Cloning and expression of the chicken c-kit proto-oncogene

Gene, 128 (1993) 257-261 0 1993 Elsevier Science Publishers

GENE

B.V. All rights reserved.

257

0378-l 119/93/$06.00

07134

Cloning and expression of the chicken c-kit proto-oncogene (Tyrosine kinase receptor; complementary

DNA; avian brain; homology to murine Kit)

Erika Sasaki”, Hiroshi Okamura Naito” and Michiharu Sakuraib

Tateki Chikamune”, Yukio Kanai”, Miho Watanabec, Mitsuru

b* ,

“Institute ofAgriculture and Forestry, University of Tsukuba, Tsukuba, lbaraki 305, Japan. Tel. (SI-298/53-6687; bNational Institute of Animal Health, Tsukuba, Ibaraki 305, Japan; and ‘National Institute of Animal Industry. Tsukuba, Ibaraki 305, Japan. Tel. (81-298)38-8622 Received by T. Sekiya: 11 November

1992; Revised/Accepted:

21 December/S

January

1993; Received at publishers:

1 March 1993

SUMMARY

The Kit protein is a cell-surface tyrosine kinase receptor encoded by the c-kit proto-oncogene. cDNA clones encoding chicken Kit were isolated from a chicken brain cDNA library, and the nucleotide (nt) sequence of a cDNA clone containing the entire protein-coding region was determined. The deduced amino acid (aa) sequence of chicken Kit shows 63% identity to mouse Kit, and suggests that chicken Kit shares common structural and functional features with mouse Kit. RNA blot analysis indicated that the expression pattern of the chicken c-kit transcript in chicken organs was similar to that of mouse c-kit in mice, suggesting that chicken Kit has biological roles analogous to those of mouse Kit.

INTRODUCTION

The proto-oncogene c-kit encodes a transmembrane tyrosine kinase (TyK) receptor, the Kit protein, composed of three segments: extracellular ligand-binding, transmembrane, and intracellular segments (Qiu et al., 1988; Yarden et al., 1987). Its TyK domain located in the intracellular segment is split into two domains by a hydrophilic insert sequence. A growth factor, SCF/KL/MGF, is a ligand for the Kit protein (Witte, 1990). The demonstration that mouse c-kit and the mouse dominant white spotting (W) locus are allelic (Chabot et al., 1988; Geissler Correspondence

to:

Dr.

M.

Sakurai,

Laboratory

of

Molecular

Immunology, National Institute of Animal Health, 3-l-l Kannondai, Tsukuba, Ibaraki 305, Japan. Tel. (81-298)38-7790; Fax (81-298)38-7880. *Present address: The Chemo-Sero-Therapeutic Research Institute, 668 Okubo

Shimizu,

Abbreviations:

Kumamoto aa,

amino

860, Japan.

Tel. (81-96)344-1211.

acid(s); bp,

base

pair(s);

cDNA,

DNA

complementary to RNA; dNTP, deoxyribonucleoside triphosphate; kb, kilobase or 1000 bp; Kit, cell-membrane TyK receptor; c-kit, gene encoding Kit; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCl/O.OIS TyK, tyrosine kinase; Xaa, any aa.

et al., 1988) together with studies on the W mutants (Geissler et al., 1981; Russell, 1979) have revealed that mouse Kit plays essential roles in various aspects of hematopoiesis and proliferation and/or migration of primordial germ cells and melanoblasts during embryogenesis. In addition, studies employing a monoclonal antibody to mouse Kit showed that this protein is also functionally required for spermatogenesis and melanocyte activation at later stages of development (Nishikawa et al., 1991; Yoshinaga et al., 1991). In order to obtain insights into the possible roles of c-kit-encoded protein in chickens, we cloned chicken c-kit cDNA and examined the expression of the RNA transcript in chicken organs.

