Vol. 187, No. 2, 1992 September
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Pages
STRUCTURE OF THE HUMAN HEPARIN-BINDING PLEIOTROPHIN
1113-1122
GROWTH FACTOR GENE
Shoupeng Lai, Frank Czubayko, Anna Tate Riegel and Anton Wellstein’ V.T. Lombardi Cancer Center and Department of Pharmacology Georgetown University, Washington DC 20007 Received
August
7, 1992
ABSTRACT: We report that the human gene coding for the heparin-binding growth factor pleiotrophin (PTN) spans more than 65 kb and contains at least 7 exons. Analysis of human genomic DNA fragments showed that the open reading frame (ORF) is located on 4 exons. The splice sites in the ORF coincide with the boundaries of functional domains in the human PTN protein and appear to be conserved in the mouse PTN and in the related family of midkine genes. The 5’- and the 3’-ends in the untranslated regions of the human PTN are distinct from those of other species and are highly homologous to the antisense cDNAs of heat shock protein 70 and of ribosomal protein L7 respectively. These two regions are located on separate exons and could play a role in the posttranscriptional regulation of human PTN gene expression. n 1992Academic Press, Inc.
A novel gene family of hepatin-binding growth factors termed pleiotrophin/midkine
(PTN/MK)
has emerged in the last three years (for a review see [l]). PTN was originally described with different names reflecting the sources of the protein as well as its diverse biological activities (Heparin-Binding Heparin-Affin
Neurotrophic
Factor [2], Heparin-Binding
Regulated Peptide [4], Heparin-Binding
Osteoblast Specific Factor [8], Pleiotrophin
Neurite Promoting
Factor [3],
Growth-Associated Molecule
[5-71,
[9,10]). The PTN and closely related MK proteins
appear to play a role during the development of the neuroectoderm and the physiologic expression of the genes in the adult is found only in very restricted areas of the nervous system [l]. On the other hand, we reported very recently that PTN is secreted as an active growth l To whom correspondence should be addressed. Abbreviations: PTN, pleiotrophin; ORF, open reading frame; MK, midkine; bp, base pair; kb, kilo bases; PFU, plaque forming units.
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factor from human breast cancer cells and in addition to this we detected very high levels of gene expression in human melanoma cells [l 1J. We hypothesized that PTN could function as a tumor growth factor in addition to its role during development. To investigate the mechanisms involved with the regulation of PTN gene expression, we studied the structure of the PTN-gene and we report for the first time here the genomic organization of the human gene. METHODS Screening of a human genomic libraryThe human genomic library of Caucasian male placenta in the X FIX II vector was obtained from Stratagene. The genomic library was screened by plaque hybridization using standard procedures [12]. Plaques at a density of 4 X 10’ plaques/plate (15 cm diameter) were transferred onto BA85 nitrocellulose membranes (Schleicher and Schuell). DNA on the membrane was denatured with 0.2 M NaOH/l.S M NaCl and hybridized with a cDNA containing the complete open reading frame of human pleiotrophin (nucleotides 455-1005; EMBWGenbank accession # M57399; [l 11) or synthetic oligonucleotides corresponding to the antisense sequences of nucleotides l-80 and 100-189 [l l] respectively. The cDNA probe was excised from pRcICMV (Invitrogen; San Diego CA) into which the respective cDNA fragment had been cloned after reverse transcription and PCR amplification of human PTN from mRNA as described recently [l 11. The excised fragment was purified in 1.O % low melting temperature agarose gel, labeled with [ar-32P]dATP and random primed labeling kit (Boehringer Mannheim) at about 5 X 108 cpm/pg DNA, and applied at 2-5 X lo6 cpm/ml in hybridization buffer. Prehybridization and hybridization were carried out at 42 “C in 5 X SSPE, 5 X Denhardt’s solution, 0.1 mg/ml denatured salmon sperm DNA, 0.5 % SDS, and 50 % formamide [12]. After hybridization the membranes were washed in 2 X SSC/O. 1 % SDS at room temperature for 15 min, at 42 “C for 15 min, and at 65 “C for 30 min. The final washing was in 0.1 X SSC/O.l% SDS at 65 “C for 30 min. When the oligonucleotides were used as probes to screen the library, they were labeled at the 5’-end by T4 polynucleotide kinase (NEB) and [Y-~*P]ATP (NEN) to a specific activity of about l-3 X 108 cpm/pg and applied at about 10’ cpm/ml in hybridization buffer. The conditions of prehybridization, hybridization and washing were the same as with the cDNA probe except that 20 % formamide was included in the prehybridization and hybridization buffer for the 90-mer probe and final washing was at 55 “C for the 90-mer and 42 “C for the 80-mer probe. The membranes were exposed overnight to XAR-5 film with intensifying screens. Positive clones were then plaque-purified by two additional rounds of plaque hybridization with the same probe. Restriction enzyme mapping of genomic clones- The DNA of each positive XFIX II clone was completely digested with Not1 to excise out the human genomic DNA insert carrying T7 and T3 promoter sequences at its ends and then partially digested with other restriction enzymes. The partially-digested restriction fragments of the genomic DNA were separated by agarose gel electrophoresis, transferred onto Nylon membranes, and probed with a 5’-end [“PI-labeled synthetic oligonucleotide corresponding to the T7 or T3 promoter. In some additional experiments the DNA of positive clones was partially digested with one restriction enzyme, annealed with a 5’-end [32P]-labeled 12-mer oligonucleotide (Promega) complementary to the left or right COS end of the X phage, separated on an agarose gel, blotted onto Nylon membrane, and visualized by autoradiography on XAR-5 film. Subcloning of genomic DNA fragmentsThe genomic DNA fragments that contain open reading frame of human pfeiotrophin were recognized by Southern-blot analysis of the restriction fragments of positive genomic clones with an [cr-32P]dATP-labeled cDNA insert or 5’-end labled oligonucleotide probe from the human PTN open reading frame. The fragments were separated
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by agarose gel electrophoresis, recovered by electro-elution from gel slices onto NA45 DEAEnitrocellulose membranes (Schleicher and Schuell), eluted from the membranes by 1.5 M NaC1/2mM EDTARO mM Tris @H 7.8) and ligated into pBluescript II SK+ (Stratagene) or pGEM 72+ (Promega). Determination of DNA sequences and of the exon/intron boundariesPlasmid DNA was prepared using the QIAGEN plasmid DNA purification kit (Qiagen). Double-stranded DNA sequencing was done by the dideoxy chain termination method with Sequenase Version 2.0 (USB) after denaturation by 0.2 M NaOH10.2 mM EDTA-Na, and annealing with the sequencing primers at 37 “C for 30 min. To define the exon/intron boundaries, synthetic oligonucleotides corresponding to the sense or anti-sense sequences in the PTN open reading frame were used as sequencing primers. The lengths of introns were determined by their positions in the restriction maps of the genomic clones. Sequence comparisonsWe used the University of Wisconsin Genetics Computer Group Program package for homology searches and sequence comparisons [ 131.
