the of chemistry vol. no. of 8, 10891-10898, 1994 1994 for ... · the journal of biological...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 269, No. 14, Issue of April 8, pp. 10891-10898, 1994 Printed in U.S.A.

Characterization of the Chromosomal Gene and Promoter for Human Insulin-like Growth Factor Binding Protein-5”

(Received for publication, September 24, 1993, and in revised form, December 29, 1993)

To better understand the regulation of insulin-like growth factor binding protein-5 (IGFBP-5) expression, we cloned the IGFBP-5 gene from human genomic librar- ies and identified a region in the 5’ flanking sequence which functions as a promoter. The human IGFBP-6 gene is divided into four exons which, primarily due to a first intron of -25 kilobases, span -33 kilobases of DNA. Southern analysis identified a single copy of the IGFBP-5 gene in the haploid human genome, and several independent mapping strategies found this gene tightly linked with, and in opposite transcriptional orientation to, the IGFBP-2 gene at chromosomal region 2q33-34. Primer extension studies identified the IGFBP-5 mRNA cap site 772 base pairs (bp) 5’ to the first nucleotide of the translation start codon. Analysis of the 5”flanking sequence identified a potential TATA element beginning 33 bp 5’ to the mRNA cap site. When a DNA fragment containing this cap site and 461 bp of upstream sequence was placed 5’ to the chloramphenicol acetyltransferase reporter gene and transfected into MDA-MB-468 human breast cancer cells, it directed chloramphenicol acetyltransferase expression in an orientation-specific manner, suggesting that this region contains elements essen- tial for IGFBP-5 promoter activity.

Insulin-like growth factor binding protein-5 (IGFBP-5)’ was originally identified in and purified from rat serum (11, human bone (21, and medium conditioned by human osteoblast-like cells (3) as a protein which bound IGF peptides with high af- finity. Isolation and characterization of human and rat IG- FBP-5 cDNA clones confirmed that the sequence of this binding protein is unique but homologous to sequences encoding the

* This work was supported by Caroline Weiss Law Karolinska-Baylor Research Fellowship (to S. V. A.), National Institutes of Health Grant R 0 1 DK-38773 (to D. R. P.), the Swedish Medical Research Council, the Swedish Cancer Foundation, the Swedish Medical Society, the Magnus Bergvall Foundation, the Claes Grochinsky Foundation, the Nilsson- Ehle Foundation, “Svenska Sallskapet for Medicinsk Forskning“ and “Forenade Liv“ Mutual Group Life Insurance Company. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted

27560. to the GenBankmIEMBL Data Bank with accession number(s) L27556-

Richmond, CA 94804. /I Present address: LXR Biotechnology, Inc., 1401 Manna Way South,

Hospital, Clinical Care Center, MC#3-2482,6621 Fannin St., Houston, ** To whom correspondence should be addressed: Texas Children’s

TX 77030. The abbreviations used are: IGFBP-5, insulin-like growth factor

binding protein-5; h, human; PCR, polymerase chain reaction; kb, kilo- base(s); bp, base paifis); PFGE, pulsed field gel electrophoresis; CAT, chloramphenicol acetyltransferase.

other five members of the IGFBP family (1,4). Additional studies found that IGFBP-5 was expressed by a wide variety of tissues (1, 4). Similar to other IGFBPs, IGFBP-5 may potenti- ate (2) or inhibit (5 , 6) IGF action, with different effects probably depending on post-translational modifications of IGFBP-5 and on the specific cell targets of IGF action.

The ability of IGF peptides to affect a target tissue may be regulated by multiple mechanisms, among them the local regulation of tissue IGFBP levels. In the case of IGFBP-5, this mechanism may be important since physiologic andor hormonal influences strongly regulate IGFBP-5 expression in certain tissues or cells (7-11). In some instances IGFBP-5 protein levels appear to be regulated by post-translational events (9, 11). However, in other cases IGFBP-5 expression is clearly regulated at the level of mRNA abundance (7-11). Thus, factors regulating IGFBP-5 mRNA abundance in a particular tissue may profoundly influence the effect of IGF peptides on that tissue.

Multihormonal regulation of IGFBP-5 mRNA levels may be controlled at the level of gene transcription, similar to the regulation of IGFBP-1 expression by multiple hormones (7-15). As a first step toward analyzing how IGFBP-5 gene transcription may be regulated by hormonal and other influences, we have characterized the gene organization and mRNA cap site for human IGFBP-5 (hIGFBP-51, and we have identified a region of 5”flanking sequence with structural and functional characteristics typical for a eukaryotic gene promoter.

EXPERIMENTAL PROCEDURES General Methods-Methods used are from Maniatis et al . (16) unless

otherwise stated. Oligonucleotides for sequencing, primer extension, and polymerase chain reaction (PCR) amplifications were synthesized by National Biosciences (Plymouth, MN), DNA International (Lake Os- wego, OR), Symbicom AB (Umel, Sweden), or the Department of Clini- cal Genetics, Karolinska Institute (Pharmacia Gene Assembler).

cDNA Clones-The hIGFBP-5 cDNA clones BP-6.1 and BP-6.12 (4) were used as hybridization probes and for DNA sequencing. Baaed on consensus nomenclature for IGFBPs (17), these cDNA clones will be referred to here as BP-5.1 and BP-5.12, respectively. The 5.4-kb BP-5.12 clone was cleaved with BgZII; cDNA fragments of 3.8, 1.0, and 0.6 kb were isolated and used as hybridization probes after 32P-labeling. The 3.8-kb 3”fragment was subcloned into pSP73 and then sfquenced by the dideoxy chain termination method (18).

Isolation of Genomic Clones-The 1.7-kb cDNA clone BP-5.1 (4) was labeled with 32P and used to screen a human leukocyte genomic library constructed in phage EMBL 3 (Clontech, Palo Alto, CA) as described previously (19). A single AIGFBP-5 clone, hgBP5-17, was plaque purified and subjected to Southern analysis using as probe a 32P-labeled, 455-bp BgZII fragment of the BP-5.1 cDNA clone, which contains 43 bp of 5’-untranslated sequence and the first 408 bp of hIGFBP-5-coding sequence. Fragments of interest were subcloned, and DNA sequence analysis was performed as described above.

The genomic clone phBP5-BS6.6, derived from the AIGFBP-5 clone hgBP5-17, was 32P-labeled and used as hybridization probe to screen a

10891

10892 hIGFBP-5 Gene and Promoter

human cosmid (pWE15) library prepared from placental DNA (20) which was kindly provided by Dr. Glen Evans, the SALK Institute, San Diego, CA. Cosmid DNA was isolated from purified positive clones by a mini-lysate procedure (21) and subjected to Southern analysis using the three BglII fragments from the BP-5.12 cDNA as probes. Selected cosmid fragments hybridizing with the 32P-labeled BglII probes were subcloned in pGEM3Zff+), and areas of interest in the subclones were sequenced as described above.

