may have been evolutionarily advantageous. In light of its apparent roles in wound healing (Garing et al., 1985; Kupper et al., 1986) and adjuvanticity (Staruch and Wood, 1983), IL-l may be a useful immunopharmacological agent not only in humans, but also in economically important species such as cattle. Bovine IL-l activities have been reported previously (Mastro et al., 1986), and we have found that bovine alveolar macrophages release IL-l in response to stimulation with lipopolysaccharide (LPS). Thus, a lgtl0 cDNA library was prepared from mRNA isolated from LPS-stimulated bovine macrophages and screened for IL-l sequences with human probes. Bovine IL-la and IL-l/I cDNAs were isolated and biologically active proteins were synthesized using an E. coli recombinant expression plasmid.

INTRODUmION

The cytokine interleukin-1 (IL-l) displays a remarkable array of immunoregulatory and inflammatory activities in animals (reviewed by Durum et al., 1985; Oppenheim et al., 1986). Molecular biology studies have demonstrated two distinct IL-l genes in both humans and mice (Auron et al., 1984; March et al., 1985; Lomedico et al., 1984; Gray et al., 1986; Furutani et al., 1985). These two genes, designated IL-la and IL-lp (March et al., 1985), encode intracellular precursors of mol. wts of approx. 31,000, which are post-translationally processed to products of mol. wt 17,500, representing the C-terminal portion of the precursor. These are the predominantly secreted forms of IL-l (Auron et al., 1984; March et al., 1984; Lomedico et al., 1984; Gray et al., 1986; Furutani et al., 1985). Both human IL-l& and IL-lb bind to the same receptor with similar affinities (Dower et al., 1986). Despite their similarities in size, function, and binding specificity, IL-lu and IL-lfi exhibit only limited amino acid homology (less than 30%) (March et al., 1985; Gray et al., 1986). However, significant amino acid homologies exist across

MATERIALS

Construction

*Author to whom correspondence should be addressed at: Immunex Corporation, 51 University St., Seattle, WA 98101, U.S.A. Abbreviations: SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; mRNA, messenger RNA; cDNA, complementary DNA; IL- 1, interleukin 1; LPS, lipopolysaccharide; BAM, bovine alveolar macrophages; U, unit; kb, kilobase; b.p., base pair; mol. wt, molecular weight. 429

AND METHODS

and analysis of cDNA

library

Bovine alveolar macrophages (BAM) were cultured in RPM1 1640 plus 10% fetal bovine serum for 16 hr with 20 pg/ml Salmonella typhimurium lipopolysaccharide in order to elicit maximal IL- 1 specific messenger RNA production. Procedures for mRNA purification and cDNA synthesis have been described (March et al., 1985; Cosman et al., 1984). BAM cDNA, representing mRNA from approx. 1.2 x lo8 cells, was modified with EcORI linkers, cloned into

CHARLESR. MALISZEWSKIet

430

IgtlO, packaged in vitro, and used to infect E. coli strain C600 hfl- according to Huynh et al. (1985). (IgtlO, packaging kits, and bacterial hosts purchased from Stratagene). Independent plaques (50,000-100,000) were blotted onto nitrocellulose filters (Schleicher and Schuell, Keene, NH), and hybridized with 32P-labeled human IL- 1 probes (Maniatis et al., 1982). The human IL-la probe corresponded to nucleotides 49-896 and the IL-ID probe corresponded to nucleotides 23-645 of the published sequences (March et aI., 1985). Filters were incubated for 16 hr at 60°C in 6 x SSC (0.9 M NaC1/0.09 M sodium citrate, pH 7) containing 0.1% sarcosyl, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin, 0.5% Nonidet P-40, 0.1% Ficoll, lOO~gg/ml salmon sperm DNA, and probe at 5 x lO’cpm/ml. Filters were washed extensively in 6 x SSC at room temp, then washed in 6 x SSC for 1 hr at 60°C prior to autoradiography. After rescreening positive plaques, cDNA inserts were isolated and subcloned into pGEMBL 18 for DNA sequencing (Hattori and Sakaki, 1986; Sanger et al., 1977). Plasmid pGEMBL 18 is a derivative of pEMBL 18 (Dente et al., 1983), in which the promoters for Sp6 and T7 polymerases flank the multiple cloning site. Sequence analysis was done by programs designed by the University of Wisconsin Genetics Computer Group (Devereux et al., 1984). Expression

