6
GENOMICS 12, 497-502 (19%) Contiguous Localization of the Genes Encoding Human Insulin-like Growth Factor Binding Proteins 1 (IGBPI) and 3(IGBP3) on Chromosome 7 EWA EHRENBORG, * CATHARINA LARSSON, * INGRID STERN,* MARIE JANSON,* DAVID R. PowEu,t AND HOLGER LUTHMAN*,’ *Department of Clinical Genetics, Karolinska Institute, P.O. Box 60 500, Karolinska Hospital, S-104 07 Stockholm, Sweden; and tDepartment of Pediatrics, Baylor College of Medicine, Diabetes Research Center, Houston, Texas 77054 Received July 8, 1991; revised October 21, 1991 In extracellular fluids the insulin-like growth factors (IGFs) are bound to specificbinding proteins (IGBPs). The genes for two members of this protein family have been mapped, the IGBPl geneto human chromosomal region 7p14-p12 and the IGBPS gene to region 2q33-q34. In this study, somatic cell hybrid analysis indicated that IGBPB is also located on chro- mosome 7. Pulsed-field gel electrophoresis was used to demon- strate the close physical linkage between IGBPl and IGBPB. Overlapping cosmid clones encompassing these genes were isolated, and restriction endonuclease mapping showed that the genes are arranged in a tail-to-tail fashion separated by 20 kb of DNA. Further characterization of the IGBPl DNA se- quencedisclosed a duplication of the intron 3-exon 4 junction within the third intron. In addition, we report RFLPs for ApaL and ToqI in the IGBPl hcus. o 1992 Academic POW. IIIC. INTRODUCTION Insulin-like growth factors (IGF) I and II are insulin- related peptides with metabolic as well as mitogenic ef- fects (Froesch et al., 1985). In extracellular fluids the majority of IGF I and II are bound to high-affinity bind- ing proteins (IGBPs). In addition to being transporter molecules, these binding proteins also modulate the function of IGF in uivo (Cascieri et al., 1988) and in vitro (Burch et al., 1990; Conover, 1990; De Mellow and Baxter, 1988; Ritvos et al., 1988; Ui et al., 1989). At pres- ent, cDNA clones for four different human binding pro- teins have been isolated (Brinkman et al., 1988a; Brewer et al., 1988; Julkunen et al., 1988; Lee et al., 1988; Luth- man et al., 1989; Binkert et al., 1989; Wood et al., 1988; Shimasaki et al., 1990; LaTour et al., 1990), and the geno- mic sequences for human IGBPl, IGBPB, and IGBP3 have been determined (Brinkman et al., 1988b; Cubbage et aZ., 1989, 1990; Ehrenborg et al., 1991). Structurally, Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession No. M74587. r To whom correspondence and reprint requests should be addressed. the IGF binding proteins are related to each other but not to any other known protein, thus forming a separate protein family. Despite their structural similarities, how- ever, the IGF binding proteins differ in their pattern of expression. The IGBPS gene is expressed in most tissues (Shimasaki et al., 1989), while expression of IGBPl and IGBPB is more tissue-specific (Brinkman et aZ., 1988a; Julkunen et al., 1988; Binkert et aZ., 1989). The protein levels of IGBP3 in serum and mRNA levels in the liver are high during puberty and adult life (White et al., 1981; Liu et al., 1991), while the serum protein and liver mRNA levels of IGBPl and IGBPB reach the highest levels during fetal life and infancy (Binkert et al., 1989; Brinkman et al., 1988a; Liu et al., 1991). Furthermore, the IGBP3 protein levels increase in the presence of an- abolic hormones such as growth hormone, IGFI (Clem- mons et al., 1989; Hardouin et aZ., 1989), and insulin (Conover, 1990), while the IGBPl and IGBPB levels de- crease in the presence of these hormones (Clemmons et al., 1989; Conover, 1990; Hardouin et al., 1989; Margot et al., 1989; Ooi et al., 1990; Seneviratne et al., 1990; Unter- man et al., 1990). Considering the structural homologies of the IGF binding proteins and their different expression patterns, the genomic organization of this gene family is of consid- erable interest. It is known that members of the same gene family are sometimes adjacent. We therefore inves- tigated whether this is also true for the genes for human IGBPs. The IGBPl gene has been localized to chromo- somal region 7p14-p12 (Alitalo et aZ., 1989; Ekstrand et al., 1990), and the IGBPB gene was recently mapped to chromosomal region 2q33-q34 (Ehrenborg et al., 1991). In this report, it is demonstrated that the genes encoding IGBPl and IGBP3 are juxtaposed on human chromo- some 7. MATERIALS AND METHODS DNA probes and cell lines. The following DNA clones were used as hybridization probes: cDNA clone phIGFBPl-103 (Luthman et al., 1989), cDNA clone phIGFBP3-cDNA897 (Cubbage et al., 1990), the 497 0888-7543/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

Contiguous localization of the genes encoding human insulin-like growth factor binding proteins 1(IGBP1) and 3(IGBP3) on chromosome 7

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GENOMICS 12, 497-502 (19%)

Contiguous Localization of the Genes Encoding Human Insulin-like Growth Factor Binding Proteins 1 (IGBPI)

and 3(IGBP3) on Chromosome 7

EWA EHRENBORG, * CATHARINA LARSSON, * INGRID STERN,* MARIE JANSON,* DAVID R. PowEu,t AND HOLGER LUTHMAN*,’

*Department of Clinical Genetics, Karolinska Institute, P.O. Box 60 500, Karolinska Hospital, S-104 07 Stockholm, Sweden; and tDepartment of Pediatrics, Baylor College of Medicine, Diabetes Research Center, Houston, Texas 77054

