Cloning of the bovine interleukin-3-encoding cDNA

Gene, 162 (1995) 309-312 ©1995 Elsevier ScienceB.V. All rights reserved. 0378-1119/95/$09.50

309

GENE 09077

Cloning of the bovine interleukin-3-encoding cDNA (Colony-stimulating factor; cytokine; PCR; nucleotide sequence; RACE)

S i m o n M u s y o k a M w a n g i ", L i n d a L o g a n - H e n f r e y a, C o l i n M c l n n e s b a n d B e a M e r t e n s a a International Livestock Research Institute (ILRI), Nairobi, Kenya; and b Moredun Research Institute, Edinburgh, EH17 7JH, UK. Tel. (44-131) 664-3262

Receivedby T. Sekiya:22 December 1994;Revised/Accepted:12 April/16 April 1995;Recievedat publishers: 18 May 1995

SUMMARY Interleukin-3 (IL-3) is one of the cytokines that act during the early and late stages of blood cell formation. To enable the study of the role of IL-3 in bovine haemopoietic stem cell differentiation, the polymerase chain reaction was used to amplify an I L - 3 cDNA from first-strand cDNAs prepared from RNA isolated from 4- and 5-hour concanavalin-Astimulated peripheral blood lymphocytes (PBL) from N'Dama cattle. An analysis of the cDNA sequence reveals that it contains a 432-nucleotide (nt) open reading frame which codes for 144 amino acids (aa). Cleavage of the putative signal peptide consisting of the first 17 aa yields the mature form of the protein (14.5 kDa). Comparisons of the bovine I L - 3 sequence with the sheep, human and mouse I L - 3 sequences show that the bovine sequence shares 90.7, 55.8 and 51.9% nt identity, respectively, in the coding region, and 85.4, 35 and 27.7% aa identity, respectively.

INTRODUCTION Interleukin-3 (IL-3), also known as multi-lineage colony-stimulating factor (multi-CSF), is a cytokine belonging to the family of growth factors referred to as the colony stimulating factors (Clark and Kamen, 1987). IL-3 is expressed by mitogen or antigen-activated T lymphocytes and natural killer cells and by a number of continuous cell lines (Ihle et al., 1982; Niemeyer et al., 1989; Wimperis et al., 1989). IL-3 was originally identified by its ability to induce the synthesis of 20~-steroid dehydrogenase (20~-SDH) in splenic lymphocytes of nude Correspondence to: Dr. B. Mertens, ILRI, P.O. Box 30709, Nairobi, Kenya. Tel. (254-2) 630-743; Fax (254-2) 631-499; e-mail: [email protected]

Abbreviations: aa, amino acid(s); Bo, bovine;bp, base pair(s); cDNA, DNA complementary to RNA; Con, concanavalin(s); CSF, colonystimulating factor(s); dNTP, deoxyribonucleosidetriphosphate; Hu, human; IL-3, interleukin-3;IL-3, gene (DNA) encodingIL-3;kb, kilobase(s) or 1000bp: Mu, murine; nt, nucleotide(s);ORF, open reading frame;Ov, ovine;PBL, peripheral blood lymphocyte(s);PCR, polymerase chain reaction; RACE,random amplificationof cDNA ends; UTR, untranslated region(s). SSDI

0378-1119(95)00359-2

mice (Ihle et al., 1981). Subsequent work has revealed that IL-3 has a very broad spectrum of activities as a haemopoietic CSF. In vitro, IL-3 supports the growth and differentiation of early haemopoietic progenitors and acts synergistically with more restricted cytokines to promote erythroid, myeloid, mixed and megakaryocytic colonies (Migliaccio et al., 1988; Schrader, 1992; Mclnnes et al., 1993). The genes coding for mouse, rat, human, gibbon, rhesus monkey and sheep IL-3 have been cloned and the recombinant proteins expressed (Fung et al., 1984; Cohen et al., 1986; Yang et al., 1986; Dorssers et al., 1987; Burger et al., 1990; McInnes et al., 1993). Comparisons of these genes have revealed a more rapid evolutionary divergence, and hence a more pronounced species specificity, than has been observed for other haemopoietic growth factors (Cohen et al., 1986; Burger et al., 1990). In view of this, to facilitate studies on the role of IL-3 in bovine haemopoietic stem cell differentiation and the aetiology of the anaemia associated with bovine trypanosomiases in cattle, we have isolated and sequenced a cDNA which contains the full coding region of bovine I L - 3 .