and analysis of recombinant

IL - 1

Escherichia coli expression vectors were constructed that were designed to synthesize mature bovine IL-lcr and IL-1B. This was accomplished by inserting a 1070 b.p. Bsm I/Pst I fragment from pBIL-la.7, encoding amino acids 136-268, into pPL3 with the aid of synthetic oligonucleotides encoding amino acids 120-135 (Fig. 1). Similarly, a 540 b.p. NheI/BglII fragment from pBIL- Ip.9, encoding amino acids 136266, was inserted into pPL3 with synthetic oligonucleotides encoding amino acids 114135 (Fig. 2). The resulting recombinant plasmids, pPL3BIL-lo! and pPL3BIL-Ifi, were used to transform E. coli strain GM-l (Coulondre and Miller, 1977) containing plasmid pRK248cIts (ATCC No. 33766) which has a gene encoding a thermolabile repressor of the 1 phage PL promoter (Fig. 3). Plasmid pPL3 is composed of a BglII/KpnI fragment isolated from pN02680 (Gourse et al., 1985), containing the i phage PL promoter; a PvuII/SphI fragment from pGem1 (Promega Biotec) containing the ampicillin resistance gene and origin of replication from pBR322; a SphI/Hind III fragment from pKK223-3 (Pharmacia) containing the rnnB T,T, terminators; and synthetic oligonucleotides with KpnI/HindIII complementary ends containing a ribosome initiation site and multiple cloning sites as follows: 5’-GGGTACCAAGTTCACGTAAAAA GGGTATCGATACTTATGGACTACAAAGATGACGATATCCATGGAATTCGAGCTCGCCCGGGGATCCTCTAGAGTCGACCTGCAGCCCAAGCTTGG-3’.

al.

Expression of bovine IL-la and IL-l/J in E. coli and sample preparation for IL- 1 assays were conducted as previously described (March et al., 1985). Samples were lysed in SDS sample buffer and analyzed by gradient (l&20% acrylamide) SDS-PAGE. Protein bands were visualized by silver staining. IL-1 assay Bovine IL-l activity was monitored by a modification of the thymocyte costimulator assay (Lachman et al., 1977). Purified bovine thymocytes were cultured in the presence of a submitogenic concn of phytohemagglutinin-M (0.3%) and serial 3-fold dilutions of samples to be tested for IL-l activity. After incubation at 37°C for 68 hr, cells were cultured for 4 hr in the presence of tritiated thymidine (20 Ci/mmol; New England Nuclear) and harvested. Incorporation of label was determined by scintillation counting. One unit (U) of IL-l activity was defined as the amount of lymphokine that induced 50% of maximal proliferation in 100 ~1 cultures. For example, if a sample induced 50% of maximal proliferation at a dilution of 1:20, then 1U was said to be contained in one-twentieth of 100 ~1, or 5 ~1, and the sample was said to contain 200 U/ml (Grabstein et al., 1986). RNA

analysis

Poly-A+ mRNA from the Jurkat human T cell line and from LPS-stimulated BAM cells was electrophoresed on 1.1% agarose gels containing 6.7% formaldehyde, blotted onto nylon membranes (Gene Screen Plus, New England Nuclear), and hybridized with “P-labeled RNA probes transcribed with SP6 polymerase. The IL- 1GIRNA probe was derived from the 245 b.p. Hind III fragment of pBIL-la.7 [Fig. l(A)] and the IL-l/l probe was derived from the 250 b.p. Hind III fragment of pBIL-I/?.9 [Fig. 2(A)]. Thus, both probes represented the 5’-most coding regions of their respective cDNAs. Hybridization and washing conditions were as described (Cosman et al., 1984). Genomic DNA

