Deer antler tissue contains two types of bone morphogenetic protein 4 mRNA transcripts

BB

ELSEVIER

Biochimica et Biophysica Acta 1263 (1995) 163-168

Biochim~ie~a et BiophysicaA~ta

Short Sequence-Paper

Deer antler tissue contains two types of bone morphogenetic protein 4 mRNA transcripts Jian Q. Feng, Di Chen, Javier Esparza, Marie A. Harris, Gregory R. Mundy, Stephen E. Harris * Unil~ersity of Texas Health Science Center at San Antonio, Medicine / Endocrinology, 7703 Floyd Curl Driue, San Antonio, TX 78284- 7877, USA Received 12 February 1995; accepted 12 May 1995

Abstract

Previously we isolated a bone morphogenetic protein 4 (BMP-4) cDNA from human prostate cancer cells and found that the 5' noncoding exon 1 of this BMP-4 cDNA was different from that of human bone cell BMP-4 cDNA. Recently we identified two alternate exon Is, 1A and IB, for BMP-4 gene by reverse transcription-polymerase chain reaction (RT-PCR) assays from fetal rat calvarial osteoblasts. In order to further examine alternate exon 1 usage in the BMP-4 gene, we screened deer antler tissue cDNA library. We isolated two types of cDNA clones encoding BMP-4 from this deer antler cDNA library. Sequencing of these clones have revealed a single open reading frame encoding a 408 amino acid protein. Comparison of 5' noncoding exon 1 portion of these cDNA sequences with those of human bone and prostate BMP-4 cDNA sequences and mouse BMP-4 genomic DNA sequence demonstrated that deer antler tissue expresses both exon 1A and 1B containing BMP-4 mRNA transcripts. This suggests that BMP-4 gene may contain alternate promoters or alternate splicing sites in deer antler tissue. Keywords: Deer antler; Bone morphogenetic protein 4; mRNA transcript; Noncoding exon

The bone morphogenetic proteins (BMPs) are a group of related proteins originally identified by their presence in bone-inductive extracts of demineralized bone [ 1,2]. BMP-4 is a member of BMP family. BMP-4 is synthesized as a large precursor, as are other BMP family members, and is processed to a mature dimer form which is then secreted into the extracellular matrix [3]. Recombinant BMP-4 stimulates new cartilage and bone formation when implanted near long bone [4]. Recently we have demonstrated that BMP-4 is expressed in primary cultures of fetal rat calvarial osteoblasts. The expression of BMP-4 m R N A coincides with the expression of other markers of bone cell differentiated function, such as osteopontin, osteocalcin and alkaline phosphatase in prolonged primary cultures of fetal rat calvarial osteoblasts [5]. We have also found that BMP-4 mRNA is expressed in normal and neoplastic human prostate tissues and human prostate cancer cell line [6]. Previously we have isolated a BMP-4 c D N A clone from a human prostate cancer cell line, PC-3. We found that the

* Corresponding author. Fax: + 1 (210) 5676693. 0167-4781/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 I 6 7 - 4 7 8 1 ( 9 5 ) 0 0 1 0 6 - 9

5' noncoding exon 1 of human prostate BMP-4 cDNA was clearly different from that of human osteosarcoma BMP-4 cDNA, although the coding regions were identical. This suggested the possibility of alternate promoters for the BMP-4 gene [7]. To determine which 5' noncoding exon 1 is utilized in normal bone cells, we then examined the normal bone cell BMP-4 cDNA sequence by screening fetal rat calvarial osteoblast cDNA library. We found that the 5' noncoding exon 1 of normal rat bone cell BMP-4 had a similar nucleotide sequence (77% identity) with that of human bone osteosarcoma cell BMP-4 cDNA and had a very different nucleotide sequence (23% identity) with that of human prostate cancer cell BMP-4 cDNA [7]. In order to understand the regulation of the BMP-4 gene expression, recently we have isolated the BMP-4 gene from a mouse genomic library and characterized the exonintron structure and promoter [8]. We also demonstrated that the two alternate exon ls, 1A and IB, for the BMP-4 gene are utilized by normal rat bone cells (fetal rat calvarial osteoblasts) by RT-PCR assays. Northern blot analysis using exon 1A, 1B and exon 2 - 4 probes and quantitative competitive PCR results indicate that the IA containing transcript is the primary BMP-4 m R N A expressed in FRC