Southern Analysis-Total human DNA was isolated from peripheral leukocytes by standard methods (22). Samples of total human DNA, of cosmid and A genomic DNA, or of DNA from somatic human-rodent cell hybrids and from normal Chinese hamster and mouse cells (NIGMS Coriell Cell Repositories) were cleaved with restriction endonucleases; cleaved DNA was electrophoresed and transferred to filters, and the filters prehybridized, hybridized, and autoradiographed as described (22). Filters were washed in 0.1 x SSC, 0.1% SDS at 65 "C after hybridization to cDNA or genomic probes, and washed in 6 x SSC at 65 "C after hybridization to oligonucleotide probes.

PCR Analysis-PCR used 0.25 p~ of each primer, 0.2 m~ of each dNTP, and 1.5 units of Taq-polymerase (Perkin-Elmer Cetus) in 45 mM KCl, 1.0 mM MgCI,, 10 mM Tris-HCI, pH 8.4 at 70 "C, 0.1% Tween 20; final volume was 50 pl. PCR products, analyzed in and isolated from agarose gels, were used to estimate the size of introns or of the 3'- untranslated region of IGFBP-5 or as 32P-labeled probes.

To determine chromosomal localization, PCR was performed in the above buffer; 50 ng of human DNA, 50 ng of hamster DNA, and 75 ng of DNA from somatic human-rodent cell hybrids served as templates in separate reactions, while oligonucleotides BP5-9 and BP5-18 (see Figs. 1 and 3) served as primers for each reaction. Samples were incubated for 3 min at 95 "C followed by 35 cycles at 95 "C for 1 min, 55 "C for 1 min, and 72 "C for 3 min. PCR products were separated in 1% agarose gels next to DNA length markers.

Fluorescent in Situ Hybridization-Slides with human metaphase chromosomes, prepared using lymphocyte cultures from normal con- trols (23), were prehybridized and hybridized as described (24); plasmids phBP5-E4.6, phBP5E2.5, and phBP5-E5.6 (Fig. 2) were used as probes after labeling with biotin-16-dUTP (Boehringer Mannheim) by nick translation. The three plasmids were pooled in a mixture of 50% formamide, 2 x SSC, 1% Tween 20,10% dextran sulfate, 1 pg of human Cot-I DNA (Life Technologies, Inc.), and 9 pg of salmon sperm DNA were denatured at 75 "C for 5 min and then preannealed a t 37 "C for 30 min. Hybridization was performed at 37 "C overnight. The signal was made fluorescent, amplified, and the chromosomes counterstained as described (23). Results were analyzed in a confocal laser scanning mi- croscope (Leica) followed by destaining of the metaphase chromosomes and subsequent quinacrine fluorescence by quinacrine (QFQ)-banding.

Pulsed Field Gel Electrophoresis (PFGE) and PFGE Probes- Cultured lymphoblastoid cells from two normal humans were embedded in agarose plugs and proteinase K treated as described (25) and then cleaved with restriction enzymes BssHII, EagI, MluI, NaeI, NarI, NotI, NruI, SacII, or SfiI. Fragments and size markers were separated using PFGE as described (25); PFGE was performed a t 170 V and pulse time

was 180 x 180 s for 20 h and then 90 x 90 s for a further 20 h. Southern analysis was performed as described above, using 32P-labeled 5' and 3' regions of the hIGFBP-2 and -5 genes in succession to probe the same filter. AnXbaIISmaI fragment from the phIGFBP2-XE6.4 subclone (26) was the Fj"IGFBP-2 probe, the phBP5E4.6 subclone was the 5'-IG- FBP-5 probe, and PCR products from cosmid clone chBP2-2.4 (26) and subclone phBP5-E2.5 served as 3'-IGFBP-2 and 3'-IGFBP-5 probes, respectively. Before rehybridization, the filter was incubated in 0.1 x SSC, 0.1% SDS for 3 x 10 min at 100 "C and autoradiographed to document removal of previous probe.

Plasmid Construction-Plasmid phBP5-HBl1.1, derived from AIG- FBP-5 clone hgBP5-17, was cleaved with HindIII and PstI. A 484-bp fragment spanning from -461 to 23 bp relative to the mFWA cap site was inserted in the sense orientation at the PstI and HindIII sites 5' to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene in the promoterless pCAT(An) plasmid (27), creating p461CAT. The promoter fragment was then cut out of p461CAT with BamHI and inserted into the pCAT(An) cloning cassette at the same BamHI sites to create the antisense promoter construct p461CAT(AS). Both constructs were sequenced to determine orientation.

Cell Culture and DNA Zkansfection-MDA-MB-468 human breast cancer cells, obtained from Dr. Douglas Yee, University of Texas Health Science Center at San Antonio, were grown in Dulbecco's modified Ea- gle's medium containing 10% fetal calf serum in a 5% CO, atmosphere (28). For transfection, MDA-MB-468 cells were seeded a t 1-2 x lo6 cells/60-mm plate and transfected 18 h later by calcium phosphate precipitation (29) as described (30). Each plate of cells was transfected with 10 pg of plasmid DNA, and 3 pg of pRSVL plasmid containing the Rous sarcoma virus promoter 5' to the luciferase gene was cotransfected to control for transfection efficiency (13, 30, 31). After incubation in serum-containing medium for 22 h, cells were collected and their cytosol assayed for protein content, and for CAT and luciferase activity, as described (32).

Chloramphenicol Acetyltransferase and Luciferase Assays-CAT assays were performed according to the method of Gorman et al. (33), and luciferase assays were performed after the method of de Wet et al. (31), as previously described (30).

Primer Extension of Native IGFBP5-mRNA-MDA-MB-468 cells, seeded a t a density of 2 x lo6 cells/100 mm plate, were harvested after a 40-h incubation in serum-containing medium; total RNA was then isolated using the acid guanidinium thiocyanate-phenol-chloroform ex- traction method (34). Primer extension reactions were performed using a kit (Promega) and following the recommendations of the manufac- turer except for modifications mentioned below. Fifty pg of MDA-MB- 468 total RNA was denatured at 85 "C for 5 min and mixed with 1 pmol of oligonucleotide primer 5:PE (Fig. 6) which had been end-labeled with 32P by T4-kinase. The mixture was incubated at 80 "C for 5 min, cooled over 15 min to 55 "C, and then kept at 27 "C for 10 min. The 5:PE primer was extended a t 42 "C for 30 min using 30 units of avian myeloblastosis virus reverse transcriptase per reaction. The sample was further treated with RNase A and 0.5 M EDTA followed by ethanol precipitation as described (19), and was then analyzed on a 6.5% poly-