analysis

Genomic DNA was isolated from bovine peripheral blood leukocytes as previously described (Maniatis et al., 1982). DNA (10 fig) was digested with restriction endonucleases, electrophoresed in a 0.7% agarose gel, blotted onto nitrocellulose and hybridized with 32P-labeled bovine IL-l probes. The IL-la probe was obtained by nick translating the 640 b.p. Hind III/BstE II fragment isolated from pBIL-la.7 and the IL-ID probe was prepared from the 454 b.p. Pvu II fragment isolated from pBIL-lB.9. RESULTS

Cloning and sequence of bovine IL - 1a Lipopolysaccharide-stimulated bovine alveolar macrophages produce IL- 1 messenger RNA, as deter-

431

Cloning of bovine IL-1

A.

HindlII I

BsmI

Bst EII

PstI

H

pBIL-1Q .7

160 bp

Fig. 1. Restriction map and nucleotide sequence of bovine IL-la cDNA. (A) Partial restriction map of the cDNA insert in pBIL-la.7. The coding region for the precursor protein is boxed and the coding region for mature IL-la is shaded. (B) Nucleotide sequence and predicted amino acid sequence of bovine IL-la. DNA and protein sequences are numbered beginning with the initiator Met. The amino terminus (Ser 120) of recombinant mature IL-la is marked with a star. A potential N-glycosylation site is marked with a triangle. The 3’ polyadenylation/maturation signal (dashed line), AT-rich regions (boxed), and the ATTTA sequence motif (underline) are indicated.

mined by Northern blot analysis with human IL-la and IL-la RNA probes (data not shown). Accordingly, a bovine alveolar macrophage cDNA library was packaged into the hgtl0 vector system, screened with a ‘*P-labeled human IL-la DNA probe, and positive clones were subcloned into pGEMBL 18. The nucleotide sequence and partial restriction endonuclease map for the cDNA insert in pBIL-la.7 are shown in Fig. 1. The 2.1 kb insert includes a long

stretch of A residues corresponding to the poly(A) tail of mRNA, which is preceded by the polyadenylationjmaturation signal, AATAAA [Fig. l(B), dashed line]. The 3’ region also includes AT-rich sequences containing several copies of the ATTTA sequence motif [Fig. l(B), boxed], features which have been reported to affect the stability of cytokine and proto-oncogene mRNAs (Shaw and Kamen, 1986; Cosman, 1987). An open reading frame, begin-

432

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et al.

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1166 1247 ,326 ,405 14n4 1563 $642 1677

Fig. 2. Restriction map and nucleotide sequence of bovine IL-ID cDNA. (A) Partial restriction map of the cDNA insert in pBIL-lP.9. The coding region for the precursor protein is boxed and the coding region for mature IL-I/I is shaded. (B) Nucleotide sequence and predicted amino acid sequence of bovine IL-IB. DNA and protein sequences are numbered beginning with the initiator Met. The predicted amino terminus (Ala 114) of mature IL-lb is marked with a star. A potential N-glycosylation site is marked with a triangle. The 3’ polyadenylation/maturat~on signal (dashed line), AT-rich regions (boxed) and the ATTTA sequence motif (underline~overline) are indicated.

ning with an initiator Met codon at nucleotide 1 and ending with a termination codon at nucleotide 804, codes for 268 amino acids with a predicted mol. wt

of 30,820. Comparison of the amino acid sequence with that of human IL-la, indicates that the amino terminal amino acid of mature bovine IL-la may be

Cloning

of bovine

IL-l

433

123

BOV IL-IQ

Fig. 3. Structure of E. coli expression plasmids pPL3BIL-1~ and pPL3BIL-l/I. The plasmids contain sequences derived from pBR322 including the origin of replication (ori) and the ampicillin resistance gene (Amp). Regions containing the IP, promoter used to direct transcription of IL-l and the rrnB transcription terminators T, and T, are indicated. Waved lines represent synthetic oligonucleotides used to fuse the coding regions for IL-IX and IL-lb (boxed) to pPL3.