164

J.Q. Feng et al. / Biochimica et Biophysica Acta 1263 (1995) 163-168

osteoblasts [9]. Existence of multiple promoters in murine BMP-4 gene was also reported by other laboratory [10]. To further examine BMP-4 transcript utilization and gene expression, we screened deer antler tissue cDNA library. We isolated two different types of BMP-4 cDNA sequences which contain exon IA and 1B in 5' noncoding regions from this tissue. The antlers of Texas Fallow deer were obtained from Heart-Bar Deer Farm (Hondo, TX). Total RNA of deer antler tissue was extracted and sent to ClonTech for cDNA library construction (ClonTech, Palo Alto, CA). From poly A RNA of deer antler tissue, double-stranded cDNA was synthesized and then cloned into the B a m H I / X b a I site of the pDR2 vector (ClonTech, Palo Alto, CA). The resulting deer antler cDNA library was screened with a human BMP-4 full length cDNA probe (1.7 kb) which was previously isolated from human prostate cancer PC-3 cells in our laboratory. Random-primed probe labeling, phage lifts, and filter hybridization were carried out using standard procedures [11]. Hybridization conditions were: 6 X SSC (1 X S S C = 0 . 1 5 M NaC1, 0.015 M sodium citrate, pH 7.0), 5 X Denhardt's, 0.5% sodium dodecyl sulfate (SDS), 10 mM EDTA, 10 /zg/ml poly(A) tRNA, and 1 5 0 / ~ g / m l salmon sperm at 68°C for 24 h. Filter washing was carried out at 55°C in a solution of 2 × SSC and 0.1% SDS for 30 min, and in a solution of 0.1 × SSC + 0.1% SDS for 10 min. All DNA sequence analysis was performed by the dideoxynucleotide chain-termination method [12]. The cDNA inserts were cloned into Bluescript SK vectors from the pDR2 vectors and sequenced directly in both orientations of the SK vector with Sequenase (Version 2.0, USB, Cleveland, OH) using universal and reverse primers and synthetic primers. Four independent BMP-4 clones were isolated from the deer antler cDNA library. Three of them are identical and contain exon 1A in 5' noncoding region (BMP-4A, Table 1). The longest antler BMP-4A cDNA is 1700 base pairs (bp) with the ATG start codons at bp 355-357 and the TAG stop codon at bp 1579-1581. One clone contained exon IB in 5' noncoding region (BMP-4B, Table 1). The intact cDNA of BMP-4B is 2324 bp with the ATG start codons at bp 860-862 and the TAG stop codon at bp 2084-2086 (Fig. 1). The exon 2 and coding regions of these two types of cDNA are identical (Table 2). The deduced protein of deer antler BMP-4 is 408 amino acid residues in length. The nucleotide similarity of the deer