310 M S S L S I L H L L L L L ~TCATCCTGCCAGCCCAAACATGAGCAGTCTCTCCATCTTGCACCTCCTTCTGCTCCTG

13 60

EXPERIMENTAL AND DISCUSSION

L A L H A P Q A K G L P V V T S R T P Y CTTGCACTCCATGCTCCTCAGGCAAAGGGGTTGCCTGTCGTGACATCGAGGACTCCATAT

33 120

S M L M K E I M D D L K K I T P S P E G TCTATGTTGATG~GGA~TTATGGATGACCTAAAGAAAAT~CTCCGAGTCCAG~GGC

53 180

S L N S D E K N F L T K E S L L Q A N L TCCTTG~CTCAGATGAG~G~TTTCCTGACGAAAGAGAGCCTTCTGCAGGCAAACCTG

73 240

K V F M T F A T D T F G S D S K I M K N AAAGTATTCATGACATTTGCCACAGATACTTTTGG~GCCATTCAAAAATCATGAAAAAT

93 300

L K E F Q P V L P T A T P T E D P I F I I I 3 CTT~GG~TTCCAGCCAGTGCTGCCCACAGCCACACCCACGG~GATCC~TTTTTATT

360

E N K N L G D F R M K L E E Y L V I I R GAG~C~G~CTTGGGCGATTTCCGGATGAAATTGG~G~TATTTGGTTATCATTAGA

133 420

N Y L K S K N I W F S ~CTATCTG~GTCC~G~CATATGGTTTTCCT~CATCTG~GTCTG~GCCCAGTTC

144 480

approx. 550 b p which was the expected size. This p r o d u c t

CCTCTCTG~GCCTCAG~TATCAGTGAC~CAGACTGTCCTAAACTTCTTCGATGTTTC

540

was purified, cloned into the p G E M - T vector (Promega,

TCACATGGTCCAGGCCTGG~G~TT~TTTTCTCCTGTGGAGTCAGATG~TCGTTGAT

600

M a d i s o n , WI, USA) a n d sequenced in b o t h directions to

TA~T~TTCCTGATATGTGTGACCCT~TTTGTCCTTTTGGGATTATGTTTCTCATTTT

660

determine its identity. To extend analysis into the 3' U T R , total R N A prepared from 4- a n d 5-h C o n A - s t i m u l a t e d

(a) Cloning of the bovine IL-3 eDNA F i r s t - s t r a n d bovine l y m p h o c y t e c D N A s prepared from R N A isolated from 4- a n d 5-h C o n A - s t i m u l a t e d P B L from N ' D a m a cattle (Bos taurus) were subjected to 35 cycles of P C R using 5' a n d 3' primers based o n the 5' a n d 3'-UTR, respectively, of the cloned sheep IL-3 e D N A sequence ( M c l n n e s et al., 1994). Agarose-gel electrophoresis of the P C R reactions revealed a single b a n d of

T~TCTATTGAGACTTATTTATTTATGTATGTA~TA~ATTACCTTGTGCAAATGTGA

720

AGGGTATTTATTTTAGCAGAGGAGTCATGTTCTGCTCCTTCTGGACAAAACTT~GATGG

780

P B L from N ' D a m a cattle was reverse transcribed as

GGACTTGGG~TATGTTCATTTGTACTTCAGGTTTGAAACTGAGGTACACATCTGTGAAA

840

described in the R A C E protocol for amplifying e D N A 3'