RESULTS The human PTN gene spans at least 65 kb-
PTN was initially
The strategy for genomic cloning of human
to screen a library with a cDNA probe containing the open reading frame
(ORF) of the human PTN gene [ll].
We opted to use a probe only comprising the ORF since
the S- and 3’-untranslated regions of the human PTN cDNA contain long stretches of sequences highly homologous to other human genes and we wished to avoid picking clones containing these latter genes. The S-end of human PTN shares a stretch of 97 nucleotides homologous to the human heat shock protein 70 cDNA (hsp70; EMBL/Genbank
accession # Ml 1717) and a stretch
of 277 nucleotides at the 3’-end of the PTN cDNA is homologous to the human ribosomal protein L7 cDNA (EMBL/Genbank
accession # X52967) (for details see below; Fig. 3).
After screening of about 2 X 106 PFU of the human library, 10 positive clones were obtained and plaque purified Physical maps of these clones were constructed by partial EcoRI digestion. Since there is no EcoRI site in human PTN cDNA, complete exons should be contained in the EcoRI fragments hybridizing with a cDNA probe. EcoRI mapping and Southern-blotting analysis of these clones revealed that they belonged to two separate groups which were not overlapping with each other and spanned a region of more than 35 kb. Clones PTN211 and PTNl comprised the longest ones in the respective groups and their relative positions are illustrated in Figure 1A. After sequencing of the most 5’-end fragment among clone PTN211 and PTNl (Fig. lB), it was 1115
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PTN53
restriction map of the human PTN gene. A: Restrictionmapof genomic clones.“-‘I showsthe positionof the respectivehumanFTN genomicclone. E = EcoRI. “II” indicatesa gapbetweenclones. Closedvertical barsrepresents the positionof exonsin the ORF andthe shadedvertical bar represents the putativepositionof an exon containing part of the S-untranslatedregion. B: Sequencing strategyfor the exon/intronboundaries of the ORF. E =EcoRI, EV =E.coRV. Shadedvertical boxesshowthe lengthandpositionof exonsin the ORF (01, 02, 03, 04). The arrowsshowthe directionandpositionof each sequencing reaction. Figure 1. Partial
EXON
INTRON
hPTN mPTN hMK hPTN mPTN hMK hPTN mPTN hMK hPTN mPTN hMK hPTN mPTN hMK
. ..ATTCCTGG . ..TACT-CA. ..CCC-TCA^AATGCAGGCTCAA..(116bp)..AAAGAGAAACCAG MetGlnAlaGln . . . . . . . . . ..LysGluLysProG A----TC-T-C--G..(117bp)..-----------T* SerSer * . . .. . .. . .. . * * * "G------CAC-G-...(77bp)..GCCA-A--GAA-* * HisArg . . . . . . . . . ..AlaLys * UGTGAAG..(174bp)..AAGCAATTTGGCG 1uLysLysValLys.. . . . . . . . ..LysGlnPheGlyA -------G-----A..(174bp)..--------------A* * * * * . . .. .. . . .. . * * * -T--GGTGAA----..(158bp)..---G-G-----Asp * ValLys * . . . . . . . . . . . * Glu *
GTAAGCAG..(ll29bp)..CCATTCAG . . . . . . . . . . . . . ..(?)..-TT-G--*
--T-TGG-...(160bp)..-GTTGT-LysA GTAAGCTC..(2071bp)..GCCTTTAG --C--TCT.......(?)..T-A----*
*
*
*
--G--GCG...(lZZbp)..CT-GCC--
CGGAGTGCAAATAC..(162bp)..CCCAAACCTCAAG 1aGluCysLysTyr . . . . . . . . . ..ProLysProGlnA -T--------G---..(162bp)..-----G------* * * * * . .. . .. . .. . . * * * * -C--C-----G---..(162bp)..G-A--GG-CA--* Asp * * * . . . . . . . . . ..Ala * AlaLys* CAGAATCTAAGAAG..(183bp)..TAAGCAAAAACAA 1aGluSerLysLys.. -G--G--A------......(?) * * * * *.. -CA-GAAAGG----..(156bp)..-C-ATC-TGC-CC * LysLysGly * . .
Figure 2. Exon/intron
boundaries
*
GTAAGTCC...(>20kb)..TAATACAG -----AAG.......(?).......... * --C--CGA...(717bp)..-TT----AAAGAAAA...
of the open reading frame of the human PTN gene
@PTN). Datafrom the humanMK gene(hMK) [14] andmouseFTN gene(mPTN) [15] are shownfor comparison.Only partial boundarysequences are presented.” - ’ represents nucleotides identicalto the sequence of the hFTN gene,” * ” represents aminoacidsidenticalto thosein the hPTN protein, ” A ” indicatesa gapin the nucleotidesequence. 1116
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0 L r PTN
cDNA
b PTN
cDNA
m
cDNA
PTN
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900 I
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A
regions hamalogy
h PTN
cDNA
h PTN
gene
PTN53 I 0
PTN33 I 10
PTNLI
/ 20
I 30
40
1
PTNI I 50
I 60
kb
Figure 3. Comparison of PTN cDNAs from different species with the organization of the human PTN gene. A: F’TN cDNAs were aligned using sequence data in EMBWGenbank [13] (r = rat, accession # M55601 [6]; b = bovine, # X52945 [9]; m = mouse, # D90225 [8]; h = human, # M57399 [19]). The homology in different overlapping regions relative to hPTN is given. The solid bars indicate regions whose antisense strands are homologous to human heat shock protein 70 cDNA (hsp; 90.7 %; # M11717) and human ribosomal protein L7 cDNA (L7; 98.6 %; # X52967). The boundaries of the 5’ and 3’ ends of the open reading frame in the FTN cDNAs are aligned as indicated by the dashed vertical line. Domains in the F’TN protein are indicated by sig ( = secretory signal peptide), cys ( = cysteine-rich domain) and nut ( = nuclear translocation signal). B: Genomic clones (pTN53, PNT33, F7'NZll and PTNI) obtained from a human library and the position and relative size of four exons containing the OF@ and one 5’ exon are shown.