BMP-4A sequence with human and rat bone cell BMP-4 sequences are 92 and 89%, respectively, and the nucleotide similarity of the BMP-4B sequence with human and rat bone cell BMP-4 sequences are 79 and 78%. The deduced amino acid sequence encoded by deer BMP-4 cDNA is 97% identical to that deduced from human bone osteosarcoma cell BMP-4 cDNA (13 amino acid differences) and is 96% identical to those deduced from normal rat bone cell BMP-4 cDNA (16 amino acid differences) and from mouse BMP-4 genomic DNA (18 amino acid differences) (Fig. 2). The BMP-4 mature regions are the same among these four mammalian species. The identities of exon 1 portion of deer BMP-4A cDNA with exon 1 portion of human bone cell BMP-4 cDNA and exon IA of mouse BMP-4 genomic DNA are 88 and 80%. The identities of exon 1 portion of deer BMP-4B cDNA with exon 1 portion of human prostate cell BMP-4 cDNA and exon I B of mouse BMP-4 genomic DNA are 79 and 73% (Fig. 3, Table 1). The size of exon 1B is various among these four mammalian species (Fig. 4). The length diversification of exon 1B suggests that there may be an extra exon between exon 1B and exon 2, or alternate 5' splice sites in intron 1 between exon 1B and exon 2 in deer BMP-4 gene. The formation of new bone during embryonic skeletal development and bone remodeling is a well regulated process under the control of osteogenic factors. These factors include transforming growth factor /3 (TGF-/3), fibroblast growth factors (FGFs) and insulin-like growth factors (IGFs). BMP-4 is a member of a BMP family in the extended TGF-/3 superfamily. BMP-4 is widely expressed in embryonic life, particularly in developing limb buds, suggesting an important role in morphogenesis [ 13,14]. Recombinant BMP-4 stimulates bone formation in vivo and in vitro. BMP-4 mRNA expression can be detected by in situ hybridization in the early phase of fracture after the onset of fracture before new cartilage or bone formation in mice [15]. BMP-4, with other growth factors, may play important roles in embryonic skeletal development, normal bone formation and in fracture repair process. Although the effects of BMP-4 on bone formation and bone cell differentiation have been demonstrated in recent years, the effects of BMP-4 on other tissues and the regulation of BMP-4 gene expression is still largely unknown. Recently, we have demonstrated that normal human prostate tissue and human prostate cancer cell line

Table 1 Human osteosarcomacells, human prostate cancer cells and deer antler tissue utilize different Exon 1 (Exon IA and Exon IB) of the BMP-4 gene (% of identity in Exon 1A and Exon 1B of the BMP-4 gene) Human bone Human prostate Deer antler Deer antler U2-OS cell cDNA PC-3 cell cDNA tissue cDNA tissue cDNA (Exon 1A) (Exon 1B) (BMP4A) (Exon 1A) (BMP4B)(Exon 1B) Mouse genomic DNA (Exon 1A) Mouse genomicDNA (Exon 1B)

78 27

51 66

80 35

33 73

165

J.Q. Feng et al. / Biochimica et Biophysica Acta 1263 (1995) 163-168

androgen-regulated. BMP-4 may also play some roles in deer antler tissue growth and morphogenesis. Two different exon 1 containing BMP-4 cDNAs found in prostate and bone cells in human and rat, and in deer antler tissue

PC-3 express BMP-4 mRNA predominantly [6]. BMP-4 may play important roles in normal prostate growth, morphogenesis and neoplastic transformation. Deer antler is a fast growing bone-like tissue and its growth is highly