2342 ACATGTGCAT ATTTCATTCC CCAGGCAGAC ATTTTTTAGA AATCAATALA TGCCCCMTA TTGGAAAGAC TTGTTCTTCC ACGGTGACTA CAGTACATGC TGAAGCGTGC CGTTTCAGCC 2462 CTC-T TCAATTTGTA AGTAGCGCAC GAGCCTCTGT GGGGGAGGAT AGGCTGAAAA AAAAMGTGG GCTCGTIZP ATCTACAGGA CTCCATATAG TCATATATAG GCATATAAAT 2502 CTATGCTTTT TCTTTGTTTT TTTCTTTCTT CCTTTCTTTC AAAGGTTTGC ATTMCTTTT CAAAGTAGTT CCTATAGGGG CATTGAGGAG CTTCCTCATT CTGGGAAAAC TGAGAAAACC 2702 CATATTCTCC T M T A C M C C CGTAATAGCA TTTTTGCCTG CCTCGAGGCA GAGTTTCCCG TGAGCAATAA ACTCAGCTTT TTTGTGGGGC ACAGTACTGG ATTTGACAGT GATTCCCCAC 2822 GTGTGTTCAT CTGCACCCAC CGAGCCAGGC AGAGGCCAGC CCTCCGTGGT GCACACAGCA CGCGCCTCAG TCCATCCCAT TTTAGTCTTT AAACCCTCAG GAAGTCACAG TCTCCGGACA 2942 CCACACCACA TTGAGCCCAA CAGGTCCACG ATGGATCCAC CTAGTCCCAC CCCAGCCTTT TTCTTTCATC TGAACAGAAT GTGCATTTTT GGRAGCCTCC CTCACTCTCC ATGCTGGCAG 3062 AGCAGGAGGG AGACTGMGT MGAGATGGC AGAGGGAGAT GGTGGCAAAA AGGTTTAGAT GCAGGAGAAC AGTAAGATGG ATGGTTCCGG CCAGAGTCGA TGTGGGGAGG RACAGAGGGC 3102 TGAAGGGAGA GGGGGCTGAC TGTTCCATTC TAGCTTTGGC ACMAGCAGC AGAAAGGGGG MAAGCCAAT AGAAATTTCC TTAGCTTCCC CACCATATGT ATTTTCRTGG ATTTGAGAGG

3422 TCAGTCTAAA CTGGCCATGC TTTGGMGGG ACAAGACTAT GTGCTCCGCT GCCCACCTTC AGCCTGCAAT GAGGGACTGA GGCCCACGAG TCTTTCCAGC TCTTCCTCCA TTCTGGCCAG 3302 AAAGAGAGGA AMTGGGGGA ATGGGTTGCA MATAGAAAT GAGCTTMTC CAGGCCGCAG AGCCAGGGRA GGTGAGTAAC CTTAGGAGGG TGCTAGACTT TAGAAGCCAG ATAGGAAGAA

3542 TCCCTGCATC CTCCCTGGGG TGGAGGATGG AAGGMAGCT GGGACAAGCA GGGAACGCAT GATTCAGGGA TGCTGTCACT CGGCAGCCAG ATTCCGAAAC TCCCATTCTC CAATGACTTC 3662 CTCAACCMT GGGTGGCCTT GTGACTGTTC TTTAAGGCTG AAGATATCCA GGAAAGGGGG CTTGGACACT GGCCAAGGAG ACCCCTTCGT GCTGTGGACA CAGCTCTCTT CACTCTTTGC 3182 TCATGGCATG ACACAGCGGA GACCGCCTCC AACAACGAAT TTGGGGCTAC GMGAGGAAT A G C G A M M G CAAATCTGTT TCAACTGATG GGAACCCTAT AGCTATAGAA CTTGGGGGCT 3902 ATCTCCTATG CCCCTGGACA GGACAGTTGG CTGGGGACAG GAGAAGTGCT CAATCTTCAT GAGACRAAGG GGCCCGATCA AGGCAGCCAC AAGGCCTTGA CCTGCCGAGT CAGCATGCCC 4022 CATCTCTCTC GACAGCTGTC CCCTAAACCC MCTCACGTT TCTGTATGTC TTAGGCCAGT ATCCCAAACC TCTTCCACGT CACTGTTCTT TCCACCCATT CTCCCTTTGC ATCTTGAGCA 4142 GTTATCCAAC TAGGATCTGC CRAGTGGATA CTGGGGTGCC ACTCCCCTGA GAAMGACTG AGCCAGGMC TACAAGCTCC CCCCACATTC CTCCCAGCCT GGACCTAATT CTTGAGAGGG 4262 GCTCTCTCTT CACGGACTGT GTCTGGACTT TGAGCAGGCT TCTGCCCCTT GCGTTGGCTC TTTGCTGCCA GCCATCAGGT GGGGGATTAG AGCCTGGTGT AAGTGCGCCA GACTCTTCCG 4382 GTTTCCAAAG TTCGTGCCTG CGAACCCAAA CCTGTGAGTC TCTTCTGCAT GCAGGAGTTT CTCCTGGGCA GCTGGTCACT CCCCAGAGAA GCTGGGCCTT CATGGACACA TGGAACTAAG 4502 CCTCCCAAAT GGGAGTTCTG GCTGAGCCCA GGGTGGGGAG ATCCTGGGAA GGGAGGCACT GGAGGAAGAC GGCACCTCTT CCCCCATGGC AGGGTGTGAG GGAGGCAGGT TTGGAATGGT

4142 GCCCACTCCC ATGCTCACAC CCACAGAAGG TCTTCCCATC CCCTTTAGAT TCGTGCCTCA CTCCACCAGT GAGGAAGATG CCTCTGTCTT TCCCACGACT GCCAGGAGAT AGGGAAGCCC 4622 GCGAGTATGG CAATCTAAGC AGGGGTCTGG TCTCTTTGAC TCCAGGCTCG CTTTGGCCGA CTGTCTGCTC ACCCAGAGAC CTTGGACTCC GGACTATCCA TGGCTCCGAA TCTAAGTGCT