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and

et al., 1985;

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would

a predicted

Cameron

be composed mol.

et al., 1986). of 150 amino

wt of 17,210.

Cloning and sequence of bovine IL-l/I Several positive clones were selected from the bovine alveolar macrophage cDNA library following screening with a 32P-labeled human IL-lb DNA probe. A partial restriction endonuclease map and the DNA sequence of the 1.8 kb pBIL-l/I.9 insert are shown in Fig. 2. The 3’ region of the IL-lp gene includes the polyadenylation/maturation signal [Fig. 2(B), dashed line], a short poly(A) tail, and several AT-rich sections [Fig. 2(B), boxed]. The open reading frame, beginning at nucleotide 1 and ending at nucleotide 798, codes for 266 amino acids with a predicted mol. wt of 30,760. Comparison of bovine and human IL-lb sequences (March et al., 1985; Kronheim et al., 1985) indicates that Ala 114 corresponds to the amino terminal amino acid of the mature protein. Thus, mature bovine IL-lb would consist of 153 amino acids and would have a predicted mol. wt of 17,732.

Fig. 4. Induction of bovine IL-l synthesis in E. coli. Aliquots of E. coli cultures containing the following plasmids were centrifuged and prepared for SDS-PAGE. Lane 1. pPL3BIL-l/3; lane 2, pPL3BIL-la; lane 3, pPL3 lacking IL-l sequences. The positions of protein mol. wt markers are indicated as mol. wts x 10-l. Arrows indicate positions of recombinant bovine IL-la and IL-lb.

were grown at 30°C before heat induction of the P, promoter by shifting the cultures to 42°C for 3 hr. The proteins synthesized by the cultures were analyzed by SDS-PAGE (Fig. 4). Major new protein bands can be seen for both IL-1s~ (Fig. 4, lane 2) and IL-la (Fig. 4, lane 1). The apparent mol. wt of 18,000 for IL-lx is in good agreement with the mol. wt of 17,210 as deduced from the DNA sequence. The apparent mol. wt of IL-lb (19,500) is, however, larger than the predicted mol. wt of 17,732. The above cultures were assayed for IL-l activity as judged by the thymocyte co-mitogenesis assay using bovine thymocytes (Table 1). The results indicate that biologically active bovine IL- la and IL-lb were synthesized by the appropriate bacterial cultures. No IL-l activity was detected in a culture containing the control plasmid. Analysis

of bovine IL -1 mRNA

Poly(A)-RNA rophages was

Table

Expression

of recombinant

bovine IL-lu

I.

and IL-l/I

To determine if the cDNA inserts in pBIL-la.7 and pBIL-lb.9 encode proteins with IL-l activity, DNA fragments encoding bovine IL-la (amino acids 12&268) and IL-l/? (amino acids 114-266) were inserted into an E. coli expression system under transcriptional control of the 1P, promoter. Cells, transformed with plasmids pPL3BIL-lcr and pPL3BIL-l/I (Fig. 3), or the control plasmid pPL3,

Plasmid” pPL3BIL-lor pPL3BIL-Ip pPL3

from LPS-stimulated alveolar machybridized with bovine IL-la and

BOViIK thymocyte costimulator units/ml/O.D. of E. coli” Experiment 8 12,693 98,581 0

I’

Experiment

assay: 2

223,903 69,632 0

“Activity is expressed in U/ml of induced E. co/icultures, normalized to O.D., = I. ‘Detergent lysates of E. cd; harboring the indicated plasmids were prepared and assayed as described in Materials and Methods. CNumbers represent means of duplicate samples.

434

CHARLES R. MALISZEWSKI et al. DISCUSSION

.

-28s .