GCGCAGAGGTCGAGCGCTGGCCGAAA•CTGTTCACGGTTTTCACGACTCCTGGGAATGCGGTGGGATTTCCTTTCTGCGGCGGGTCGGGAGTTGTAAAAC

100

CTCCGCGACCTTGAGACCTGAAACATGTGATGCG•CTTTTCTCAGGAGACGCCTCTTTTCGAGTCTGTCGCGAGCCTTGCGTCCCCGCGCCCCGCCGCCG

200

CGTCCTTGAATGGCCCGCGGACCACTTTCTTTGGGGATTTGCTTTGCTTTTGGCGGGGGATCGCCGTAGTGCGGAGTTTGTCTCCGTAAAGGAACTGGAG

300

GAAGGA•CGTAGCGGCGGCTGCCTCATCACCTTCCCACACC•ATAACCGCCTAAATATCTCCCCCTCCGCCCCGCGCGACAGGGTCCGCAGCTTTGGGCT

400

GGGAGCTAAA•CGGCTGTCCAGTTCTTCTGGACATTCTCACGGTCCGCCAGGTTGTGACTAGCTGTCACTGGGAGCAGTATTTAGCCTGATCCGAGACCC

500

TGGGGAGGAAGTGGGTGCGCGCGGTGTGTGTGTTTGTGTGTCCCTCAGCGCTTTCCTTTTTAAATGACCCTCGGTGGTGAGGCTTTCGGCGG~TGAGACT

600

CCG•GGCCCCCTATCCCGCCCCCACTGGAGCCGGCTCGCGCCGAAAGCGCAGCCCCTACGGTACTCCCCAGACCCTTGGCTT•AGGCGCTGGTGCGAGGG

700

Exon 1 B - ~ E x o n

2

CCCAGTTCGACGCCCGGAAGGCTACAGGAGCCATTCCGTAGTGCCATCCAGAGCAACGCACTGCCGCAGCTCCTCTGAGCCT TTCCAGCAAGTTTGTTCA Exon 2 - - - - - ~ o n

3

AGATTGGCTGTCAAGAATCATGGACTGTTATTATATGCCTTGTTTTCTGTCAAGACACCATGATTCCTGGTAACCGAATGCTGATGGTCGTTTTATTATG N

!

P

G N

R 14 L

14 V

V

L

L

L

L

G G A

T

H A

S

L

[

P

E

T

G K K

K V

A

E

!

Q G

H A

G G R

900

C

CCAAGT•CTGCTAGGAGGCGCGACGCATGCTAGTTTGATACCTGAGACGGGGAAGAAAAAAGT•GCCGAGATTCAGGGCCA•G•GGGAGGACGC•GCTCA Q V

800

R

1000

S

•GGCAGAGCCATGAGCTCCTTCGGGACTTCGAGGCCACACTTCTGCAGATGTTCGGGCTGCGCCGCCGCCCGCAGCCTAGCAAGAGCGCAGTCATCCCGG 1100 G Q

S

H E

L

L

R D

F

E

A

T

L

L

Q 14 F

G

L

R R

R P

Q P

S

K

S

A

V

|

P

D

ATTACATGCGGGATCTTTACCGGCTTCAGTCTGGGGAGGAGGAAGAGGAAGAGCAGATCCAGGGCATCGGTCTGGAGTATCCTGAGCGCCCCGCCAGTCG 1200 Y N R D L Y R L Q S G E E E E E E 0 ! 0 G l G L E Y P E R P A S R

GG~ACCGT~G~G~TT~CGAAGAA~T~TG~GAACATC~CAGGGACCAGCGAAAACT~TGCTTTTCGTTT~TCTTT~CCTCAGCAT~ A

N

T

V

R

S

F

H

H E

E

H

L

E

N

|

P

G T

S

E

N

S

A

F

R

F

L

F

N

L

S

1300

!

CCA~G~C~GGT~TCTCCACTGCC~GCTTCGG~CTTTCGG~GCAGGTG~CCAGGGCCCTGATTGGGAAAGGGGCTTCCATCGTATAAACATTT 1400 P

E

N

Q V

|

S

T

A

E

L

R D

F

R

E

Q V

D

Q

G P

D M E

R G

F

H R

I

N

l

Y

AT~GGTTATGAAGCCCCC~GAA~CGGTGCCTGGGC~CT~T~CAC~CTACTG~C~CTGGTC~CCAC~TGT~CGCGGTGGGAAAC E

V

N

K P

P

A

E

A

V

P

G

H L

|

T

R

L

L

D

T

R

L

V

H

H N V

T

R W E

1500

T

TTTT~TGT~GCCCTGCAGTCCTTCGCTG~CCCGGGAGAAGCAGCCCAACTACGGGCTGGC~TTGAGGT~CCCACCTCCATCA~CACGGACCCAC 1600 F

D

V

S

P

A

V

L

R W T

R

E

K Q

P

N

Y

G L

A

]