4862 AGCCAGGACT GACCCTCCTT CCTCCAGCCT GCCCTGACCC ACCTGGCAAA GCAGGGCACA TGGGGAGGAA GAGACTGGAA CCTTTCTTTG ACAGCCAGGC CTAGACAGAC AGGCCTGGGG 4902 ACACTGGCCC ATGAGGGGAG GAAGGCAGGC GCACGAGGTC CAGGGAGGCC CTTTTCTGAT CATGCCCCTT CTCTCCCACC CCATCTCCCC ACCACCACCT CTGTGGCCTC CATGGTACCC 5102 CCACAGGGCT GGCCTCCCCT AGAGGGTGGG CCTCAACCAC CTCGTCCCGC CACGCACCGG TTAGTGAGAC AGGGCTGCCA CGCAACCGCC AAGCCCCCCT CAAGGTGGGA CAGTACCCCG 5222 GACCCATCCA CTCACTCCTG AGAGGCTCCG GCCCAGAATG GGAACCTCAG AGAAGAGCTC TAAGGAGAAG AAACCCChTA GCGTCAGAGA GGATATGTCT GGCTTCCAAG AGAAAGGAGG 5342 CTCCGTTTTG CAAAGTGGAG GAGGGACGAG GGACAGGGGT TTCACCAGCC AGCAACCTGG GCCTTGTACT GTCTGTGTTT TTAAAACCAC TAAAGTGCAA GAATTACATT GCACTGTTTC 5462 TCCRCTTTTT ATTTTCTCTT AGGCTTTTGT TTCTATTTCA AACATACTTT CTTGGTTTTC TAATGGAGTA TATAGTTTAG TCATTTCACA GACTCTGGCC TCCTCTCCTG AAATCCTTTT 5502 GGATGGGGAA AGGGAAGGTG GGGAGGGTCC GAGGGGAAGG GGACCCCAGC TTCCCTGTGC CCGCTCACCC CACTCCACCA GTCCCCGGTC GCCAGCCGGA GTCTCCTCTC TACCGCCACT 5702 GTCACACCGT AGCCCACATG GATAGCACAG TTGTCAGACA AGATTCCTTC AGATTCCGAG TTGCTACCGG TTGTTTTCGT TGTTGTTGTT GTTGTTTTTC TTTTTCTTTT TTTTTTTGAA 5022 GACAGCAATA ACCACAGTAC ATATTACTGT AGTTCTCTAT AGTTTTACAT ACATTCATAC CATAACTCTG TTCTCTCCTC TTTTTTGTTT TCAACTTTAA AAACAAAAAT AAACGATGAT 5942 AATCTTTACT GGTGAAAAGG A T G G . Y I U Y T C A A C A A ATGCAACCAG TTTGTGAGY y

FIG. 1.3'-Untranslated sequence of hIGFEiP-6 cDNA. Sequence from hIGFBP-5 cDNAclone BP-5.12 begins with the nucleotide that follows the last thymidine residue reported previously (4). Nucleotides are numbered on the left; numbers refer to the distance, in bp, 3' to the mRNA cap site. An AAATAAA polyadenylation signal, poly(A) motif, and two ATITA motifs are underlined and in bold print. Within the sequence, oligonucleotides BP5-19 and BP5-21 span from bp 3117 to 3137 and from bp 4445 to 4462, respectively, while BP5-16, BP5-18, and BP5-20 are complementary to sequence from bp 5979 to 5999, bp 3157 to 3175, and bp 4485 to 4504, respectively.

hIGFBP-5 Gene and Promoter 10893

hgBP5-17 )-',-I

phBP5-BS6.6 - phBPS-E8ll.l H

chBP5-9.2 I I

phBP5-El. 6

phBP5-E2.5

phBP5-E5.6 - hIGFBP-5 Gene I

I 1 1 1

El E2 E3 E4

0.4 4.6 1.1 EcoRI cleavage

95 195 2 5 5.6

I, e.1

FIG. 2. Structure of the hIGFBP-5 chromosomal gene. Inserts of genomic A-clone hgBP5-17 and cosmid clone chBP5-9.2 appear as bold lines. Fragments subcloned from hgBP5-17 to create phBP5-BS6.6 and phBP5-HBI1.l, and fragments subcloned from chBP5-9.2 to create phBP5-E4.6, phBP5-E2.5 and phBP5-E5.6 appear as fine lines. A schematic map of the hIGFBP-5 gene presents exons 1 4 (El-E4) as filled boxes and introns 1-3 as lines between exons. An EcoRI restriction map of the chBP5-9.2 cosmid insert is at the bottom; numbers represent fragment lengths in kb.

FIG. 3. Sequence of the hIGFBP-5 gene. DNA sequence of coding and 3'-untranslated regions appears as upper case letters while sequence of introns appears as lower case letters. The estimated size of each intron is shown, as is the size of an unsequenced gap in the 3'-untranslated reqion which spans 3095 bp in hIGFBP5 cDNA BP-5.12 (4). Amino acids, given in three-letter code above the DNA sequence, are numbered on the left. The sequence of oligonucleotide 5-9 is underlined. The 3'- poly(A) motif appearing in bold print is the last sequence shared by hIGFBP-5 cDNA and genomic clones.

-20

15

49

8 3

93

127

161

170

204

210

244

acrylamide sequencing gel alongside the sequencing reaction products from the 5:PE primer and phBP5-HBll.l.

Primer Extension of mRNA Expressed by p46lCAT-Total RNA was isolated from MDA-MB-468 cells harvested 22 h after transfection with p461CAT. Sixty pg of this RNA was used in individual primer extension experiments as described above, except that 1 pmol of oligonucleotide CATPE (5'-TATCAACGGTGGTATATCCAGTGAT-3') served as primer, and samples were incubated a t 70 "C for 5 min followed by cooling over 15 min to 55 "C. Samples were analyzed on a 6.5% polyacrylamide sequencing gel alongside sequencing reaction products from the CAT PE primer and p461CAT.

Northern Analysis of hZGFBP-5 mRNA-Total RNA, isolated from MDA-MB-468 cells as described above, was used for Northern analysis as described previously (13,30). The 455-bp BglII fragment of the BP- 5.1 cDNA clone was labeled with 32P, and lo7 countdmin were used as probe.

RESULTS

Complete Sequence of hZGFBP-5 cDNA-Much of the 3'-untranslated region of hIGFBP-5 cDNA clone BP-5.12 was not originally sequenced (4). This sequence is shown now in Fig. 1; the first nucleotide presented here immediately follows the last thymidine residue of the published sequence (4). Two A m A motifs, which may promote mRNA degradation when located in the 3"untranslated region of an mRNA transcript (35), are identified and complement two additional ATITA motifs present in the previously reported 3'-untranslated sequence (4).

Isolation and Characterization of the hIGFBP-5 Gene-The A clone hgBP5-17 was isolated by screening 6 x lo5 plaques from


A A B C D E F G H I J K L

- 4 . 0 - 3 . 0 - 2 . 0 - 1.6 - 1.0

I3 ll

c,

FIG. 4. Localization of the hIGFBP-6 gene to chromosome 2. A, PCR amplifications from a human-rodent cell hybrid; representative analysis of PCR products generated by amplification of total genomic DNA (A), total hamster DNA ( K ) , and somatic human-rodent cell hybrids containing human chromosomes 1-9 (BJ). The length in kilobases of the PCR products is given to the left, and of the size marker (L) to the right. B , fluorescence hybridization of genomic exon-containing

both chromatids of each chromosome 2 are indicated by arrows. subclones to human metaphase chromosomes. Fluorescent signals for

a human leukocyte genomic library using the hIGFBP-5 cDNA clone, BP-5.1, (4) as probe. When analyzed by Southern blotting after cleavage with BamHI, BglI, HindIII, SacII, and/or SalI, the hgBP5-17 clone hybridized only to a BgZII fragment which spans the 5'-most 455 nucleotides of the BP-5.1 cDNA. Two A fragments, a 6.6-kb BamHI-Sal1 fragment and a 1.1-kb Hin- dIII-BglI fragment, were subcloned in pGEM-3 and pSP73 to create phBP5-BS6.6 and phBP5-HB1l.l, respectively (Fig. 2).