found that the most 5’-end exon in clone PTN211 began at one base pair upstream of the translation initiation codon of the ORF of human PTN (see Fig. 2). To obtain upstream regions of the PTN gene, the human genomic library was screened with a 90-mer antisense oligonuclcotide Corresponding to nucleotides 100-189 (region II; see Fig. 3A) of the human PTN cDNA. Among approximately 4 X 106 PFU of the human library, two additional positive clones, PTN33 and PTN53, were obtained and mapped with EcoRI. As shown in Fig. 1A they are overlapping with each other and span about 30 kb. From a combination of 1117
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(Fig. IA), we conclude that the human PTN gene
spans at least 65 kb. The ORF of the human PTN is contained on 4 exons and their exon/intron conserved in the PTN/MK
gene family-
boundaries are
To examine the exonlintron organization in the ORF
of the human PTN gene, the DNA of positive genomic clones was digested with restriction enzymes and exon-containing fragments were detected by Southern-blotting
with the cDNA
probe containing the ORF of human PTN. These fragments were subcloned into a plasmid vector and oligonucleotide
sequencing primers corresponding to sense or antisense sequences of the
PTN cDNA were used to sequence the exon/intron boundaries. Approximately 5 kb of a 5’-end fragment in clone PTN 2 11 that hybridized with our cDNA probe were sequenced completely and 3 exons in the ORF of the human PTN gene (01, 02 and 03) were detected in this fragment (Fig. 1B). The fourth exon (04) containing the 3’-end of the ORF of human PTN was found in a 3.4 kb EcoRI-fragment in the middle of clone PTNl (Fig. 1A and 1B). These 4 exons contained the complete ORF of the human PTN gene as evidenced by our sequence data (see Fig. 2). The length of the intron between exons 01102 and 02/03 were 1129 bp and 2071 bp respectively (Fig.2). The intron between exons 03/04, however, was more than 20 kb in length (Fig. 1A and 2). We conclude from these data that the exons of the ORF in the human PTN gene span at least 24 kb. A comparison of exon/intron boundaries of human PTN (Fig. 2) with the known boundaries of the human MK gene [14] and the mouse PTN gene [15] shows that the positions of the exon/intron boundaries within the ORF were conserved in the PTN/MK
gene family among
different species (Fig. 2). For example, the exon/intron boundaries in the ORFs between human and mouse PTN genes were identical, between human PTN and MK genes were very similar, and the major variations were in the lengths of their introns (Fig. 2). Another interesting finding from the comparison was that the four exons conserved in the ORF coincided with potential functional domains in the PTN proteins (see Fig.3 and Discussion). 1118
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The S- and 3’-untranslated The S-region
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regions of the human PTN gene differ from other species.
Sequence comparisons revealed that the antisense strand of the first 97
nucleotides of the human PTN cDNA was 90.7% homologous with a stretch in the 3’untranslated region of the human hsp70 cDNA (region I; Fig. 3A). This region I in the human PTN cDNA was absent in the bovine, rat and mouse PTN cDNAs whereas the adjacent region II of the human PTN cDNA was partly homologous to the 5’-untranslated regions of the cDNAs reported for rat (82.8 % , 274 bp overlap), mouse (79.3 % , 217 bp overlap) and bovine PTN (72.3%, 184 bp overlap). The respective data are shown in Fig. 3A. As mentioned above, we had picked two clones (PTN33 and PTN53) from the genomic library using an oligonucleotide
probe corresponding to the S-end of region II (nucleotides 100-189).
We identified a 4.5 kb E!co RI fragment that hybridized with oligonucleotide
probes matching
sequences in the 5’- as well as in the 3’-ends of region II and we conclude that at least one additional exon containing the region II is located in this fragment. Furthermore, the length of the intron between this exon and the first exon containing ORF (01) was at least 12 kb (Fig. 1A). No cross-hybridization of clones PTN33 and PTN53 with an oligonucleotide
probe from
region I (nucleotides l-80) was observed. Consistent with this, 30 clones that were picked out of about 106 PFU of the human library using the region I probe (nucleotides l-80) did not hybridize with the above probe from region II (nucleotides 100-189). From this, we conclude that the 5’ untranslated region of human PTN consists of a minimum of two additional exons separated by more than 12 kb of an intronic sequence and that the most 5’-end exon containing the region I is unique in the human PTN gene. The 3’-region
-
In addition to the differences observed in the 5’ region of PTN from different
species, the 3’-regions diverge even more obviously. A data base search showed that an antisense stretch of 277 nucleotides at the extreme 3’-end of the human PTN cDNA was 98% homologous to the ORF of human ribosomal protein L7 (region IV; Fig. 3A). This homologous region includes the translation initiation site and the N-terminal 90 amino acids out of the 248 1119
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amino acids of the L7 protein. Most strikingly, the splicing site between the exon containing the 3’-end of the ORF of human PTN and the following exon located exactly to the beginning of the L7 homologous region IV. This region IV in the human PTN cDNA was missing in PTN cDNAs from other species based on sequence comparisons (Fig. 3A). On the other hand, a sequence comparison between the mouse and rat 3’-untranslated regions corresponding to the human domain IV revealed a 87% homology in a 337 nucleotide overlap. A 66% homology in a 290 nucleotide overlap was found between the respective regions of the mouse and bovine PTN cDNAs. From this, we reason that the 3’-untranslated region IV in the human PTN cDNA consists of at least one exon and that this exon is distinct from other species (Fig. 3A). Altogether, we conclude that the human PTN gene consists of at least 7 exons, four of which contain the ORF (Fig. 3).