M Nz citrate

pH 7.6;

EXPERIMENTAL

AND DISCUSSION

(a) Cloning of the chicken c-M cDNA Two oligo primers were designed from the mouse c-kit cDNA sequence (Qiu et al., 1988) to amplify an internal portion of putative chicken c-kit cDNA by PCR. A cDNA pool synthesized using poly(A)+RNA from a chicken (White Leghorn) brain was then utilized as a template in

258 PCR with the two primers as described in the legend to Fig. I. The nt sequence of the PCR amplified fragment showed an extensive homology to the corresponding part of the mouse cDNA, suggesting that this fragment (CCKIT probe) was indeed a part of the chicken cDNA. A chicken brain cDNA library was constructed and screened with CCKIT probe. 23 hybridizing clones were obtained and cDNA inserts of the clones were subcloned into the pBluescript plasmid vector. Physical mapping of the inserts (2.4 kb to 5.2 kb) by employing EcoRI and Hind111 (data not shown) suggested that all these cDNA inserts originated from one mRNA species, although the possibility of the presence of small deletions, insertions, and/or nt changes was not excluded. The nt sequence of the longest cDNA insert (clone 3) was determined. The sequencing strategy is shown in Fig. I.

Ikb IC 3’

5’ -,44-_,-*~-~4--+ +--,-4-e-+ -CC

c

C-C

ccc

*tCC--t*

+---i--w-CtC

CCKIT

EcoRl EcoRl Fig. I. Schematic

EcoRl I

Hindlll I

II

I

diagram

of a chicken

I

c-kit cDNA clone. The top line

represents the cDNA insert in clone 3 (see section a). The ORF for the chicken Kit protein is depicted by a box, in which, the regions encoding the putative

extracellular

(EC), transmembrane

(TM, closed box), and

intracellular (IC) segments of the protein are indicated. The regions corresponding to the TyK domain of the protein are represented by hatched

boxes.

arrows.

(b) Analysis of the nt and aa sequences In the complete nt sequence of the cDNA (Fig. 2), an ORF extends from an ATG codon at nt 2-4 to a TGA codon at nt 2882-2884. The 3’ untranslated region from nt 2885 contains two putative polyadenylation signals AATAAA at nt 494994954 and 5024-5029, and is followed by a poly(A) tail. The ATG codon at nt 2-4 appears to be the start codon for the putative chicken Kit protein because the stretch of 24 aa encoded by the nt starting at the ATG codon shows a distinct identity (8/24 identical residues) to the N-terminal aa sequence of the mouse Kit protein (Qiu et al., 1988). The N-terminal 21-aa sequence of the mouse protein has been suggested to constitute a signal peptide essential for the processing of membrane glycoproteins (Qiu et al., 1988). The 24-aa sequence of the chicken protein has all the features characteristic of a signal peptide. Indeed, calculation according to von Heijne (1986) predicts the cleavage site for the signal peptidase to be Gly**-Ser 25. The fact that the ATG codon is preceded only by one deoxycytidine residue in the cDNA suggests that the 5’ end of the mRNA is not included in this cDNA, although no other 5’ sequence was found in any other clones obtained in this study. The aa sequence of the protein deduced from the ORF (Fig. 2) consists of 960 aa, and shows an extensive identity

TM

EC

The strategy

The position

On the bottom

is shown

above

below

by

probe (see section a) is also shown.

line, the sites for some restriction

The scale (I kb) is indicated obtaining primer:

of the sequencing

of the CCKIT

enzymes

are depicted.

the top line. Methods:

(a) PCR for

CCKIT probe: The nt sequences of two PCR primers 5’-TCAAATGAATTCATGGACATGAA-3’; R-primer;

(L5’-

ATCCGGGATCCTTCCTT?%TCAT-3’) are identical to the mouse -cDNA sequence nt 217442196 (sense) and nt 2688-2666 (antisense) (Qiu et al., 1988), except for nt changes (underlined) introduced for generating an EcoRI site in L-primer and a BamHI site in R-primer. Total RNA was extracted thiocyanate

from method

a 12-week

old chicken

(Sambrook

et al., 1989). and then poly(A)‘RNA

was prepared using Oligotex-dT30 scription was primed by R-primer then used as a template buffer