E

V

T

H L

H Q

T

R

T

H

~GGGCCAGCATGT~G~TTAGCCGATCGTTACCTC~GG~GTGGG~TT~GCCCAGCTCCGGCCCCTCCTGGT~CCTTTGGCCAT~TGGCCGGG 1700 Q G Q

H V

R

]

S

R

S

L

P

Q

G S

G D

~

A

Q

L

R

P

L

L

V

T

F

G

H D

G R

G

~TGCCTT~CCC~CACCGGAGGGCC~GCGTAGCCCC~G~TCACCCA~GGGCACGGAAGAAG~T~GAACTGCCGGCGCCACTCGCTCTA 1800 H A L T R H R R A K R S P K H H P Q R A R K K N K N C R R H S L Y

TGTG~CTT~GT~TGTGGGCTGC~AAC~CTG~TTGTGGCCCCACCAGGCTACCA~CCTTCTACTGC~CGGG~CTGCCCCTTTC~GCTGGCC~C 1 ~ 0 V

D

F

S

0

V

G U

N D

U

l

V

A

P

P

G Y

Q A

F

Y

C

H G D

C

P

F

P

L

A

D

~CCTC~CTC~CAACCACGCCATTGTGCA~CCCTCGTC~CTCTGTC~CTC~GCATCCCCAAAGCCTGTTGTGTTCC~CCGAACT~GCGCCA 2000 H L

N

S

T

N

H A

|

V

Q

T

L

V

N

S

V

N

S

S

|

P

K A

C C

V

P

T

E

L

S

A

!

TCTCCATGCTGTACTTG~T~GTAT~C~GGTGGTTCTGAAAAATTATCAGGA~T~TAGTG~GG~TGT~GTGCCGCT~TCAGGCCTTCCT S

N

L

Y

L

D

E

Y

D

K

V

V

L

K

N

Y

Q

E

N

V

V

E

G C G'C

R

2100

*

TGGG~CACA~CACACA~CA~CACACCC~TCCACTACT~CCCA~CACTA~CA~CTGCTTCCTTATAGCTG~CTTTTATCTTAAAA

2200

~~AAAAAAAATAAACATTCACCTT~CCTTATTTAT~CTTTACGTGCAAATGTTTT~CCATATTGATCATATATTTT~CAAA

2300

GTATATTTAT~CTACATATTAAA

2324

Fig. |. N u ¢ | e o t i d e a n d ~ d u c e d a ~ o ~ ¢ ~ d s e ~ c n ¢ ~ s o ~ t ~ e c D ~ ¢ o d J n g o ~ e ~ r ~ P - ~ . ~ h e nu¢|~otz~e~ e q u e n c e i ~ e ~ d deducedami~acidsequence~h~wnbe~w~Theputativeex~b~undaries~emar~edab~vethenuc~e~tidesequenc~.

|-2324wire,he

J.Q. Feng et al. / Biochimica et Biophysica Acta 1263 (1995) 163-168

166

Table 2 Human osteosarcoma cells, human prostate cancer cells and deer antler tissue utilize the same Exon 2 of the BMP-4 gene (% of the identity in Exon 2 of the BMP-4 gene)

Mouse genomic DNA (Exon 2)

Human bone cell U2-OS cell cDNA (Exon 2)

Human prostate PC-3 cell cDNA (Exon 2)

Deer antler tissue cDNA (BMP4A) (Exon 2)

Deer antler tissue cDNA (BMP4B) (Exon 2)

95

95

92

92

suggest that BMP-4 gene may contain alternate promoters or alternate splicing sites in these species. In summary, we demonstrated two types of BMP-4 mRNA transcripts, containing exon 1A and 1B, in deer

Deer Human

Rat Mouse

Deer Human

Rat Mouse

Deer Human

Rat Mouse

Deer Human

Rat Mouse

Deer Human

Rat Mouse

antler tissue by cDNA library screening and sequencing. These two types of deer BMP-4 mRNA transcripts are homologues to bone and prostate BMP-4 mRNA transcripts of human, rat and mouse.