The phBP5-BS6.6 clone was used as hybridization probe to screen 8 x lo5 colonies of a human cosmid library. One plaque- purified positive clone, chBP5-9.2, was recognized by each of the three BgZII fragments from the BP5.12 cDNA, EcoRI cleavage of chBP5-9.2 released nine fragments, of which the 4.6-, 2.5, and 5.6-kb fragments were shown to contain exon sequence by hybridizing with one or more of the BP-5.12 BgZII fragments. These three EcoRI fragments from chBP5-9.2 were subcloned in pGEM3Zf(+) to generate phBP5E4.6, phBP5- E2.5, and phBP5E5.6, respectively (Fig. 2) and were used, along with the A fragment phBP5-BS6.6, to sequence the coding and 3"untranslated regions and exodintron borders of the hIGFBP-5 gene (Fig. 3). The 3'-untranslated region of genomic hIGFBP-5 was not completely sequenced, leaving a gap equiv- alent to 3095 bp of the hIGFBP-5 cDNA clone BP-5.12 (Fig. 1). However, the 3"untranslated region was studied by PCR amplifications using three primer pairs: BP5-9/BP5-18, BP5-19/ BP5-20, and BP5-21/BP5-16 (see Figs. 1 and 3 legends). The PCR product from each primer pair was of identical length when either total human DNA, the cosmid subclone phBP5- E5.6, or the hIGFBP-5 cDNA BP-5.12 served as template (data not shown), suggesting strongly that no additional introns are

present in the 3'-untranslated region of the hIGFBP-5 gene. The order of the nine EcoRI fragments from the chBP5-9.2

cosmid insert was mapped by the method of Wahl et aZ. (36) to further characterize the introdexon organization of the hIG- FBP-5 gene (Fig. 2). Briefly, Southern analysis used chBP5-9.2 DNA that was completely cleaved with SfiI and then partially cleaved with EcoRI; SfiI was chosen since this enzyme cleaves the cosmid insert at a single site found in the 4.6-kb (exon 1-containing) EcoRI fragment. Hybridization with oligonucleotide probes 5735 and 5:PE, located 5' and 3' to the SfiI cleavage site, respectively (see Fig. 6), determined the relative order of the nine EcoRI fragments of the cosmid insert. The order of these EcoRI fragments suggested by the above studies was confirmed by additional PCR and hybridization experiments (data not shown).

The exact location of each exon within subclones and the cosmid clone was determined by PCR amplifications with exon- specific oligonucleotides and specific vector primers. The sizes of introns 2 and 3, determined by PCR amplifications, are 0.6 and 1.3 kb, respectively. Based on the EcoRI restriction map and additional PCR amplifications within subclones, intron 1 has a length of -25 kb.

The authenticity of the cosmid fragments was confirmed by hybridizing the 32P-labeled cosmid clone to EcoRI-cleaved total human DNA. Of the nine genomic EcoRI fragments detected, seven had lengths corresponding to the seven internal cosmid fragments shown in Fig. 2, while the others measured -12 and -16 kb, much longer than the outer two fragments of the cosmid insert. The 32P-labeled phBP5-BS6.6 clone, which contains the 5"flanking region of the hIGFBP-5 gene, hybridized with -16-, 1.0-, and 4.6-kb fragments of EcoRI-cleaved total human DNA, while 32P-labeled phBP5-E5.6, which represents the 3' end of the cosmid clone, hybridized only with a -12-kb fragment ofEcoRI-cleaved total human DNA(data not shown). This analysis indicates that the haploid human genome contains a single copy of the hIGFBP-5 geqe.and that this gene is orga- nized as presented in Fig. 2.

Chromosomal Localization of the hIGFBP-5 Gene-The hIG- FBP-5 gene was localized using a panel of somatic cell hybrids, each carrying one human chromosome on a rodent background. PCR reactions, using primers from the coding and the adjacent 3'-untranslated regions of exon 4 amplified an expected 1.7-kb fragment from total human DNA and from the cell hybrid carrying human chromosome 2, but not from any other hybrid, nor from hamster or mouse DNA (Fig. 4A). Chromosomal localization was also investigated by fluorescent in situ hybridization which showed that, for all analyzed metaphases, only chromosome 2 hybridized with exon-containing probes (Fig. 4B ). QFQ- banding placed the IGFBP-5 gene on the long arm at q33-q34 (data not shown), the same region where the IGFBP-2 gene was previously localized (26).

Human DNA was cleaved with different rare cutting restriction enzymes, separated by PFGE, transferred to a filter, and then hybridized successively with four DNA probes derived from the 5' and 3' regions of the IGFBP-2 and -5 genes, respectively. As shown in Fig. 5A, the 3' probes for IGFBP-5 and -2 hybridized to BssHII, EagI, NaeI, NarI, SacII, and SfiI fragments of the same length; most common fragments were -90 kb or less in size, indicating a close linkage between the two genes. In contrast to the 3' probes, the 5' probes for IGFBP-5 and -2 hybridized to fragments of the same length only when DNA samples were cleaved by NarI and SfiI; additionally, for DNA samples cleaved with either BssHII or NaeI, both 5' probes hybridized to different PFGE fragments than did their respective 3' probes. The results of the Southern blots, presented in Fig. 5B, indicate that the hIGFBP-2 and -5 genes are transcribed convergently and are separated by -20-40 kb of


FIG. 5. Physical linkage of the hIG- FBP-5 and -2 genes. A, Southern analysis of DNA fragments separated by PFGE

the 3' regions of the IGFBP-5 and -2 and hybridized with probes taken from

genes. Each lane contains DNA, prepared from lymphoblastoid cell lines, that was cleaved with the restriction endonuclease indicated below. The position of the 90-kb DNA size marker is indicated on the left. B, table summarizing the size, in kb, of restriction fragments separated by PFGE and then recognized on Southern analysis by IGFBP probes. Enzymes used are shown at the top and the 5'- or 3'-IG- FBP-5 or -2 probe used for the Southern blot is shown on the left. The shortest DNA size marker was 90 kb; thus, a fragment presented as <<90 kb may be as short as -10 kb. Boxes surround fragments identified by more than one IGFBP probe. C, restriction maps and relative positions of the hIGFBP-5 and -2 genes. Gene sizes and structures, including the positions of exons 1 4 (EI-E4), are from this report and from Ehrenborget al. (26). The location of the four probes used for Southern analysis of the PFGE filter are shown at the top. Analyzed restriction

l ine represents the 20-40 kb of DNA sites are indicated by arrows. The dotted

which separates the genes.