(Perkin-Elmer

units of Taq DNA polymerase

by the guanidium

(Takara, Japan). The reverse tranto make a cDNA pool, which was

in a 50-cycle Cetus)/0.2

brain

PCR [I x Taq DNA polymerase

mM

dNTPs/O.Z

(Perkin-Elmer

uM

Cetus)].

each

primer/5

One cycle was

1min at 94’C, 2 min at 47°C and 3 min at 72°C. DNA fragment amplified in the PCR was cloned

in the pUCl8

vector. (b) Construction

and

screening of a chicken cDNA library: A cDNA library was constructed in hZAPI1 vector (Stratagene, La Jolla, CA, USA) using sizefractionated

(>2.5 kb) poly(A)‘RNA

as a template and a cDNA synthesis nants were screened under stringent

from a IO-day old chicken

brain

kit (Pharmacia). I.2 x IOh recombiconditions (Sambrook et al., 1989)

using the “P-labeled CCKIT probe. cDNA inserts in positive clones were rescued by subcloning into the Bluescript vector using the helper phage

R408 (Stratagene).

(63%) to that of mouse Kit (Qiu et al., 1988). Based on comparison to the mouse aa sequence, the putative chicken Kit protein is predicted to contain an extracellular segment (aa l-505), a transmembrane segment

Fig. 2. The nt sequence of the chicken c-kit cDNA and deduced aa sequence. Double-stranded plasmid DNA containing the chicken c-kit cDNA was alkali denatured and sequenced by the dideoxy method (Sanger et al., 1977) using [a-32P]dCTP and Sequenase Ver. 2.0 (US Biochemical, Cleveland, OH, USA). Synthetic oligos generated to a newly derived sequence were used to prime overlapping sequences. The sequence of both DNA strands was confirmed. The nt sequence of CCKIT probe (see section a) was identical to nt 2131-2559 of this sequence. The deduced aa sequence starting from the putative initiation codon at nt 2-4 is indicated above the corresponding codons. The nt and aa residues are numbered at the end of every lane. Stop codon is marked with an asterisk. The predicted cleavage site for the signal peptidase is indicated by an arrow. Potential N-linked glycosylation sites are overlined; Cys residues in the extracellular segment are encircled, and the transmembrane segment is indicated by a bold horizontal line under the nt sequence. The aa conserved among the catalytic domains of protein kinases are indicated by double dashes (=). and Tyr”’ and Tyr9” implicated in the regulation of the kinase activity by symbols # (see section b). Putative polyadenylation signals are underlined. Sequence data reported here will appear in the DDBJ, EMBL and GenBank Nucleotide Sequence Databases with the accession number Dl3225.

259 (aa 506-5~) and an intracellular segment (aa 531-960) (see Fig. 1). Moreover, in the intracellular segment, a putative protein TyK domain (aa 563-671 and 8