DISCUSSION
The amino acid sequence of PTNs is extremely conserved across species [l] and this conservation is reflected in a very similar organization of the exon/intron structure comprising the open reading frame at least of the mouse and human PTN and MK genes (see Fig. 2, 3 and 114,151). We suggest that functional domains in the PTN protein are the underlying structural feature that conserved the exon/intron structure in the ORF. Each of the four exons in the ORF of human PTN gene comprises one putative functional unit of the PTN protein as depicted in Fig. 3A. For example, the putative secretory N-terminal signal peptide (“sig” in Fig.3A) [l l] is contained in the first exon of the ORF (01) and the splicing site between 01 and 02 is at the 7”’ amino acid downstream of the signal peptide cleavage site (Fig. 2 and 3). A core region of the PTN protein contains 10 cysteine residues (cys in Fig. 3A) that are conserved in the PTN/MK
family [l] and form stable disultide bridges [3]. This core region is split into two
exons 02 and 03 containing 6 and 4 cysteine residues respectively (Fig. 2 and 3). Exon 04 comprises the C-terminus of the PTN protein that contains a putative nuclear translocation signal based on its homology with histone Hl (“nut” in Fig. 3A). A comparison with the MK genes 1120
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supports this conclusion inasmuch as signal peptide and cysteine-rich domains are separated along the same boundaries as those in the PTN genes (Fig 2). In the 5’- and 3’-untranslated regions, however, the human PTN gene diverges clearly from the other known PTN genes. The most 5’-sequences of PTN cDNAs reported for the other species start more than 200 nucleotides downstream of the human cDNA (Fig. 3A). The known mouse cDNA of 1498 base pairs appears to reflect the complete mRNA closely since Northern blots gave a 1.5 kb [8] band. The same is true for the human PTN cDNA which contains 1393 base pairs [9] and results in a 1.4 kb band on a Northern blot. However, mapping of the 5’-ends by primer extension studies will be required to reveal the precise differences between species. Whether alternative splicing in the 5’-end and the use of alternative promoters as described for the MK gene [16,17] also applies to the PTN gene will be the subject of future studies. Differences in the 3’-ends of the PTN cDNAs are even more apparent since the splice junction of the exon containing the 3’-end of the ORF also defines the point of divergence between species. Only in the human gene an antisense piece from the L7 DNA appears to be spliced in at this point (Fig. 3A). The three known PTN cDNAs from other species are homologous among each other in their 3’-ends but share no sequences with the human cDNA. To what extent this may affect mRNA stability or is used as a regulatory element will have to be studied with mutational analysis of this region. It is tempting to speculate how the striking sequence homologies of the human PTN gene with the hsp70 and L7 antisense could regulate gene expression at the posttranscriptional Theoretically,
level.
the human PTN 3’-end mRNA (region IV) could hybridize with the 5’-end of the
L7 mRNA and function as a very specific inhibitor of L7 translation without affecting PTN translation. Vice versa, hsp70 mRNA could regulate PTN translation by hybridizing with the 5’end of PTN. This kind of regulation has been described for another growth factor that acts during development, basic fibroblast growth factor (bFGF) [18]. During different Xenopus developmental stages an antisense transcript to bFGF blocks production of the growth factor and it is believed that this is a mechanism of regulation of growth factor activity. Interestingly, only 1121
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the human PTN cDNA seems to contain these regulatory domains and their potential functional role remains to be studied. The role that PTN plays during development may warrant these complex possibilities of regulation.
ACKNOWLEDGMENTS: This work was supported by the National Cancer Institute grant UOI CA51908. F.C. was supported by a grant from the Institute of Clinical Pharmacology, University of Bonn, Germany.
REFERENCES
1. 2.
3. 4.
5. 6. 7. 8.
9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Biihlen, P. and Kovesdi, I. (1991) Prog. Growth Factor. Res. 3, 143-157. Biihlen, P., Miiller, T., Gautschi-Sova, P., Albrecht, U., Rasool, C.G., Decker, M., Seddon, A., Fafeur, V., Kovesdi, I., and Kretschmer, P. (1991; Growth Factors. 4, 97-107. Kuo, M.D., Oda, Y., Huang, J.S., and Huang, S.S. (1990) J. Biol. Chem. 265, 18749-18752. Courty, J., Dauchel, M.C., Caruelle, D., Perderiset, M., and Barritault, D. (1991) B&hem. Biophys. Res. Commun. 180, 145-151. Rauvala, H. (1989) EMBO J. 8, 2933-2941. Merenmies, J. and Rauvala, H. (1990) J Biol. Chem. 265, 16721-16724. Hampton, B.S., Marshak, D.R., and Burgess, W.H. (1992) Mol. Biol. Cell 3, 85-93. Tezuka, K., Takeshita, S., Hakeda, Y., Kumegawa, M., Kikuno, R., and Hashimoto-Gotoh, T. (1990) B&hem. Biophys. Res. Commun. 173, 246-251. Li, Y.S., Milner, P.G., Chauhan, A.K., Watson, M.A., Hoffman, R.M., Kodner, C.M., Milbrandt, J., and Deuel, T.F. (1990) Science 250, 1690-1694. Li, Y.S., Gurrieri, M., and Deuel, T.F. (1992) Biochem. Biophys. Res. Commun. 184, 427-432. Wellstein, A., Fang, W.J., Khatri, A., Lu, Y., Swain, S.S., Dickson, R.B., Sasse, J., Riegel, A.T., and Lippman, M.E. (1992) J. Biol. Chem. 267, 2582-2587. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular cloning. A laboratory manual, Cold Spring Harbor Laboratory Press, New York. Devereux, J., Haeberli, P., and Smithies, 0. (1984) Nucl Acid Res 12(l), 387-395. Uehara, K., Matsubara, S., Kadomatsu, K., Tsutsui, J., and Muramatsu, T. (1992) J. B&hem. (Tokyo) 111, 563-567. Naito, A., Yoshikura, H., and Iwamoto, A. (1992) Biochem. Biophys. Res. Commun. 183, 701-707. Matsubara, S., Tomomura, M., Kadomatsu, K., and Muramatsu, T. (1990) J Biol. Chem. 265, 9441-9443. Kadomatsu, K., Huang, R.P., Suganuma, T., Murata, F., and Muramatsu, T. (1990) J Cell. Biol. 110, 607-616. Kimelman, D. and Kirschner, M.W. (1989) Cell 59, 687-696. Kretschmer, P.J., Fairhurst, J.L., Decker, M.M., Chan, C.P., Gluzman, Y., Biihlen, P., and Kovesdi, I. (1991) Growth Factors. 5, 99-114.