MIPG~LLCQVLLGGA~ASLIPETGKKKVAEIQGHAGGRRSGQS MIPGNRMLMVVLI/2QVLLGGAISHJASLIPETGKKKVAEIQGHAGGRRSGQS MIPGNRMLMVVLIAZQVLLGGAITD[ASLIPETGKKVAEIQGHAGGRRSGQS MIPG~LLCQVLLGGAIRDIASLIPETGKKKVAEIQGHAGGRRSGQS HELLRDFEATLLQMFGLRRRPQPSKSAVIPDYMRDLYRLQSGEEEEE HELLRDFEATLLQMFGLRRRPQPSKSAVIPDYMRDLYRLQSGEEEEE HELLRDFEATLLQMFGLRRRPQPSKSAVIPDYMRDLYRLQSGEEEEE HELLRDFEATLLQMFGLRRRPQPSKSAVIPDYMRDLYRLQSGEEEEE 1 IGLEYPERPASRANTVRSFHHEEHLENIPGTSE TGLEYPERPASRANTVRSFHHEEHLENIPGTSE TGLEYPERPASRANTVRSFHHEEHLENIPGTSE TGLEYPERPASRANTVRSFHHEEHLENIPGTSE

~

232 232 233 233

~

Rat Mouse

Deer Human

Rat Mouse

Deer Human

Rat Mouse

145 144 145 145

~ HNVT ~ WETFDVS PAVLRWTREKQPNY IHI HNVT IRI WETFDVSPAVLRWTREKQPNY [R IHNVT IR[ WETFDVSPAVLRWTREKQPHY [H IHNVT IQI WETFDVSPAVLRWTREKQPNY

Deer Human

101

186 186 187 187

Mouse

Rat

~

i01 100 i01

GFHRINIYEVMKP GFHRINIYEVMKP GFHRINIYEVMKP GFHRINIYEVMKP

GLAIEVTHLHQTRTHQGQHVRISRSLPQGSG GLAIEVTHLHQTRTHQGQHVRISRSLPQGSG GLAIEVTHLHQTRTHQGQHVRISRSLPQGSG GLAIEVTHLHQTRTHQGQHVRISRSLPQGSG

Deer Human

~ SA~R~ FNL INI SA IFI R IFLI FNL ISI SA IF[ R [FF [FNL ISI S A W a IWRI FNL

FREQVDQGPDWE sIP~N I VISISI~ILI FREQVDQGPDWE sIPE. I VIS ISl~U~lLI FREQVDQGPDWE SIPEN L WSl lAE, W FREQVDQGPDWE SIPEN I

PAE ~ VPGHLITRLLDTRLV PAE IV[ VPGHLITRLLDTRLV PAE IMI VPGHLITRLLDTRLV PAE IMI VPGHLITRLLDTRLV

EQIQ -QIH EQSQ EQSQ

50 50 50 50

~ WAQLRPLLVTFGHDGRGH INI WAQLRPLLVTFGHDGRGH INI WAQLRPLLVTFGHDGRGH IDI WAQLRPLLVTFGHDGRGH

282 282 283 283

~ QR ~ RKKNKNCRRHSLYVDFSDVGWND IS IQRI ~ RKKNKNCRRHSLYVDFSDVGWND IP IQRI -¢ RKKNKNCRRHSLYVDFSDVGWND [P IQR ~ RKKNKNCRRHSLYVDFSDVGWND