E L A H 1 1 w E L A CATGGAGCCCGCCLACCTCCCCiCCClGCTGGCCCICGECGTC~TG~TG~lGAGCCTC~T

H

A

VLLLSLI

P A 1; C I S V P H E E 5 CCCCCCALCTCCTTCAGTCCCTCATCAAG~ATCCTCCCTCGTT~TGAAC&AACGG~ACGA

S

t

V

L R L k iG N E E C P V CTTAACCCTGAACTCCAATCAGLAACCACCCLTGICTTCCAATTT~~4~AACT~ACATCC

1

W

NFUhSOP

20 60

V

NLLGACT ICCPTLV GAATCTACTlCCACCTTCCACTIriCGAC~~CCC&CCCTGClC&TTACAGA&T&TT~CTG

ITEYCC

660 1980

40 120

VCDLLNFLRRKRDSF CTATGCCCATC:CTTAAATTTCCTLA~GCGGAACCGAGATTCATT~ATTTGTCCAAAGCA

60 180

E E H A E A A V V E N TCAACACCACCCACAACCACCTCTTTTATCAGAA~CTTTTCCACCAGGCAGAGCCCACAGC

80 240

DAVNEYMDNKPCVSYAVPPK CCATCCTCTCAATCAGTACATCCACATCAAACCACCACTCTCATATCCTGTTCCCCCAAA

720 2160

100 300

A DXXRPVYSCSYTDPDYTLS AGCTCATAAAAAAAGGCCGETC&AATCTG~ATCTTA~&CCGATCACG&lGTTACCCTTTC

740 2220

Y KDPNYLfLVDSLlYGKEOS TCTTAAAGATCCAAATETCCT~TTCCTTCTTGATTClCTG&TCTATGCGAAACAA~AC&C

I20 360

NLEDDELALOVEDLLSFSVP CATGTTCGAACATEACCAACTTGCTCTACATCTTGAACATCT&TTAACCTTCTCTT&~CA

760 2280

0 I L 1 v ‘3 P LTDPDVLNFTLRK TCACATCCTCCTCCTCTCCCCACTAACACATCCACATCTTTTTAAACTTCACCCTGAGAAA

140 420

V A W G W S f L A S KNCIHROLAA GCTCCCAAACCCCATGAGCTTCCTCCCCTCTAAAAACTCCATTCATAGGLATCTG~CACC

S A K T RISNEKEWHT ATCCCCAAAAACAACC&TTTCCAATGAGAhCGAATGGCACACCAA&AATCCAACAATCAC D 1 G R Y E (@;I X S K C AC&CATACCCAGAT&TCAETCCAAAAGCA&A~CCACTATT~TCA&CT~TTTCT&TGTTTT

NKCEE

aa 748-910) intervened by a hydrophilic insertion sequence (aa 672-747) is assigned. Features of this sequence quite similar to that of the mouse Kit protein,

KNATIR

S

/

V

N

S

f

Y

V

f

L

L

H

Q

160 480

C

P

K

H

680 2040

A

E

P

T

A

700 2100

790 2340 T

~~DCKPLPKNMTFIPNPUKCI A’~CCCATCCCAAACCTCTCCCCAAAAACATGACATTCATTCCCAATCCACACAACCCTAT

I

:

:

L L T H C R I TKICOFCLAR R N I AACAAATATTCTTCTCACTCATGCTCCAATAACAAAAATCTCTCACTTTGCTCTGCCAAC

BOO 2400

1

I I K N V 0 R S f K C FATTATAAAGA&CCTACACACCTCATTCAACGCCTC~IAC~ACTGTTTCGCAAACCATAA

6

Y

U

‘;;J

L

C V E K I S E P I FLNVRPV TCE&CTTCAGA&AATATCAGACCACAlTTTCCTCA&T~TCACACCA~TTCACAAA&CTCT

A

K

H

N

160 540

DIRNDSNYVVKCNARLPVKW ACATATAACGAATCACTCAAATTACCTGCTTAAAGCAAAT~CTCGTCTC~CTGTGAAGTG

H

K

A

L

200 600

M A FNCVYT P E s I CATGCCALCICAAAGCATTIT~AA~l~CGTTTA~A~~TTC~A~A~TGATCTCTGGTCTT&

220 660

G I LLWELFSLGSSPYPGKPV TCCAATATTECTTTCCGAACTCTT~TCCTT&CGAACCACCCCTTACCCACC~&TCCCCCT

860 2580

240 720

KECYRMFSPECS DSXFYKMI CCACTCCAACTTCTATAAAATGATCAACCACCCATATCC~ATCTTCAGCCCCCAGTGCTC

880 2640

260 760

P P E N V D I NKSCWDAOPLQRP ACCCCC~CAAATCTAOCACATAATGAA~ACTT~~TGGCATGCCGATCCCTTGCAGA~A~C

900 2700

280 840

T F f U 1 VULIEQPLSDNAPRV CACATTLAAACACATCCiCCA~CTGATACAACA~CA~CTTTCC~ATA&T~CCCCC~GGGT

920 2760

P V I TLSKSYELLKECEEFEV TCCAGTCATTACCTT&TCCAAAACCTATGAGCTTCTCAAAG&AGGGGAAGAATTTCAAGT T@l ITDVDSSVKASWI TACATCCATAAfCACCC&TCTGCATAGCAGCCTAAAAGCTAGTTGGATTTCTTACAAAAC

S

Y

K

S

A I V T SKSRNLCDYGYERKLT TCCGATTCTTACAAiCAAAAGCAGAAATTTCCCTCITTACCGATACCAAACCAAATTAAC L N IRSVCV NDSCEF ATTCA&CATCCCTTCACTCEG~GTTA&T~ATTCTGCAGAATTCACA7GCCAAGCA~ACAA

T

:T,

R

A

E

N

620 2460 F

E

S

D

V

W

S

Y

840 2520

x PFGXTNATVTLKALAKCFVR fCCTTTCGGAAA&ACCAATCCCICCCTAA~~JTGAAAGCACTAC~TAAA~C&TTTGTCCC NNTTIDINA L F A T TTTCTTTOCAACAATCAATACClOllTTClCdTIlATAAATCCACCACAAAAT~GAAATTTAAC

c

u

N

6

N

L

T

V PKPkEEVWMVMNET E V E A V ACTTCAATATGACCOCTATCCAAAACCAAACCAA~AAGT~TGGATGTACATCAACCAAAC

300 900

YALFSTPYSIULNATDHSVR GTATGCAAACTTTTCCACiCCCC~?TCllGGCIliCCTACACATCAllCCGTGA~

320 960

INSVCSSA S S T CATTAACTCACTCCGTACCAGCGCTTCATCTACTCACCCC~TCCTGCTACGCG&AGATGT

340 IO20 VLYNSYT

LUNSSOHYVKFKT ATTCCACAATTCATCCCACCATTATGTCA4GTTCAAGACT~TGCC~A4T&A~AGTTATAC

360 IO80

TECCIYTFFV SELHLTRLKG A.AGCG&ACTTCACClTACCCGATT&AAAGCAACACAC~~ACGCATTTACACATTTTTTGT

380 iI40

SNSDASSSVTFNVYVkTKPE GTEC&ACTCAGATCCCAGCTCCTCTGTAACA?TTA&TCTCTAC~TGA&AAC&AAACCACA

400 1200

ILTLOKLCNOI CATCCTTACCTTGCATATCCTCGGCAATCACATTCTTCACTCTCTCCCAACTCCATTCCC

t

A

UOV

T

C

F

P

440 1320

YWYF@PCTEPA@LDSP A P T I ACCCCCTACCATTTACTCCTATTTTTCCC~A~GAACTtiAACA~AGGTGTTTACACTCAC~ ISPMOVKVSYTNSSV T AACCATATCCCCCAfCCATCTCAAACTCACTT4CACAAA~TCATCCCTCCCATCCTTTGA S L V f S T V N A R I GCGG&TCCTCCTCG&GAGCACTGTGAACGCCICfllCTTCA&GACC&CTGGTACCAT~TC OEASSNCDKSSV CTCTCACCC&TCCACiAATCCTC&CAAGAGCTCTGTTTTCTTTAACTTTG~TATTAAAGA QIRTHTLFTPLLIA GCAAATCCCTACCC&CACCCTTTTCACACCTTTACTAATCGCATTTGGCCTCCCCGCTGC