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STRUCTURE OF THE HUMAN HEPARIN-BINDING PLEIOTROPHIN
1113-1122
GROWTH FACTOR GENE
Shoupeng Lai, Frank Czubayko, Anna Tate Riegel and Anton Wellstein’ V.T. Lombardi Cancer Center and Department of Pharmacology Georgetown University, Washington DC 20007 Received
August
7, 1992
ABSTRACT: We report that the human gene coding for the heparin-binding growth factor pleiotrophin (PTN) spans more than 65 kb and contains at least 7 exons. Analysis of human genomic DNA fragments showed that the open reading frame (ORF) is located on 4 exons. The splice sites in the ORF coincide with the boundaries of functional domains in the human PTN protein and appear to be conserved in the mouse PTN and in the related family of midkine genes. The 5’- and the 3’-ends in the untranslated regions of the human PTN are distinct from those of other species and are highly homologous to the antisense cDNAs of heat shock protein 70 and of ribosomal protein L7 respectively. These two regions are located on separate exons and could play a role in the posttranscriptional regulation of human PTN gene expression. n 1992Academic Press, Inc.
A novel gene family of hepatin-binding growth factors termed pleiotrophin/midkine
(PTN/MK)
has emerged in the last three years (for a review see [l]). PTN was originally described with different names reflecting the sources of the protein as well as its diverse biological activities (Heparin-Binding Heparin-Affin
Neurotrophic
Factor [2], Heparin-Binding
Regulated Peptide [4], Heparin-Binding
Osteoblast Specific Factor [8], Pleiotrophin
Neurite Promoting
Factor [3],
Growth-Associated Molecule
[5-71,
[9,10]). The PTN and closely related MK proteins
appear to play a role during the development of the neuroectoderm and the physiologic expression of the genes in the adult is found only in very restricted areas of the nervous system [l]. On the other hand, we reported very recently that PTN is secreted as an active growth l To whom correspondence should be addressed. Abbreviations: PTN, pleiotrophin; ORF, open reading frame; MK, midkine; bp, base pair; kb, kilo bases; PFU, plaque forming units.
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factor from human breast cancer cells and in addition to this we detected very high levels of gene expression in human melanoma cells [l 1J. We hypothesized that PTN could function as a tumor growth factor in addition to its role during development. To investigate the mechanisms involved with the regulation of PTN gene expression, we studied the structure of the PTN-gene and we report for the first time here the genomic organization of the human gene. METHODS Screening of a human genomic libraryThe human genomic library of Caucasian male placenta in the X FIX II vector was obtained from Stratagene. The genomic library was screened by plaque hybridization using standard procedures [12]. Plaques at a density of 4 X 10’ plaques/plate (15 cm diameter) were transferred onto BA85 nitrocellulose membranes (Schleicher and Schuell). DNA on the membrane was denatured with 0.2 M NaOH/l.S M NaCl and hybridized with a cDNA containing the complete open reading frame of human pleiotrophin (nucleotides 455-1005; EMBWGenbank accession # M57399; [l 11) or synthetic oligonucleotides corresponding to the antisense sequences of nucleotides l-80 and 100-189 [l l] respectively. The cDNA probe was excised from pRcICMV (Invitrogen; San Diego CA) into which the respective cDNA fragment had been cloned after reverse transcription and PCR amplification of human PTN from mRNA as described recently [l 11. The excised fragment was purified in 1.O % low melting temperature agarose gel, labeled with [ar-32P]dATP and random primed labeling kit (Boehringer Mannheim) at about 5 X 108 cpm/pg DNA, and applied at 2-5 X lo6 cpm/ml in hybridization buffer. Prehybridization and hybridization were carried out at 42 “C in 5 X SSPE, 5 X Denhardt’s solution, 0.1 mg/ml denatured salmon sperm DNA, 0.5 % SDS, and 50 % formamide [12]. After hybridization the membranes were washed in 2 X SSC/O. 1 % SDS at room temperature for 15 min, at 42 “C for 15 min, and at 65 “C for 30 min. The final washing was in 0.1 X SSC/O.l% SDS at 65 “C for 30 min. When the oligonucleotides were used as probes to screen the library, they were labeled at the 5’-end by T4 polynucleotide kinase (NEB) and [Y-~*P]ATP (NEN) to a specific activity of about l-3 X 108 cpm/pg and applied at about 10’ cpm/ml in hybridization buffer. The conditions of prehybridization, hybridization and washing were the same as with the cDNA probe except that 20 % formamide was included in the prehybridization and hybridization buffer for the 90-mer probe and final washing was at 55 “C for the 90-mer and 42 “C for the 80-mer probe. The membranes were exposed overnight to XAR-5 film with intensifying screens. Positive clones were then plaque-purified by two additional rounds of plaque hybridization with the same probe. Restriction enzyme mapping of genomic clones- The DNA of each positive XFIX II clone was completely digested with Not1 to excise out the human genomic DNA insert carrying T7 and T3 promoter sequences at its ends and then partially digested with other restriction enzymes. The partially-digested restriction fragments of the genomic DNA were separated by agarose gel electrophoresis, transferred onto Nylon membranes, and probed with a 5’-end [“PI-labeled synthetic oligonucleotide corresponding to the T7 or T3 promoter. In some additional experiments the DNA of positive clones was partially digested with one restriction enzyme, annealed with a 5’-end [32P]-labeled 12-mer oligonucleotide (Promega) complementary to the left or right COS end of the X phage, separated on an agarose gel, blotted onto Nylon membrane, and visualized by autoradiography on XAR-5 film. Subcloning of genomic DNA fragmentsThe genomic DNA fragments that contain open reading frame of human pfeiotrophin were recognized by Southern-blot analysis of the restriction fragments of positive genomic clones with an [cr-32P]dATP-labeled cDNA insert or 5’-end labled oligonucleotide probe from the human PTN open reading frame. The fragments were separated
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by agarose gel electrophoresis, recovered by electro-elution from gel slices onto NA45 DEAEnitrocellulose membranes (Schleicher and Schuell), eluted from the membranes by 1.5 M NaC1/2mM EDTARO mM Tris @H 7.8) and ligated into pBluescript II SK+ (Stratagene) or pGEM 72+ (Promega). Determination of DNA sequences and of the exon/intron boundariesPlasmid DNA was prepared using the QIAGEN plasmid DNA purification kit (Qiagen). Double-stranded DNA sequencing was done by the dideoxy chain termination method with Sequenase Version 2.0 (USB) after denaturation by 0.2 M NaOH10.2 mM EDTA-Na, and annealing with the sequencing primers at 37 “C for 30 min. To define the exon/intron boundaries, synthetic oligonucleotides corresponding to the sense or anti-sense sequences in the PTN open reading frame were used as sequencing primers. The lengths of introns were determined by their positions in the restriction maps of the genomic clones. Sequence comparisonsWe used the University of Wisconsin Genetics Computer Group Program package for homology searches and sequence comparisons [ 131.