A 3' ICFBP-5

BssHll Eagl Nael Narl Sacn Sfil

3' ICFBP-2

I e. 3' ICFBP-2 90 I U

4 5' ICFBP-2 225

c 5' IGFBP-5 3' ICFBP-5

H H

El E2E3 U

3' IGFBP-2 H

U W E 2

L ,j 90

5' ICFBP-2 I

El . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I I A t

L Sfil Nscl Sac11 BnHl l

I NscI

ICFBP-5 "- ICFBP.2

-33 kb 20-40 kb -32 kb

-461 MGCTTAGGMGATTTCTTGGGCACGGTATATCCAGTTGGCTMTM~TACGTCTCCCTTCAGCCTGTGCCTTGACTACTTARAGGATAGG ---T-GGM-T--C-------- T-G ------ CA-.... --------------- GT-A----TAA-----A---T-CG-G---G-- ..........

-180 MGGGGAGCCCCCTTGTGTCTAGMGGCCTCTCCCCACCCCCACCCCGTGTGAGTTTGTACTGCMGCTCCTTGGCATCCTTGCCTGAGTTGGG

. n 11 G~CTTCTGCAGGGG... .. ~GAGCTAGG~GAGCTGCMGCAGTGTGGGCTTTTT.....CCCTTTTTTGCTCCTTTTCATTA ..CCC

"""""-C""G~T"""""""""""""-T"T"""""TTccc"""""T"""-TG"-Tc"-

4 94 CTCCTCCGTTTTCACCCTPCTCCGGACTTCGCGTAGAACCTGCGAATTTCGAAGAGGAGGTGGC~GTGGGAG~GAGGTGTTAGGGTTTGG

oligo 5:PE

6 3 0 GGGGGAGGGCACCTGCTCTACCTGCCAGMTTTT~C~C~C~TCTCCGGGGGCCCTCTTGGCCCCTTTATCCCTGCAC "A"-CA""""-T"""-A"G"A"A" . . . . . . . TTTT""-A""""""A"cT""""""""c"-AcA"-

1 2 5 TCTCGCTCTCCTGCCCCACCCCGAGGTMAGGGGGCGACTAAGAGAAGA'X

FIG. 6. Sequence of the hIGFBP-5 gene 5' to the ATG translation start codon. DNA sequence from the human gene appears above homologous sequence from the rat gene (39); sequences were aligned by the method of Smith and Wa- terman (50). Gaps in either sequence are indicated by a period (.), and nucleotides conserved between the two sequences appear in the rat sequence as a dash (-1. A potential TATA element, and the adeno-

moter, are boxed. The ATG translation sine identified as cap site in each pro-

bers on the left refer to the distance, in bp, start codon appears in bold print. Num-

5' (negative) or 3' (positive) to the human mRNA cap site (bp 1). Primer extension oligonucleotide 5:PE, complementary to sequence from bp 106 to 130, is located beneath the arrow. Oligonucleotide 5:735 is complementary to sequence from bp 711 to 735.

DNA, this is shown schematically in Fig. 5C. was used in primer extension studies to map the native hIG- hZGFBP-5 Gene Organization 5' to the ATG Tkanslation Start FBP-5 mRNAcap site. MDA-MB-468 human breast cancer cells

CodonSequence of the 1233 bp immediately 5' to the first served as source of RNAfor primer extension experiments since nucleotide of the hIGFBP-5 ATG translation start codon is they express high levels of IGFBP-5 mRNA (28). As shown in shown in Fig. 6, as is the location of oligonucleotide 5:PE which Fig. 7A, a single -6-kb hIGFBP-5 mRNA transcript is seen by

10896 hIGFBP-5 Gene and Promoter

A AC

AC

5'

' T A T T

MDA C. A T C

468 MR

/

3'

FIG. 7. Localization of the hIGFBP-5 mRNA cap site by primer extension. A, Northern analysis: 10 pg of whole RNA from MDA-MB- 468 cells was electrophoresed, transferred to a nylon membrane, and hybridized with lo7 countdmin of a g"P-labeled, 455-bp BglII fragment from the BP-5.1 cDNA clone. The size of the IGFBP-5 mRNA transcript is on the left. B , primer extension: oligonucleotide 5:PE was labeled with :'eP, hybridized with 50 pg of whole RNA from MDA-MB-468 cells, and then extended with avian myeloblastosis virus reverse transcriptase. 5:PE also primed DNA sequencing of phBP5-HBI1.l; the products of this reaction (lanes G , A, T, and C ) and of the primer extensions were run in the same sequencing gel. Sequence of the TATA promoter element appears on the right, as does sequence surrounding the 5' end of the IGFBP-5 mRNA, the A_ in this latter sequence identifies the adenosine which serves as primary cap site for IGFBP-5 mRNA transcripts.

Northern analysis of MDA-MB-468 RNA. As shown in Fig. 6 and 7B, the major primer extension product ofthis hIGFBP-5 mRNA transcript terminates at the adenosine located 772 bp upstream of the first nucleotide of the ATG translation start codon. This mRNAcap site is 33 bp downstream from the start of an AT-rich sequence (TATA element) which may potentially bind TFIID.

Functional Characterization of the hZGFBP-5 Promoter-To test whether the region 5' to the hIGFBP-5 mRNA cap site exhibits promoter activity, a 484-bp fragment containing the cap site and 461 bp of 5"flanking sequence was inserted upstream to the CAT reporter gene in either sense or antisense orientation to create p461CAT or p461CAT(AS), respectively. Based on three independent transfections into MDA-MB-468 cells, this DNA fragment was -50-fold more active in directing CAT expression when inserted in sense rather than antisense orientation (Fig. 8).

Chimeric IGFBP-SEAT mRNA, transcribed from p461CAT after transient transfection into MDA-MB-468 cells, was extended with an oligonucleotide primer complementary to sequence located in the 5' region of the CAT gene. As shown in Fig. 9, the adenosine identified to be the primary cap site for native hIGFBP-5 mRNA is also the primary cap site for the chimeric mRNA produced by the hIGFBP-5 promoter/CAT reporter gene construct.

DISCUSSION

The hIGFBP-5 gene contains four exons which span -33 kb of DNA, a size due primarily to intron 1 which extends for -25

K A T P461 P461 (As)

0 100 2i11 FIG. 8. Demonstration of hIGFBP-5 promoter activity. A DNA

fragment containing 461 bp of IGFBPd promoter was placed 5' to the CAT reporter gene in either sense or antisense orientation to create p461CAT ( ~ 4 6 1 ) and p461CAT(AS) (p461fAS)), respectively. These constructs, and the promoterless vector ($AT), were transiently transfected into MDA-MB-468 cells. After 22 h, cells were harvested and cellular protein assayed for the ability to acetylate ['"Clchlorampheni- col. Reaction products were separated by thin layer chromatography and autoradiographed. Relative promoter activities are shown a t the bottom; pCAT and p461CAT activities were arbitrarily set at 0 and 1008, respectively, for the three independent experiments, while activity of p461CAT(AS) is presented as mean f standard deviation for the three experiments. CM, non-acetylated ['4Clchloramphenicol; AC, two forms of mono-acetylated ['4Clchloramphenicol.