M

P

S

f

f

f

S

T

G

T

I

f

f

N

F

A

I

K

E

460 I380 480 1440

,E

f

FCVAAC

500 1500 520 1560 540 1620

IVKILVYIYLPKPKYE 1 K C I ACTCATCTCCATCATACTCATGATCCTCCTCTACATATATTTGCACAAACCCAAATATGA I

INGNNVVY YQWKVVEE AGTCC&CTGCAA>TGTTCAAGAA&TAAATGGAAACAACT&TGTTTACATAGA~CCAAC DHWWEFPRN Q L P Y CCAACTTCCTTATCATCACAAATGGCACTTTCCTAC&AACCCGTTGACTTTTGCTAAAAC

420 1260

RLSFCXT

D

P

T

560 1680 580 1740 600

FCKVVEATAYCLFKS 1 C A C A CCTTGCTGCTCGAGCTTTTCC&AAGCTTCTTGAAGCCACT~CTTATGGCCTATTTAAATC

I800

VKMLKPSAHLTER D A A I T V A TCATCCCCCTATGACACTACCAGTGAAAATGTTCAAACCAACCGCCCATTTAACTGAAAG

1660

KVLSYLCNHINIV Y s E L A~AA~CC:TCATCTCAC&CCTAAAA~TTCTClCTTICCTT~~TAA~CA~ATTAATATT~T

1920

620

640

940 2720 U

TTCACTCCCATCTCCAACAAGAGGACCCCACCTGTACTTCCTCTTTGTATTGTGTAGGLC CTCACAGACCCACCACTTCTATTTCTTTTACTTTTACTCACTACTACCACTGTCTACCTC AAATCTTCTTCTAATACTCTCCTTTACCACACACTCTTACACATAAACTTAATGT~CTGA CCACCCTTACACCCATCATTGCAATCTTAACTGAAGCTCTATATATTTTGCTATATTCTC TCTATTTCCACTAGACACCCAGATCTCAAGAAGAACAACA&AAAAACACA&AACAAACCC TCCGCC&CCCTCTTCAATCAGATGACTACACATTCAACCAAATCTGCAGCCGlCCTTCTC TGGATCCATGCTlGCCAACCCTGTAGTCATTTT~CACTTAATTTl~TTTTCTTGAATlT& AGAAATAAACAGATATAAAGCAAAGTGCTTT~CAAACCCCTTTCCATAT~CAGCCACACC TGAGATGCTTTCCCCGCCACACA~TCCAGCTTCG&GG~CAGGAGGG&CAGACAGTGC~TC EETCCCCAACAACTCC&AGCCAAC~CATGCACATGGCC&ATA&AACACACAACTTCC&GA CACTCTCGGGCATGACGCACATCTG~AACGCAAACACCTCAGTCTTTATTl~CTACCTC~ CCACTCACCAGTGCACACCACTCACTACCTTTCTGTGGGTGCCTGCA~CACATCCC&CTG CCATTCCACCACCCAGCATCCACCCICTCICIITCACAATCCCACCGATGAGATGCTTGGTTTGC TAACACTCATCTCCAAAACCATTCACCAAAGATACLTCCCCTCTGCTCAGCTGGTCAAAT GACACCACCACCCTTCCTTACACTAAATCGICTCTCAGACCAGCACAGGCCGTTTAACGA GACATCTTATCTTCCCACGACCAGAACCAAAAAATTCTAT~CTGAGGGCCCTTTGCTTAT CTTATCATATCCACCTCAATATTTCCCACACCCTCCCTTCCATAATGCTCTCAGCAAACG CCTCTTCCTCCCACAACTCATTTCATCTTTCTATCTCTCTA~TCGTGCTCACATCCCAG& CAACAACTTCACTCCCAACCATCCAATTTTTTAATCGAATACAACAATCTATACCTTCGG TCTTGTTTCGACCCACATATGACCCTCTCGGCTTTCTGlTGCGCCTCCCATlClCC TTAAGTATCTTATAATTCCTlTTCTTCCTGTTCGAA&~A~TCTTC~TTCTlTlTTTTlTT TTTTTTTTAACCCTTCTTCTC?CCCClTTAAATTA~TA&AT~CCTCAAGAGGA~CATTTT TCAC&CACAEAGTACCAGTTAA~TACCTATTT&TtTACAITG~&CT?AACCAClCTGlA& fTTATGTTTTACC&CGATTTTCGTTCACTTTAC&TCC&TlTTAAATGTGTAACT&&ClCC CC&A&ATGCAACkCGATTCTAAATACACACACTGGT>ACATTTTAGGTACTCTTCAAT ACCACACTCTTTATATGCCTGTCCATTTGGTTTATTGTTACAGGAATTC&TCTTT~AC&G TTACTCCAGAAGTACCCATTTCCICTGAACTTACTTAGCCTGTTGCACCTTTCC&AAGTT ACCCACTTCTTTCCACCCACTCTTATCCATACTTCTTGTTCACCATTTTCAGTTTAATGC TATAAAACCTCTTTCCTACTCTCTTCCCTTTCAGAGCAACTCT&CACAAACTACTTTT&T AATCATACATCTCTTGGTACTTTATCCICITTTAGCTGAAAAAAGTTTGGGTTTTTTCTT TCTTA&AATTTATATTTTTATAATTTGGGGGGGGTTLAA&CTTATTTTGCAATGCCTTAG TCTTTCAATCCTTTTATCCATTCTTTTTCTAAATATCCAAATCTACCAATAATCTCCTTT TCACTATTCTCACTCCTTGACTCTCAAAAATATTTATATATATATATATAT~CAAA&ACT AlATCTATAAAT4TGT&ACTCTTCAAAACTTTATCAAACACTTATC~ATATGTTCClCG~ TTGTAGAATTGEACTTCACAACAGTTCA~TCTGl&AA?&TCTATTCAGATGCTAC CCATGTCTACTATGTTAACATCCAATATTTTCATGTAAGTCTT~TTCTTCG~TT~ AACCAAAAAAAAAAAAAAkAA&AAAAAAAAAA&AAAA&A&AAA&AA&&A&hA&&AAAAh&