RESULTS The human PTN gene spans at least 65 kb-
PTN was initially
The strategy for genomic cloning of human
to screen a library with a cDNA probe containing the open reading frame
(ORF) of the human PTN gene [ll].
We opted to use a probe only comprising the ORF since
the S- and 3’-untranslated regions of the human PTN cDNA contain long stretches of sequences highly homologous to other human genes and we wished to avoid picking clones containing these latter genes. The S-end of human PTN shares a stretch of 97 nucleotides homologous to the human heat shock protein 70 cDNA (hsp70; EMBL/Genbank
accession # Ml 1717) and a stretch
of 277 nucleotides at the 3’-end of the PTN cDNA is homologous to the human ribosomal protein L7 cDNA (EMBL/Genbank
accession # X52967) (for details see below; Fig. 3).
After screening of about 2 X 106 PFU of the human library, 10 positive clones were obtained and plaque purified Physical maps of these clones were constructed by partial EcoRI digestion. Since there is no EcoRI site in human PTN cDNA, complete exons should be contained in the EcoRI fragments hybridizing with a cDNA probe. EcoRI mapping and Southern-blotting analysis of these clones revealed that they belonged to two separate groups which were not overlapping with each other and spanned a region of more than 35 kb. Clones PTN211 and PTNl comprised the longest ones in the respective groups and their relative positions are illustrated in Figure 1A. After sequencing of the most 5’-end fragment among clone PTN211 and PTNl (Fig. lB), it was 1115
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PTN53
restriction map of the human PTN gene. A: Restrictionmapof genomic clones.“-‘I showsthe positionof the respectivehumanFTN genomicclone. E = EcoRI. “II” indicatesa gapbetweenclones. Closedvertical barsrepresents the positionof exonsin the ORF andthe shadedvertical bar represents the putativepositionof an exon containing part of the S-untranslatedregion. B: Sequencing strategyfor the exon/intronboundaries of the ORF. E =EcoRI, EV =E.coRV. Shadedvertical boxesshowthe lengthandpositionof exonsin the ORF (01, 02, 03, 04). The arrowsshowthe directionandpositionof each sequencing reaction. Figure 1. Partial
EXON
INTRON
hPTN mPTN hMK hPTN mPTN hMK hPTN mPTN hMK hPTN mPTN hMK hPTN mPTN hMK
. ..ATTCCTGG . ..TACT-CA. ..CCC-TCA^AATGCAGGCTCAA..(116bp)..AAAGAGAAACCAG MetGlnAlaGln . . . . . . . . . ..LysGluLysProG A----TC-T-C--G..(117bp)..-----------T* SerSer * . . .. . .. . .. . * * * "G------CAC-G-...(77bp)..GCCA-A--GAA-* * HisArg . . . . . . . . . ..AlaLys * UGTGAAG..(174bp)..AAGCAATTTGGCG 1uLysLysValLys.. . . . . . . . ..LysGlnPheGlyA -------G-----A..(174bp)..--------------A* * * * * . . .. .. . . .. . * * * -T--GGTGAA----..(158bp)..---G-G-----Asp * ValLys * . . . . . . . . . . . * Glu *
GTAAGCAG..(ll29bp)..CCATTCAG . . . . . . . . . . . . . ..(?)..-TT-G--*
--T-TGG-...(160bp)..-GTTGT-LysA GTAAGCTC..(2071bp)..GCCTTTAG --C--TCT.......(?)..T-A----*
*
*
*
--G--GCG...(lZZbp)..CT-GCC--
CGGAGTGCAAATAC..(162bp)..CCCAAACCTCAAG 1aGluCysLysTyr . . . . . . . . . ..ProLysProGlnA -T--------G---..(162bp)..-----G------* * * * * . .. . .. . .. . . * * * * -C--C-----G---..(162bp)..G-A--GG-CA--* Asp * * * . . . . . . . . . ..Ala * AlaLys* CAGAATCTAAGAAG..(183bp)..TAAGCAAAAACAA 1aGluSerLysLys.. -G--G--A------......(?) * * * * *.. -CA-GAAAGG----..(156bp)..-C-ATC-TGC-CC * LysLysGly * . .
Figure 2. Exon/intron
boundaries
*
GTAAGTCC...(>20kb)..TAATACAG -----AAG.......(?).......... * --C--CGA...(717bp)..-TT----AAAGAAAA...
of the open reading frame of the human PTN gene
@PTN). Datafrom the humanMK gene(hMK) [14] andmouseFTN gene(mPTN) [15] are shownfor comparison.Only partial boundarysequences are presented.” - ’ represents nucleotides identicalto the sequence of the hFTN gene,” * ” represents aminoacidsidenticalto thosein the hPTN protein, ” A ” indicatesa gapin the nucleotidesequence. 1116
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0 L r PTN
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b PTN
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m
cDNA
PTN
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regions hamalogy
h PTN
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h PTN
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PTN53 I 0
PTN33 I 10
PTNLI
/ 20
I 30
40
1
PTNI I 50
I 60
kb
Figure 3. Comparison of PTN cDNAs from different species with the organization of the human PTN gene. A: F’TN cDNAs were aligned using sequence data in EMBWGenbank [13] (r = rat, accession # M55601 [6]; b = bovine, # X52945 [9]; m = mouse, # D90225 [8]; h = human, # M57399 [19]). The homology in different overlapping regions relative to hPTN is given. The solid bars indicate regions whose antisense strands are homologous to human heat shock protein 70 cDNA (hsp; 90.7 %; # M11717) and human ribosomal protein L7 cDNA (L7; 98.6 %; # X52967). The boundaries of the 5’ and 3’ ends of the open reading frame in the FTN cDNAs are aligned as indicated by the dashed vertical line. Domains in the F’TN protein are indicated by sig ( = secretory signal peptide), cys ( = cysteine-rich domain) and nut ( = nuclear translocation signal). B: Genomic clones (pTN53, PNT33, F7'NZll and PTNI) obtained from a human library and the position and relative size of four exons containing the OF@ and one 5’ exon are shown.