324 324 324 324

WIVAPPGYQAFYCHGDCPFPLADHLNSTNHAIVQTLVNSVNS S I PKACCVPT WIVAPPGYQAFY CHGDCPFPLADHLNSTNHAIVQTLVNSVNS S IPKACCVPT WIVAPPGYQAFYCHGDCPFPLADHLNSTNHAIVQTLVNSVNSS I PKACCVPT WIVAPPGYQAFYCHGDCPFPLADHLNSTNHAIVQTLVNSVNSS IPKACCVPT

376 376 376 376

ELSAISMLYLDEYDKVVLKNYQEMVVEGCGCR* ELSAISMLYLDEYDKVVLKNYQEMVVEGCGCR* E LSAI SMLYLDEY DKVVLKNYQEMVVEGCGCR * E LSAI SMLYLDEY DKVVLKNYQEMVVEGCGCR *

408 408 408 408

LTR ~ R ~ AKRSPKHH LTRI RI al RI AKRSPKHH LTRI RI al - IAKRSPKHH LTR IRI a[ - IAKRSPKHH

Fig. 2. The comparison of BMP-4 amino acid sequences deduced from deer antler tissue cDNA, human osteosarcoma cell cDNA, normal rat bone cell cDNA and mouse genomic DNA.

J, Q. Feng et al, / Biochimica et Biophysica Acta 1263 (1995) 163-168

167

& DeerBMP4A

Hu

(U2-OS)

GCCCGGAAGt'TAGGTGAGTTTGGCATCTGAGCGGAGGGACCCGAGCCTGC *********ee******** ******* **** ******* ******** GCCCGGAAGt'TAGGTGAGTGTGGCATCCGAGCTGAGGGACGCGAGCCTGA

50 90

DeerBMP4A

GACGTCG~ATCAGTGCTGAGTATCTCGCCTCTCTCCCTGATGGGA * * * * * * * * * ~ * * ** * * * * * * * * * * * * ** * * * * * * * * * * * * * *

100

nu

GACGCCGCTC-CTGCTCCG-GCTGAGTATCTAGCTTGTCTCCCCGATGGGA

139

DeerBMP4A

TTCCCGTCCGCGCCGTCTCGAGCCTGCTGCGCC-CAATCCTCGGCCCTCG ********* ** * * * * * * * * * * * * * * * * * ** * * * * * * * * * * * *

149

nu

TTCCCGTCCRAGCTATCTCGAGCCTGCAGCGCCACAGTCCCCGGCCCTCG

189

CCCAGGTTCACTGCAGCTGTACAGAGGTCCCAAGGAGCTGCTGCTGGCGA * * * * * * * * * e * * * * * * ** * * * * * * * * * * * * * * * * * * * * * * * * * * * * CCCAGGTTCACTGCAACCGTTCAGAGGTCCCCAGGAGCTGCTGCTGGCGA

199

(U2-0S)

(U2-OS)

DeerBMP4A

Hu

(U2-OS)

239

DeerBMP4A

GCCCGCTACTGCAGGGACCT *********t**********

219

Hu (u2-os)

GCCCGCTACTGCAGGGACCT

259

B

CGCAGAGGTCGAGCGCAGGCCGAAAGCTGTTCACCGTTTTCTCGACTCC

70

DeerBMP4B

CGCAGAGGTC~AGCGCTGGCCGAAAGCTGTTCACGGTTTTCACGACTCC

50

Hu

GGGGAACATC~AGCCA-TTCCGTAGTGC ***** ** • • **** , ***

97

TGGGAATGCG~TGGGATTTCCTTTCTG

78

HU

(PC-3)

****************

(PC-3)

DeerBMP4B

*****************

******

*******

C

Fig. 3. The sequence comparison of Exon 1 portions of deer antler BMP-4A and BMP-4B cDNAs with those of human bone osteosarcoma cell (U2-OS) BMP-4 cDNA and human prostate adenocarcinoma cell (PC-3) BMP-4 cDNA. (A) Exon 1A comparison. (B) Exon 1B comparison.