_

AA&AAAAAAAA

P

1

t

V

R

E

0

V

960 2680 2940 3000 3060 3120 3160 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3640 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4320 4980 5040 6100 5111

260 indicate that the protein encoded by the ORF is indeed chicken Kit protein. The putative extracellular segment of the chicken protein encompasses 13 Cys residues and I I potential glycosylation sites (Fig. 2). The extracellular segment of mouse Kit contains five immunoglobulin domains, and 12 out of the I3 Cys residues in this segment have been suspected to be involved in the immunoglobulin structure (Majumder et al., 1988; Qiu et al., 1988). AI1 12 Cys residues in the mouse protein are conserved in the chicken protein, suggesting that the chicken protein also contains the immunoglobulin domains. Reith et al. (1991) have shown that two normal isoforms of mouse Kit are coexpressed in mouse mast cells and placenta: one isoform (Kit +) is the protein encoded by the published cDNA sequence (Qiu et al.. 1988). and the other isoform (KitA +) has an in-phase insertion (Gly-Asn-Asn-Lys) within the extracellular domain. The protein encoded by the cDNA in clone 3 (Fig. 2) corresponds to the Kit + isoform. To determine whether an isoform corresponding to the KitA+ isoform is produced in chicken brain, the nt sequences of the relevent regions (equivalent to nt 1441-1609 in Fig. 2) of six other cDNA clones were determined. The nt sequence of each clone was identical to that of clone 3, indicating that the amount of the KitA+ isoform, if any, is much lower in chicken brain than in mouse mast cells and placenta. The aa sequence identity of the kinase domains between the chicken and mouse Kit proteins (94%) is most extensive. The insertion sequence within the TyK domain of the chicken protein also shows a distinct homology (63%) to that of mouse. Hanks et al. (1988) have compiled the aa sequences and conserved motifs among the catalytic domains of a number of protein kinases. These include: (I) the putative nt binding site, Gly-Xaa-Gly-Xaa-Xaa-Gly; (2) the Lys residue (corresponding to Lys ho’ in chicken Kit) involved in phosphotransfer reaction; (3) the TyK-specific consensus, AspLeu-Ala-Ala-Arg-Asn; (4) the sequence implicated in ATP binding, Asp-Phe-Gly; (5) the other TyK-specific consensus, Pro-( lle/Val)-( Lys:‘Arg)-Trp-(Thr/Met)-Ala-Pro-Glu; and (6) the Glu and Arg residues (corresponding to Glt? and ArgH9” in chicken Kit) completely conserved among the protein kinases. In the chicken protein, these aa residues and motifs have been identical to those of the mouse protein. In mouse Kit, two potential Tyrphosphorylation sites, Tyr”’ and Tyr934, are implicated in the regulation of the enzymatic activity of this protein (Qiu et al., 1988). These two Tyr residues are also conserved in the chicken protein (Fig. 2). Taken together, conservation of these features suggests that the chicken Kit protein is an active TyK, and its enzymatic activity is normally regulated in vivo.