found that the most 5’-end exon in clone PTN211 began at one base pair upstream of the translation initiation codon of the ORF of human PTN (see Fig. 2). To obtain upstream regions of the PTN gene, the human genomic library was screened with a 90-mer antisense oligonuclcotide Corresponding to nucleotides 100-189 (region II; see Fig. 3A) of the human PTN cDNA. Among approximately 4 X 106 PFU of the human library, two additional positive clones, PTN33 and PTN53, were obtained and mapped with EcoRI. As shown in Fig. 1A they are overlapping with each other and span about 30 kb. From a combination of 1117
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(Fig. IA), we conclude that the human PTN gene
spans at least 65 kb. The ORF of the human PTN is contained on 4 exons and their exon/intron conserved in the PTN/MK
gene family-
boundaries are
To examine the exonlintron organization in the ORF
of the human PTN gene, the DNA of positive genomic clones was digested with restriction enzymes and exon-containing fragments were detected by Southern-blotting
with the cDNA
probe containing the ORF of human PTN. These fragments were subcloned into a plasmid vector and oligonucleotide
sequencing primers corresponding to sense or antisense sequences of the
PTN cDNA were used to sequence the exon/intron boundaries. Approximately 5 kb of a 5’-end fragment in clone PTN 2 11 that hybridized with our cDNA probe were sequenced completely and 3 exons in the ORF of the human PTN gene (01, 02 and 03) were detected in this fragment (Fig. 1B). The fourth exon (04) containing the 3’-end of the ORF of human PTN was found in a 3.4 kb EcoRI-fragment in the middle of clone PTNl (Fig. 1A and 1B). These 4 exons contained the complete ORF of the human PTN gene as evidenced by our sequence data (see Fig. 2). The length of the intron between exons 01102 and 02/03 were 1129 bp and 2071 bp respectively (Fig.2). The intron between exons 03/04, however, was more than 20 kb in length (Fig. 1A and 2). We conclude from these data that the exons of the ORF in the human PTN gene span at least 24 kb. A comparison of exon/intron boundaries of human PTN (Fig. 2) with the known boundaries of the human MK gene [14] and the mouse PTN gene [15] shows that the positions of the exon/intron boundaries within the ORF were conserved in the PTN/MK
gene family among
different species (Fig. 2). For example, the exon/intron boundaries in the ORFs between human and mouse PTN genes were identical, between human PTN and MK genes were very similar, and the major variations were in the lengths of their introns (Fig. 2). Another interesting finding from the comparison was that the four exons conserved in the ORF coincided with potential functional domains in the PTN proteins (see Fig.3 and Discussion). 1118
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The S- and 3’-untranslated The S-region
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regions of the human PTN gene differ from other species.
Sequence comparisons revealed that the antisense strand of the first 97
nucleotides of the human PTN cDNA was 90.7% homologous with a stretch in the 3’untranslated region of the human hsp70 cDNA (region I; Fig. 3A). This region I in the human PTN cDNA was absent in the bovine, rat and mouse PTN cDNAs whereas the adjacent region II of the human PTN cDNA was partly homologous to the 5’-untranslated regions of the cDNAs reported for rat (82.8 % , 274 bp overlap), mouse (79.3 % , 217 bp overlap) and bovine PTN (72.3%, 184 bp overlap). The respective data are shown in Fig. 3A. As mentioned above, we had picked two clones (PTN33 and PTN53) from the genomic library using an oligonucleotide
probe corresponding to the S-end of region II (nucleotides 100-189).
We identified a 4.5 kb E!co RI fragment that hybridized with oligonucleotide
probes matching
sequences in the 5’- as well as in the 3’-ends of region II and we conclude that at least one additional exon containing the region II is located in this fragment. Furthermore, the length of the intron between this exon and the first exon containing ORF (01) was at least 12 kb (Fig. 1A). No cross-hybridization of clones PTN33 and PTN53 with an oligonucleotide
probe from
region I (nucleotides l-80) was observed. Consistent with this, 30 clones that were picked out of about 106 PFU of the human library using the region I probe (nucleotides l-80) did not hybridize with the above probe from region II (nucleotides 100-189). From this, we conclude that the 5’ untranslated region of human PTN consists of a minimum of two additional exons separated by more than 12 kb of an intronic sequence and that the most 5’-end exon containing the region I is unique in the human PTN gene. The 3’-region
-
In addition to the differences observed in the 5’ region of PTN from different
species, the 3’-regions diverge even more obviously. A data base search showed that an antisense stretch of 277 nucleotides at the extreme 3’-end of the human PTN cDNA was 98% homologous to the ORF of human ribosomal protein L7 (region IV; Fig. 3A). This homologous region includes the translation initiation site and the N-terminal 90 amino acids out of the 248 1119
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amino acids of the L7 protein. Most strikingly, the splicing site between the exon containing the 3’-end of the ORF of human PTN and the following exon located exactly to the beginning of the L7 homologous region IV. This region IV in the human PTN cDNA was missing in PTN cDNAs from other species based on sequence comparisons (Fig. 3A). On the other hand, a sequence comparison between the mouse and rat 3’-untranslated regions corresponding to the human domain IV revealed a 87% homology in a 337 nucleotide overlap. A 66% homology in a 290 nucleotide overlap was found between the respective regions of the mouse and bovine PTN cDNAs. From this, we reason that the 3’-untranslated region IV in the human PTN cDNA consists of at least one exon and that this exon is distinct from other species (Fig. 3A). Altogether, we conclude that the human PTN gene consists of at least 7 exons, four of which contain the ORF (Fig. 3).