-18s

Fig. 5. Autoradiograms of Northern blots of IL-l mRNA. Bovine IL-la RNA probe (lanes 1, 2) and bovine IL-Ifi RNA probe (lanes 3, 4) were hybridized to polyadenylated RNA from the Jurkat human T cell line (lanes 1 and 3; negative controls) or LPS-stimulated bovine alveolar macrophages (lanes 2 and 4). The positions of 18s and 28s rRNA bands are indicated.

IL-l/l RNA probes, as shown in Fig. 5. Bovine IL-la and IL-lp appear to be encoded by single mRNA species of approx. 2200 and 2000 nucleotides, respectively. Densitometric scanning of the autoradiograms revealed that IL-lfi is expressed at approx. IO-fold higher levels than IL-la in these stimulated cells. Both the size and abundance of bovine IL-la and IL-ID mRNA correspond to those reported for human (March et al., 1985) and murine (Gray et al., 1986) IL-l mRNAs.

Analysis

We describe the cloning, sequencing, and expression of bovine IL-la and IL-lfi cDNAs. A lgtl0 cDNA library was constructed from LPS-stimulated bovine alveolar macrophage mRNA and screened for IL-l sequences using human cDNA probes. Nucleotide sequence analysis of positive clones and the subsequent production of biologically active cytokines in an E. coli expression system confirmed the identity of the bovine IL-la and IL-lfi genes. The cDNA inserts of plasmids pBIL-la.7 and pBIL-la.9 encode proteins of 268 amino acids (mol. wt 30,820) and 266 amino acids (mol. wt 30,760), respectively. Previous studies have indicated that human (Auron et al., 1985) and murine (Giri et al., 1985) IL-1s are synthesized as precursor proteins with mol. wts of approx. 31,000. Post-translational processing removes approx. I 10 residues from the amino end of each protein, resulting in mature forms of mol. wt 17-18,000. Based upon an alignment of bovine, human and murine IL- 1 sequences, the amino termini of mature bovine IL-l a and IL-lp likely begin at Gin 119 and Ala 114, respectively. Con-

B kb

23.1 9.4 6.6

12

3

kb -23.1

4.4

2.32.0-

of bovine IL-1 genomic DNA

Bovine genomic DNA was digested with restriction endonucleases and hybridized with bovine IL-la and IL-ID DNA probes on Southern blots (Fig. 6). Two IL-la hybridizing bands were observed when genomic DNA was digested with Hind III (5.5, 0.7 kbp), Pst I (4.9, 2.8 kb.p.), and Xba I (7.8, 3.9 kb.p.) [Fig. 6(A)]. Probing with IL-ID resulted in single bands for Hind III (2.1 kb.p.), Pst I (3.0 kb.p.), and Xba I (2.8 kb.p.) [Fig. 6(B)]. The restriction fragment sizes were unique for IL-la and IL-ID, indicating that cross hybridization of probes did not occur. These results suggest that genes for IL-la and IL-lb probably exist as single copies in the bovine genome.

Fig. 6. Autoradiograms of hybridizations with (A) bovine IL-la and (B) bovine IL-l/I cDNA probes to Southern blots of bovine genomic DNA. Genomic DNA (10 pg) was digested with Hind III (lane l), Pst I (lane 2), or Xba I (lane 3), electrophoresed in a 0.7% agarose gel, blotted, and hybridized at high stringency to nick-translated j2P-labeled IL-1 cDNAs. The molecular size markers (in kb.p.) are from Hind III-digested bacteriophage E. DNA.

43.5

Cloning of bovine IL-1

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Fig. 7. Alignment of human, bovine, murine, and rabbit IL-1 amino acid sequences as deduced from their RNA sequences. Numbering is based on relative position in the total alignment starting with initiator Met. Boxed residues indicate homology at 6 or 7 residues. Arrows represent amino termini of mature IL-lu (upper arrow) and mature IL-I@ (lower arrow). Stars identify conserved basic residues at position 133 and triangles identify residue 280, the carboxy-terminal endpoint for active protein (see text).