Mouse BMP4 gene

E x o n 1A

E x o n 1B

Exon 2

N

N

0

Exon 3

Exon 4

ATG

TGA

Human U2- OS BMP4

Human PC3BMP4

Rat B M P 4 A Rat BMP4 1B-2-3 RT-PCR product Deer BMP4A

Deer BMP4B

•

Region >65% identity to mouse BMP4 exon 1B

Iiiiiiiiiiiiiiiiiiiiiiii~i~i~i)iiiiiiiii)i:!;~iiiiii~

[]

Region <25% identity to mouse BMP4 exon 1B

Fig. 4. Diagram of mouse genomic BMP-4 gene, human prostate cancer cell and bone osteosarcoma cell BMP-4, rat bone cell BMP-4 and deer antler BMP-4 transcripts, and rat bone cell BMP-4 PCR product. The mouse BMP-4 gene transcription unit is about 7 kb and contains 2 coding exons and 3 non-coding exons labeled Exon IA, Exon I B and Exon 2. BMP-4 transcripts from human, rat and deer and putative splice points, extrapolated from mouse genomic BMP-4 gene, shown in the lower panel.

168

J.Q. Feng et al./ Biochirnica et Biophysica Acta 1263 (1995) 163-168

We are grateful to Nancy Garrett for her help with the preparation of this manuscript.

References [1] Wozney, J.M., Rosen, V., Celeste, A.J., Mitsock, L.M., Whitters, M.J., Kriz, R., Hewick, R. and Wang, E.A. (1988) Science 242, 1528-1534. [2] Luyten, F.P., Cunningham, N.S., Ma, S., Muthukumaran, N., Hammonds, R.G., Nevins, W.B., Wood, W.I. and Reddi, A.H. (1989) J. Biol. Chem. 264, 13377-13380. [3] Suzuki, A., Nishimatsu, S., Shoda, A., Takebayashi, K., Murakami, K. and Ueno, N. (1993) Biochem. J. 291,413-417. [4] Shimizu, K., Yoshikawa, H., Matsui, M., Masuhara, K. and Takaoka, K. (1994) Clin. Orth. 300, 274-280. [5] Harris, S.E., Sabatini, M., Harris, M.A., Feng, J.Q., Wozney, J. and Mundy, G.R. (1994) J. Bone Miner. Res. 9, 389-394.

[6] Harris, S.E., Harris, M.A., Mahy, P., Wozney, J.M., Feng, J.Q. and Mundy, G.R. (1994) Prostate 24, 204-21 I. [7] Chen, D., Feng, J.Q., Feng, M., Harris, M.A., Mundy, G.R. and Harris, S.E. (1993) Biochim. Biophys. Acta 1174, 289-292. [8] Feng, J.Q., Harris, M.A., Ghosh-Choudhury, N., Feng, M., Mundy, G.R. and Harris, S.E. (1994) Biochim. Biophys. Acta 1218, 221-224. [9] Feng, J.Q., Chen, D., Cooney, A.J., Tsai, M.J., Harris, M.A., Dallas, S., Tsai, S.Y., Feng, M., Mundy, G.R. and Harris, S.E. (1995) J. Biol. Chem. (in press). [10] Kurihara, T., Kitamura, K., Takaoka, K. and Nakazato, H. (1993) Biochem. Biophys. Res. Commun. 192, 1049-1056. [11] Wozney, J.M. (1990) Methods Enzymol. 182, 738-751. [12] Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. [13] Lyons, K.M., Pelton, R.W. and Hogan, B.L.M. (1989) Genes Dev. 3, 1657-1668. [14] Francis, P.H., Richardson, M.K., Brickell, P.M. and Tickle, C. (1994) Development 120, 209-218. [15] Nakase, T., Nomura, S., Yoshikawa, H., Hashimoto, J., Hirota, S., Kitamura, Y., Oikawa, S., Ono, K. and Takaoka, K. (1994) J. Bone Miner. Res. 9, 651-659.