5'

p461 G A T C CAT 3'

FIG. 9. Primer extension of mRNA expressed by p461CAT. Whole RNA was isolated from MDA-MB-468 cells which had been transiently transfected with p461CAT 22 h previously. Oligonucleotide CAT PE, complementary to sequence 8-32 bp upstream of the CAT ATG translation start codon, was labeled with "P, hybridized with 60 pg of whole RNAfrom the MDA-MB-468 cells, and then extended using avian myeloblastosis virus reverse transcriptase. CATPE was also used to prime DNA sequencing of p461CAT; the products of this reaction (lanes G , A, T, and C ) and of the primer extension were run in the same

right, as does sequence surrounding the 5' end of the chimeric IGFBP- sequencing gel. Sequence of the TATA promoter element appears on the

5/CAT mRNA the A in this latter sequence identifies the adenosine which serves a's the primary mRNA cap site.

kb. The hIGFBP-2 gene is similar, spanning -32 kb and containing a -27-kb first intron (26, 37). All introdexon splice junctions of the hIGFBP-5 gene conform to consensus sequences derived from other vertebrate genes (38) and are identical to those of the rat IGFBP-5 gene (39) except for minor


differences at the intron 1 borders. Human and rat IGFBP-5 genes are similar to human IGFBP-1, -2, and -3 genes in terms of location of exon borders relative to nucleotides encoding conserved cysteine residues (26).

The coding region sequence reported here for hIGFBP-5 genomic DNA is identical to the hIGFBP-5 cDNA sequence reported by Kiefer et al. (4). The final 18 bp of 3”untranslated sequence from an additional hIGFBP-5 cDNA isolated by Shi- masaki et al. (1) is the only region where these three DNA sequences differ. The reason for the difference is unclear, but it should be noted that rat IGFBP-5 gene (39) and cDNA (1) sequences in this region are identical to the sequence reported here and by Kiefer et al. (4).

Several independent mapping strategies presented here and in past studies (26) show a tight linkage of hIGFBP-5 and -2 genes on chromosomal region 2q33-q34; this is reminiscent of studies localizing hIGFBP-1 and -3 genes to chromosomal region 7p14-pl2 where they are closely linked in a tail-to-tail orientation (401, identical to the orientation shown here for hIGFBP-2 and -5. These observations correct the preliminary localization of the hIGFBP-5 gene to chromosome 5, an assign- ment based solely on PCR analysis of a panel of somatic human-rodent cell hybrids (1). Also, localizing hIGFBP-5 at 2q33- q34 strengthens the association between human IGFBP and homeobox (HOX) gene families. Both families have genes localized to four regions: 2q31-q34 includes IGFBP-2 and -5/HOXD cluster, 7p15-pl2 includes IGFBP-1 and -3mOXA cluster, 17q12-q22 includes IGFBP-4/HOXB cluster, and chromosome 12 includes IGFBP-6/HOXC cluster (23, 40-441, with HOX clusters named using the new nomenclature (45).

HOX genes, present in organisms as diverse as yeast and man, encode transcription factors crucial to early developmen- tal programs. Available data indicate that duplication of HOX genes at a single chromosomal locus probably preceded dispersion of the resulting HOX cluster to four chromosomal loci (46). Close association of hIGFBP genes with HOX gene clusters suggests that HOX and IGFBP genes were linked prior to the first duplication of chromosomal DNA containing the ancestral HOX cluster. The presence of multiple HOX clusters in the prevertebrate chordate amphioxus and also in agnathans, which diverged from the main line of vertebrate evolution -550 million years ago, suggests that IGFBPs are ancient proteins; this hypothesis is supported by the detection of IGFBP activity in agnathan serum (47, 48).

The evolution of multiple gene clusters appears to follow a similar pattern in the HOX and IGFBP gene families. Taken together, the observations that: (i) IGFBP-1/-3 and IGFBP-2/-5 gene pairs have identical tail-to-tail orientation; (ii) IGFBP-3 and -5 are more closely related to each other than to other IGFBPs; and (iii) IGFBP-1 and -2 are more closely related to each other than to IGFBP-3 or -5, all suggest that duplication of an ancient IGFBP gene preceded dispersion of this gene pair to multiple chromosomal loci, similar to the pattern proposed for evolution of the HOX genes (40, 46, 49).

Primer extension localized the hIGFBP-5 mRNA cap site 772 bp 5’ to the first nucleotide of the ATG translation start codon. This is probably the true hIGFBP-5 mRNA cap site, since (i) it is identical to the cap site identified in the rat IGFBP-5 gene (39; Fig. 61, and (ii) it predicts a -6013-bp mRNA, consistent with the size of IGFBP-5 mRNA transcripts reported here and by others (1, 4, 7-11).

The 5’-flanking region of the hIGFBP-5 gene has structural and functional characteristics of a eukaryotic promoter: (i) the mRNA cap site is 33-bp 3’ to the start of a potential TATA promoter element; (ii) a DNA fragment containing the first 461-bp 5’ to the cap site directs expression of the heterologous reporter gene CAT in an Orientation-specific manner; and (iii)

the major cap site for chimeric IGFBP-5/CAT mRNA, produced under the direction of the 461-bp IGFBP-5 promoter region, is the same adenosine identified as cap site in the native hIG- FBP-5 gene. Each of these features is shared by the well characterized gene promoters for hIGFBP-1 and hIGFBP-3 (30,321.

Human and rat IGFBP-5 promoter sequences are quite similar, as shown in Fig. 6. Sequence of the potential TATA element is 100% conserved between human and rat promoters, and location of this element relative to the mRNA cap site is also conserved. Overall, 191 of the first 207 bp of the hIGFBP-5 promoter are conserved in the rat promoter. This conservation of structure implies conservation of important promoter functions and suggests that transcriptional mechanisms may regulate IGFBP-5 expression in many tissues, including muscle and bone where IGFBP-5 may play an important physiologic role.

REFERENCES 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20. 21. 22.

23.

24.

25.

26.

27.

28.

29. 30.

31.

32.

33.

34. 35.

36.