A T A A A C T A C A C T C T C T G ~

875

Fig. 1. The nt sequence of the bovine IL-3 cDNA and the deduced aa sequence. The asterisk (*) indicates a possible site of cleavage for removal of the signal peptide. 'ATTTA'repeat units are in bold and are underlined. Methods: Blood was collected in Alsever's solution from N'Dama cattle and PBL isolated by Ficoll-Paque density centrifugation (Pharmacia LKB Biotechnology,Uppsala, Sweden). The isolated PBL were resuspended to a concentration of 1 × 1 0 6 cells/ml in RPMI 1640 supplemented with 25 mM HEPES/2mM L-glutamine/1.5~tg streptomycin per ml/2 units penicillin per ml/10% heat-inactivated fetal calf serum (Hyclone Laboratories, Logan, UT, USA)/50 pM [3-mercaptoethanol/10 ~tg ConA per ml. The cells were then placed in 25-cm2 (50 ml) tissue culture flasks (Costar, Cambridge, MA, USA) and incubated for 4 and 5 h at 37°C in a humidified atmosphere of 7% CO2 in air. After washing twice with cold phosphate buffered saline, total cellular RNA was isolated using a guanidinium thiocyanate/phenol extraction method (WenQin and Rothblum, 1991) and 1 pg of the RNA used to prepare first-strand eDNA by use of the Reverse Transcription System kit in accordance with the manufacturer's instructions (Promega). For the PCR amplification of the first 512 nt of this eDNA, an upstream primer (5'-CGAAGGACCTGGACAAGACAG) corresponding to nt 1-21 and a downstream oligodeoxynucleotide primer (5'-GTTGTCACTGATGATCTGAGT) corresponding to nt 547 566 of the ovine IL-3 eDNA sequence (McInnes et al., 1994) were used. The remaining 363 bp were generated essentially as described in the RACE protocol for amplifying eDNA 3' ends (Frohman, 1990). The adapter-oligo(dT)17 primer and the adapter primer are the same as those described by Frohman (1990). The 5' gene-specific primer (5'-CATCGAGGACTCCATATTCT)corresponds to nt 104-123 of the bovine IL-3 eDNA sequence. All the reagents used in the PCRs, with the exception of the deoxyribonucleoside triphosphates (dNTPs) (Amersham, UK), were purchased from Promega. The PCR reactions were set up comprising of the 1 × Taq DNA polymerase buffer (Promega)/2.5 mM MgC12/200pM each of the four dNTPs/1.5 ~LM each of the primers/2.5 units Taq DNA polymerase/5 ktl single-stranded eDNA template in a total volume of 100 ~tl.These were subjected to 35 cycles of PCR each consisting of 1 min at 94°C, 2 min at 55°C and 3 min at 72°C. A final 5-min extension at 72°C was performed before cooling to 4°C. To analyse the products from each PCR, 10-~tlaliquots were taken from each reaction, separated electrophoretically on a 1.4% agarose gel stained with ethidium bromide (1 ~tg/ml), and viewed by UV translumination. The sizes of the products were estimated by comparison with the DNA Ladder (50-1000 bp) molecular weight marker (Amersham). The DNA fragments of interest were purified using the

ends ( F r o h m a n , 1990). The resulting first-strand e D N A was amplified by P C R using a 5' bovine IL-3-specific primer a n d an a d a p t e r primer (legend to Fig. 1). Agarosegel electrophoresis of the reactions revealed several products which were transferred o n t o a n y l o n m e m b r a n e by S o u t h e r n transfer a n d p r o b e d with a n i n t e r n a l bovine IL-3 e D N A labelled with digoxigenin (BoehringerM a n n h e i m , M a n n h e i m , G e r m a n y ) . The largest fragment recognized by the probe was gel purified, cloned into the p G E M - T vector (Promega) a n d sequenced. F r o m these two steps, a n 875-bp e D N A which c o n t a i n s portions of the p o l y a d e n y l a t i o n signal was generated (Fig. 1). The nt sequence of this e D N A c o n t a i n s a single 432-nt O R F coding for 144 aa. The 3' U T R u p s t r e a m from the p o l y a d e n y l a t i o n site c o n t a i n s six 'ATTTA' repeat units as reported in the h u m a n IL-3 e D N A sequence (Dorssers et al., 1987). These sequence motifs mediate selective m R N A d e g r a d a t i o n (Shaw a n d K a m e n , 1986; K r u y s et al., 1989). A c o m p u t e r search for identity with k n o w n

IL-3 sequences in G e n B a n k shows that the c D N A s coding region shares 90.7, 55.8 a n d 51.9% n t identity, respectively, with the ovine, h u m a n a n d m u r i n e IL-3 e D N A sequences, while the deduced aa sequence shares 85.4, 35 a n d 27.7% identity, respectively, with the ovine, h u m a n a n d m u r i n e IL-3 aa sequences. The low identity, especially at the aa level, is n o t surprising since it has

Magic PCR Preps DNA Purification System kit (Promega) and cloned into the pGEM-T vector (Promega). The inserts were sequenced in both directions using pUC/M13 universal primers and [~-S35]dATP (Amersham) according to the fmol DNA Sequencing System, direct incorporation protocol (Promega). To ensure that homogeneous transcripts were sequenced, six colonies were sequenced in each case. The nt sequence has been submitted to the Genome Sequence Data Base (GSDB) and the EMBL/GenBank DataBases under accession No. L31893.

311 p r e v i o u s l y been shown t h a t there is low inter-species identity for IL-3, even between very closely related species such as m o u s e a n d rat ( 7 6 % at the nt level, 59% at the a a level) ( C o h e n et al., 1986).