DISCUSSION
The amino acid sequence of PTNs is extremely conserved across species [l] and this conservation is reflected in a very similar organization of the exon/intron structure comprising the open reading frame at least of the mouse and human PTN and MK genes (see Fig. 2, 3 and 114,151). We suggest that functional domains in the PTN protein are the underlying structural feature that conserved the exon/intron structure in the ORF. Each of the four exons in the ORF of human PTN gene comprises one putative functional unit of the PTN protein as depicted in Fig. 3A. For example, the putative secretory N-terminal signal peptide (“sig” in Fig.3A) [l l] is contained in the first exon of the ORF (01) and the splicing site between 01 and 02 is at the 7”’ amino acid downstream of the signal peptide cleavage site (Fig. 2 and 3). A core region of the PTN protein contains 10 cysteine residues (cys in Fig. 3A) that are conserved in the PTN/MK
family [l] and form stable disultide bridges [3]. This core region is split into two
exons 02 and 03 containing 6 and 4 cysteine residues respectively (Fig. 2 and 3). Exon 04 comprises the C-terminus of the PTN protein that contains a putative nuclear translocation signal based on its homology with histone Hl (“nut” in Fig. 3A). A comparison with the MK genes 1120
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supports this conclusion inasmuch as signal peptide and cysteine-rich domains are separated along the same boundaries as those in the PTN genes (Fig 2). In the 5’- and 3’-untranslated regions, however, the human PTN gene diverges clearly from the other known PTN genes. The most 5’-sequences of PTN cDNAs reported for the other species start more than 200 nucleotides downstream of the human cDNA (Fig. 3A). The known mouse cDNA of 1498 base pairs appears to reflect the complete mRNA closely since Northern blots gave a 1.5 kb [8] band. The same is true for the human PTN cDNA which contains 1393 base pairs [9] and results in a 1.4 kb band on a Northern blot. However, mapping of the 5’-ends by primer extension studies will be required to reveal the precise differences between species. Whether alternative splicing in the 5’-end and the use of alternative promoters as described for the MK gene [16,17] also applies to the PTN gene will be the subject of future studies. Differences in the 3’-ends of the PTN cDNAs are even more apparent since the splice junction of the exon containing the 3’-end of the ORF also defines the point of divergence between species. Only in the human gene an antisense piece from the L7 DNA appears to be spliced in at this point (Fig. 3A). The three known PTN cDNAs from other species are homologous among each other in their 3’-ends but share no sequences with the human cDNA. To what extent this may affect mRNA stability or is used as a regulatory element will have to be studied with mutational analysis of this region. It is tempting to speculate how the striking sequence homologies of the human PTN gene with the hsp70 and L7 antisense could regulate gene expression at the posttranscriptional Theoretically,
level.
the human PTN 3’-end mRNA (region IV) could hybridize with the 5’-end of the
L7 mRNA and function as a very specific inhibitor of L7 translation without affecting PTN translation. Vice versa, hsp70 mRNA could regulate PTN translation by hybridizing with the 5’end of PTN. This kind of regulation has been described for another growth factor that acts during development, basic fibroblast growth factor (bFGF) [18]. During different Xenopus developmental stages an antisense transcript to bFGF blocks production of the growth factor and it is believed that this is a mechanism of regulation of growth factor activity. Interestingly, only 1121
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the human PTN cDNA seems to contain these regulatory domains and their potential functional role remains to be studied. The role that PTN plays during development may warrant these complex possibilities of regulation.
ACKNOWLEDGMENTS: This work was supported by the National Cancer Institute grant UOI CA51908. F.C. was supported by a grant from the Institute of Clinical Pharmacology, University of Bonn, Germany.
REFERENCES
1. 2.
3. 4.
5. 6. 7. 8.
9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Biihlen, P. and Kovesdi, I. (1991) Prog. Growth Factor. Res. 3, 143-157. Biihlen, P., Miiller, T., Gautschi-Sova, P., Albrecht, U., Rasool, C.G., Decker, M., Seddon, A., Fafeur, V., Kovesdi, I., and Kretschmer, P. (1991; Growth Factors. 4, 97-107. Kuo, M.D., Oda, Y., Huang, J.S., and Huang, S.S. (1990) J. Biol. Chem. 265, 18749-18752. Courty, J., Dauchel, M.C., Caruelle, D., Perderiset, M., and Barritault, D. (1991) B&hem. Biophys. Res. Commun. 180, 145-151. Rauvala, H. (1989) EMBO J. 8, 2933-2941. Merenmies, J. and Rauvala, H. (1990) J Biol. Chem. 265, 16721-16724. Hampton, B.S., Marshak, D.R., and Burgess, W.H. (1992) Mol. Biol. Cell 3, 85-93. Tezuka, K., Takeshita, S., Hakeda, Y., Kumegawa, M., Kikuno, R., and Hashimoto-Gotoh, T. (1990) B&hem. Biophys. Res. Commun. 173, 246-251. Li, Y.S., Milner, P.G., Chauhan, A.K., Watson, M.A., Hoffman, R.M., Kodner, C.M., Milbrandt, J., and Deuel, T.F. (1990) Science 250, 1690-1694. Li, Y.S., Gurrieri, M., and Deuel, T.F. (1992) Biochem. Biophys. Res. Commun. 184, 427-432. Wellstein, A., Fang, W.J., Khatri, A., Lu, Y., Swain, S.S., Dickson, R.B., Sasse, J., Riegel, A.T., and Lippman, M.E. (1992) J. Biol. Chem. 267, 2582-2587. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular cloning. A laboratory manual, Cold Spring Harbor Laboratory Press, New York. Devereux, J., Haeberli, P., and Smithies, 0. (1984) Nucl Acid Res 12(l), 387-395. Uehara, K., Matsubara, S., Kadomatsu, K., Tsutsui, J., and Muramatsu, T. (1992) J. B&hem. (Tokyo) 111, 563-567. Naito, A., Yoshikura, H., and Iwamoto, A. (1992) Biochem. Biophys. Res. Commun. 183, 701-707. Matsubara, S., Tomomura, M., Kadomatsu, K., and Muramatsu, T. (1990) J Biol. Chem. 265, 9441-9443. Kadomatsu, K., Huang, R.P., Suganuma, T., Murata, F., and Muramatsu, T. (1990) J Cell. Biol. 110, 607-616. Kimelman, D. and Kirschner, M.W. (1989) Cell 59, 687-696. Kretschmer, P.J., Fairhurst, J.L., Decker, M.M., Chan, C.P., Gluzman, Y., Biihlen, P., and Kovesdi, I. (1991) Growth Factors. 5, 99-114.
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