Shimasaki, S., Shimonaka, M., Zhang, H-P., and Ling, N. (1991) J. Biol. Chem. 266,10646-10653

Bautista, C. M., Baylink, D. J., and Mohan, S. (1991) Biochem. Biophys. Res. Commun. 176, 756-763

Andress, D. L., and Birnhaum, R. S. (1991) Biochem. Biophys. Res. Commun. 176,213-218

Kiefer, M. C., Ioh, R. S., Bauer, D. M., and Zapf, J. (1991) Biochem. Biophys. Res. Commun. 176,219-225

Kiefer, M. C., Masiarz, F. R., Bauer, D. M., and Zapf, J. (1991) J. B i d . Chem. 266,9043-9049

Conover, C. A,, and Kiefer, M. C. (1993) J. Clin. Endocrinol. Metab. 76,1153- 1159

Erickson, G . F., Nakatani, A., Ling, N., and Shimasaki, S. (1992) Endocrinol- ogy 130, 1867-1878

Sheikh, M. S., Shao, Z-M., Clemmons, D. R., LeRoith, D., Roberts, C. T. J., and Fontana, J. A. (1992) Biochem. Biophys. Res. Commun. 185, 1003-1010

Camacho-Hubner, C., Busby, W. H., McCusker, R. H., Wright, G., and Clem- mons, D. R. (1992) J. B i d . Chem. 267, 11949-11956

Backeljauw, P. F., Dai, Z., Clemmons, D. R., and DErcole, A. J. (1993) Endo- crinology 132, 1677-1681

Conover, C. A., Bale, L. K., Clarkson, J. T., and Tsmng, 0. (1993) Endocrinol- ogy 132, 2525-2530

Powell, D. R., Suwanichkul, A., Cubbage, M. L., DePaolis, L. A., Snuggs, M., and Lee, P. D. K. (1991) J. Biol. Chem. 266,1886%18876

Suwanichkul, A., DePaolis, L. A., Lee, P. D. K., and Powell, D. R. (1993) J. B i d . Chem. 266,9730-9736

Suwanichkul, A., Moms, S. L., and Powell, D. R. (1993) J. Biol. Chem. 266, 17063-17068

Powell, D. R., Lee, P. D. K., DePaolis, L. A,, Moms, S. L., and Suwanichku1,A. (1993) Growth Reg. 3, 11-13

Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring

1992 Report on the Nomenclature of the IGF Binding Proteins, Endocrinology Harbor, NY

130, 1736-1737 Lee, Y.-L., Hintz, R. L., James, P. M., Lee, P. D. K., Shively, J. E., and Powell,

Cubbage, M. L., Suwanichkul, A., and Powell, D. R. (1989) Mol. Endocrinol. 3, D. R. (1988) Mol. Endocrinol. 2,404411

846-851 Evans, G. A., Lewis, K., and Rothenberg, B. E. (1989) Gene (Amst.) 79,9-20 Berger, S. L., and Kimmel, A. R. (1987) Methods Enzymol. 152, 604-610 Bergerheim, U., Nordenskjold, M., and Collins, V. P. (1989) Cancer Res. 49,

1390-1396 Bajalica, S., Allander, S. V., Ehrenborg, E., Bmndum-Nielsen, IC, Luthman,

Lichter, P., Cremer, T., Borden, J . , Manuelidis, L., and Ward, D. C. (1988) Hum. H., and Larsson, C. (1992) Hum. Genet. 89,234-236

Genet. 80,224-234 Janson, M., Larsson, C., Werelius, B., Jones, C., Glaser, T., Nakamura, Y..

Jones, C. P., and Nordenskjold, M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10609-10613

Ehrenborg, E., Vilhelmsdotter, S., Bajalica, S., Larsson, C., Stem, I., Koch, J., Brendum-Nielsen, K., and Luthman, H. (1991) Biochem. Biophys. Res.

Jacoby, D. B., Zilz, N. D., and Towle, H. C. (1989) J. Biol. Chem. 264, 17623- Commun. 176, 1250-1255

17626 Figueroa, J. A., Jackson, J . G., McGuire, W. L., m c k i , R. F., and Yee, D.

Graham, F. L., and Van der Eb, A. J. (1973) Virology 52,456467 (1993) J. Cell. Biochem. 52, 196-205

Suwanichkul, A., Cubbage, M. L., and Powell, D. R. (1990) J. Biol. Chem. 266,

de Wet, J. R., Wood, K. V., DeLuca, M., Helinski, D. R., and Subramani, S.

Cubbage, M. L., Suwanichkul, A., and Powell, D. R. (1990) J. Biol. Chem. 266,

Gorman, C. M., Moffat, L. F., and Howard, B. H. (1982) Mol. Cell. Biol. 2,

Chomczynski, P., and Sacchi, N. (1987)Anal. Biochem. 162, 156-159 Peltz, S. W., Brewer, G., Bernstein, P., Hart, P.A., and Ross, J. (1991) Crit. Reu.

Wahl, G. M., Lewis, K. A,, Ruiz, J . C., Rothenberg, B., Zhao, J., and Evans, G .

21185-21193

(1987) Mol. Cell. Biol. 7, 725-737

12642-12649

1044-1051

Euk. Gene Expression 1, 99-126

10898 hIGFBP-5 Gene and Promoter A. (1987) Proc. Natl. Acad. Sci. U. S. A. 64,2160-2164

(1992) Mol. Endocrinol. 6, 82-36 45. Scott, M. (1992) Cell 71, 551653 37. Binkert, C., Margot, J. B., Landwehr, J., Heinrich, G., and Schwander, J. 44. Lu-Kuo, J., Ward, D. C., and Spritz R. A. (1993) Genomics 16, 173-179

38. Green, M. R. (1991)Annu. Rev. Cell. Eiol. 7, 559499 46. Acampora, D., D'Esposito, M., Faiella, A,, Pannese, M., Migliaccio, E., Morelli, 39. Zhu, X., Ling, N., and Shimasaki, S . (1993) Eiochem. Biophys. Res. Commun. F., Stornaiuolo, A,, Nigra, V., Simeone, A,, and Boncinelli, E. (1989) Nucleic

Acids Res. 17,10385-10402

40. Ehrenborg, E., Larsson, C., Stern, I., Janson, M., Powell, D. R., and Luthman, 47' J' w'' Nagai' B' K'' Murtha' M' T', and F' H' (1993) proc' Natl. Acad. Sci. U. S. A. 90, 63004304

41. Human Gene Mapping 10.5 (1990) Cytogenet. Cell. Genet. 55, 1-785 48. Upton, Z., Chan, S . J., Steiner, D. F., Wallace, J. C., and Ballard, F. J. (1993)

42. Shimasah, S., Gao, L., Shimonaka, M., and Ling, N. (1991) Mol. Endocrinol. 49. Lee, P. D. K., Conover, C.A., and Powell, D. R. (1993) Proc. Soc. Exp. B i d . Med. Growth Reg. 3, 2 9 3 2

43. Cannizzaro, L. A,, Cmce, C. M., Griffin, C. A,, Simeone, A,, Boncinelli, E., and 50. Smith, T. F., and Waterman, M. S . (1981) Adu. Applied Math. 2,482489 204,4-29

Huebner, K. (1987) Am. J. Hum. Genet. 41,l-15

190, 1045-1052

H. (1992) Gemmics 12,497-502

5,938-948

the of chemistry vol. no. of 8, 10891-10898, 1994 1994 for ... · the journal of biological...

Documents