(c) Expression of chicken c-kit transcripts in chicken organs

To identify sites where the chicken Kit may function, the expression of the c-kir RNA was investigated in prepubertal chickens. For this purpose, poly(A)‘RNA was prepared from various tissues and analyzed by blot hybridization using the cDNA insert of clone 3 as a probe (Fig. 3). RNA transcript was detected in the brain, bursa of Fabricius, heart, kidney. lung, spleen, thymus. ovary. and testis. The highest levels of c-kir RNA were found in the brain and testis. In all tissues. a 5.5-kb RNA species was seen. This expression pattern of chicken c-kit is basically identical to that of mouse c-kir (Nocka et al., 1989; Ray et al.. 1991), suggesting that the chicken Kit protein has biological roles analogous to those of the mouse counterpart.

18s-

‘*

:..,-

;-”

.*

p-actin FIN. 3. Expression organs.

of the chicken

Poly(A)‘RNA

bursa of Fabricius

c-kir

was prepared

RNA

from

transcript

various

in various

organs

(bursa) of 7-week old chickens (White

including

Leghorn)

the method described in the legend IO Fig. I. The poly(A)-RNA each) was electrophorezed formaldehyde

(Sambrook

on a 1.2%

membrane {GeneScreen

Plus; NEN

was prehybrldized

I

for

agarose

salmon

DNA

the “P-labeled for I6 h at 6&C.

2.2 M

IO a Nylon

Research Products). The membrane

h in hybridization

solution

SDS 10% dextran sulfate). and then hybridized lion containing

gel contaming

et al.. 1989) and then blotted

by

(5 ug

cDNA

(I M

NaCIIl%

in a hybridization

solu-

insert of clone 3 and IO0 ug ml

The membrane

was washed twice in

I%

2 x SSC: 1% SDS for I5 mm at 60 C, and then once in 0.2 x SSC SDS for I5 min at 60 C before being exposed IO Kodak for three days at -70 indicated.

Hybridization

for comparison.

C. Migration

XAR-5

of 28s and 18s ribosomal

film

RNA

is

with a chmken S-acrin probe is shown below

261 (d) Conclusions (1) cDNA clones encoding the chicken Kit protein were isolated from a chicken brain cDNA library, and the complete nt sequence of the longest cDNA clone was determined. (2) The deduced aa sequence of the chicken Kit showed a 63% identity to the mouse Kit, and aa alignment suggested that chicken Kit has structural and functional features quite similar to mouse Kit. (3) The 5.5-kb chicken c-kit transcript was detectable in various organs of chickens, at relatively high levels in the brain and testis, suggesting that chicken Kit may function in these organs.

ACKNOWLEDGEMENTS

This study was supported by the Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency of the Japanese Government.

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