(b) Cross-species comparison of I L - 3 The d e d u c e d bovine IL-3 a a sequence of 144 a a is 2, 8 a n d 22 a a shorter, respectively, t h a n the d e d u c e d ovine, h u m a n a n d m u r i n e IL-3 sequences. T h e first 17 a a of this sequence are highly h y d r o p h o b i c which is characteristic of a p r o t e i n signal peptide. Residues 18-19 consist of AlaP r o which are found at the N - t e r m i n i of several cytokines including gibbon, h u m a n , ovine a n d rhesus m o n k e y IL-3 (Yang et al., 1988; Burger et al., 1990; M c l n n e s et al., 1994). We, therefore, predict t h a t A l a is is the N t e r m i n u s of the m a t u r e protein, a n d t h a t the m a t u r e p r o t e i n w o u l d c o m p r i s e 127 a a (14.5 kDa). A c o m p u t e r - a s s i s t e d a l i g n m e n t of the bovine, ovine, h u m a n a n d m u r i n e IL-3 a a sequences (Fig. 2) reveals that like the ovine sequence, the bovine sequence lacks the Cys residues f o u n d in the h u m a n a n d m u r i n e proteins. A c o m p u t e r - a s s i s t e d s e c o n d a r y structure p r e d i c t i o n ( C h o u a n d F a s m a n , 1978), however, shows t h a t the bovine p r o tein will fold into four s-helices similar to those r e p o r t e d for the h u m a n a n d ovine p r o t e i n s ( M c l n n e s et al., 1994). T h e bovine sequence also lacks the N - l i n k e d glycosylation sites f o u n d in the o t h e r three proteins. H o w e v e r , previous studies have shown t h a t the level of glycosylation does n o t influence the biological activity of the p r o tein (Burger et al., 1990). M e a n w h i l e , some of the residues r e p o r t e d to p l a y a critical role in m o d u l a t i n g the biological activity of the h u m a n p r o t e i n such as P r o 33, Lys 11° a n d Leu 111 ( L o k k e r et al., 1991a,b) are conserved in the b o v i n e protein.

Bo

M

O~ Hu Mu

SSLSILHLLLLLL ALHAPQAKGLPVVTSRT- PYSMLMK M- SSLSILHLLLLLL SLHAPQAQGLPLRTPRT PYSSLME M-SRLPVLLLLQLLVRPGLQAPMTQTTPLKTSWV-NCSNMID MVLASSTTSIHTMLLLLLMLFHLGLQASISGRDTHRLTRTLNCSSIVK

Be Ov Hu Mu

PEGSLNSDEKNFLTKESLLQANLKVFHTFATDTFGSDSKIHKN LKEFQPVLP TA PE- - - G S L N S D E K N I L A N K S L L Q A N L K A F M T F A T D T F G S D S K I M K N - L K E F Q P V L P _ _TA PLLDFNNLNGEDQDI LMENNLRRPNLEAFNR-AVKSLQNASAIES I LKNLLPCLPLATA PELK -T D D E G P S L R N K S F R R V N L S K F V E S Q G E V D P E D R Y V I K S N L Q K L N C C L pT S A N

104 104 109 112

Be Ov Hu Mu

TPTEDPIFIENKNLGDFRM TPTEDSILIEDSNLGDFRM APTRHPIHIKDGDWNEFRR DSALPGVFIRD- LDDFRK

144 146 152 166

]Z ] IMDDLKKIT ]p ] S ]I~II M D D L K K I T ]P ]S ]E ]I I T H L K Q - P ]PI h ~ J I I G K L --- p ! E

I~IEEYLVIIRNY-LKSK - NIWFS .......... ~I~ E E Y L A T I R G Y LRHDLA AAETI ...... ~Ja T F Y L K T L E N A - - - Q A Q Q T T L S L A I F - RFYMVHLNDLETVLT SRPPQPASGSVS PNRGTVEC

50 5O 51 56

Fig. 2. Alignment of the bovine (Bo), ovine (Ov), human (Hu) and murine (Mu) IL-3 aa sequences. The sequences are shown in the singleletter code, and are aligned to maximize homology. To improve alignment where an aa is missing it is denoted by a dash (-). Asterisks indicate residues conserved in all four species while dots indicate conservative aa changes. Those aa reported to play a critical role in regulating the activity of the human protein, together with the overlapping bovine, ovine and murine residues are shown in bold and are enclosed in boxes.

(c) Conclusions F r o m the a b o v e m e n t i o n e d o b s e r v a t i o n s we c o n c l u d e t h a t the c D N A isolated in this s t u d y encodes b o v i n e IL-3. The i s o l a t i o n of this c D N A opens up a n u m b e r of new m o l e c u l a r a p p r o a c h e s to study the role of the g r o w t h factor in bovine haemopoiesis. T h e large-scale p r o d u c t i o n of r e c o m b i n a n t b o v i n e IL-3 a n d the d e v e l o p m e n t of specific a n t i b o d i e s will enable us to s t u d y its role in the a n a e m i a a s s o c i a t e d with African t r y p a n o s o m i a s e s in cattle ( A n d r i a n a r i v o et al., 1994).

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