sequently, we constructed expression piasmids containing nucleotide sequences for mature bovine IL- la and IL-lb, and found that they directed the synthesis of biologically active proteins. Comparison of IL-l amino acid sequences reveals high levels of homology between bovine and other mammalian forms of IL-la and IL-I/?. The bovine IL-la sequence is 73, 62 and 71% homologous with human, murine and rabbit IL-la sequences, respectively. Bovine IL-I/I is 62% homologous with human and 59% homologous with murine IL-IS. Despite the marked cross-species sequence conservation for each form, bovine IL-la and IL-18 share only 23% homology, consistent with a and p com-

parisons for the human (26%) and murine (22%) proteins (March et al., 1985; Gray ef al., 1986). Although the a and j3 amino acid sequences differ considerably, both molecules mediate the same biological functions (Rupp et al., 1986) and bind to the same receptor with equivalent affinities (Dower ez al., 1986), implying that structural similarities exist between IL-la and IL-I/X. In an attempt to identify amino acid residues important for activity and binding, we aligned the sequences for the seven reported mammalian IL-1s (Fig. 7) using an alignment originally proposed by Hopp et al. (1986). There is a total of 50 identities (at least six out of seven matching), representing approx. 19% of all residues. The

436

CHARLES

R. MALISZEWSKI et ~1.

region flanking the proteolytic cleavage sites preceding the amino termini of the mature proteins (Fig. 7, arrows) is conspicuously lacking in sequence homology, although there does appear to be a preponderance of basic residues. Mosley et al. (1987) reported that deletion of the amino-terminal amino acids beyond position 132 for human IL-13 and IL-ID (Fig. 7, star) resulted in a complete loss of biological and receptor binding activity. Interestingly, basic residues are conserved in six of the IL-l proteins at position 133. Deletion analysis at the carboxy-terminus indicated that constructs containing amino acids to position 280 displayed detectable biological and receptor binding activities. In all seven proteins, the tripeptide Thr-AspPhe precedes position 280, suggesting that these amino acids may be required for retention of activity. The conservation of four prolines (positions 189, 210, 220, 251) and four phenylalanines (positions 174, 234, 245, 279) in the mature proteins may contribute to the secondary/tertiary structure of IL- 1. Establishing the importance of these and other conserved residues may be accomplished by mutational analysis of IL-l at specific amino acid positions, followed by determination of binding and biological activities of the protein products. Such studies could elucidate the structural requirements for IL-1 function and lead to the development of IL-l agonists and antagonists. Several possible therapeutic applications exist for IL-l in cattle and other animals, particularly as adjuvants. A large number of cattle vaccines against enteric and respiratory viruses and bacteria are simply ineffective or require multiple immunizations before protective immunity can be demonstrated (Martin, 1983). In most cases, administration with complete Freund’s adjuvant is not feasible, primarily due to the occurrence of false positives in tuberculin testing. Since IL-1 has recently been shown to act as an adjuvant at subpyrogenic doses (Staruch and Wood, 1983) is several orders of magnitude more effective than human IL-l in activation of bovine thymocytes (unpublished observation), and would not be expected to be immunogenic in cattle, it is a logical choice as an adjuvant for a number of lessthan-optimal vaccines. The availability of sizable amounts of recombinant bovine IL-l should enable the development of reasonable treatment protocols. Acknowledgemenfs-The authors thank Steve Gimpel, June Mehl, Kathy Picha and Jeff Hesselberg for technical help; Ray Goodwin, Dirk Anderson and Janis Wignall for advice; Tom Hopp for providing preliminary sequence homologies; and Linda Troup for secretarial assistance. REFERENCES Auron P. E., Webb A. C., Rosenwasser L. J., Mucci S. F., Rich A., Wolff S. M. and Dinarello C. A. (1984) Nucleotide sequence of human monocyte interleukin 1 precursor cDNA. Proc. natn. Acad. Sci. U.S.A. 81, 7907~1911. Cameron P., Limjuco G., Chin J., Silberstein L. and Schmidt J. A. (1986) Purification to homogeneity and

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