Received July 8, 1991; revised October 21, 1991

In extracellular fluids the insulin-like growth factors (IGFs) are bound to specific binding proteins (IGBPs). The genes for two members of this protein family have been mapped, the IGBPl gene to human chromosomal region 7p14-p12 and the IGBPS gene to region 2q33-q34. In this study, somatic cell hybrid analysis indicated that IGBPB is also located on chro- mosome 7. Pulsed-field gel electrophoresis was used to demon- strate the close physical linkage between IGBPl and IGBPB. Overlapping cosmid clones encompassing these genes were isolated, and restriction endonuclease mapping showed that the genes are arranged in a tail-to-tail fashion separated by 20 kb of DNA. Further characterization of the IGBPl DNA se- quence disclosed a duplication of the intron 3-exon 4 junction within the third intron. In addition, we report RFLPs for ApaL and ToqI in the IGBPl hcus. o 1992 Academic POW. IIIC.

INTRODUCTION

Insulin-like growth factors (IGF) I and II are insulin- related peptides with metabolic as well as mitogenic ef- fects (Froesch et al., 1985). In extracellular fluids the majority of IGF I and II are bound to high-affinity bind- ing proteins (IGBPs). In addition to being transporter molecules, these binding proteins also modulate the function of IGF in uivo (Cascieri et al., 1988) and in vitro (Burch et al., 1990; Conover, 1990; De Mellow and Baxter, 1988; Ritvos et al., 1988; Ui et al., 1989). At pres- ent, cDNA clones for four different human binding pro- teins have been isolated (Brinkman et al., 1988a; Brewer et al., 1988; Julkunen et al., 1988; Lee et al., 1988; Luth- man et al., 1989; Binkert et al., 1989; Wood et al., 1988; Shimasaki et al., 1990; LaTour et al., 1990), and the geno- mic sequences for human IGBPl, IGBPB, and IGBP3 have been determined (Brinkman et al., 1988b; Cubbage et aZ., 1989, 1990; Ehrenborg et al., 1991). Structurally,

Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession No. M74587. r To whom correspondence and reprint requests should be addressed.

the IGF binding proteins are related to each other but not to any other known protein, thus forming a separate protein family. Despite their structural similarities, how- ever, the IGF binding proteins differ in their pattern of expression. The IGBPS gene is expressed in most tissues (Shimasaki et al., 1989), while expression of IGBPl and IGBPB is more tissue-specific (Brinkman et aZ., 1988a; Julkunen et al., 1988; Binkert et aZ., 1989). The protein levels of IGBP3 in serum and mRNA levels in the liver are high during puberty and adult life (White et al., 1981; Liu et al., 1991), while the serum protein and liver mRNA levels of IGBPl and IGBPB reach the highest levels during fetal life and infancy (Binkert et al., 1989; Brinkman et al., 1988a; Liu et al., 1991). Furthermore, the IGBP3 protein levels increase in the presence of an- abolic hormones such as growth hormone, IGFI (Clem- mons et al., 1989; Hardouin et aZ., 1989), and insulin (Conover, 1990), while the IGBPl and IGBPB levels de- crease in the presence of these hormones (Clemmons et al., 1989; Conover, 1990; Hardouin et al., 1989; Margot et al., 1989; Ooi et al., 1990; Seneviratne et al., 1990; Unter- man et al., 1990).

Considering the structural homologies of the IGF binding proteins and their different expression patterns, the genomic organization of this gene family is of consid- erable interest. It is known that members of the same gene family are sometimes adjacent. We therefore inves- tigated whether this is also true for the genes for human IGBPs. The IGBPl gene has been localized to chromo- somal region 7p14-p12 (Alitalo et aZ., 1989; Ekstrand et al., 1990), and the IGBPB gene was recently mapped to chromosomal region 2q33-q34 (Ehrenborg et al., 1991). In this report, it is demonstrated that the genes encoding IGBPl and IGBP3 are juxtaposed on human chromo- some 7.

MATERIALS AND METHODS DNA probes and cell lines. The following DNA clones were used as

hybridization probes: cDNA clone phIGFBPl-103 (Luthman et al., 1989), cDNA clone phIGFBP3-cDNA897 (Cubbage et al., 1990), the

497 0888-7543/92 $3.00

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

498 EHRENBORG ET AL.

genomic clones phIGFBPl-B3.4 (3.4-kb BamHI fragment from the 3’ IGBPl gene), phIGFBPl-E4.0 (4.0-kb EcoRI fragment from the 5’ IGBPI gene), and phIGFBPl-E5.2 (5.2-kb EcoRI fragment from the 3’ IGBPl gene), as well as p4.0hgBP3 and p6.2hgBP3, corresponding to the 5’ and the 3’ ends of the IGBP3 gene (Cubbage et al., 1990). The T3 primer, 5’-ATTAACCCTCACTAAAG, and the T7 primer, 5’- CTATAGTGAGTCGTATT, flanking the cloning site in the cosmid vector pWE15, were also used as hybridization probes after labeling with 32P by T4 kinase.

The mouse-human hybrid cell-line Cl21 carries chromosome 7 as its only human chromosome (Lund et al., 1983), and Tp-336MG was established from a human anaplastic astrocytoma grade III (Collins, 1983).

Isolation and characterization of genomic XlGFBPl clones. The X clone XhBPl-2:l was isolated from a human genomic Charon 4A li- brary (ATCC) by hybridization with phIGFBPl-103 according to standard procedures (Berger and Kimmel, 1987).

Minipreparations of X DNA were performed as follows: LE 392 cells were infected and grown until lysis (Grossberger, 1987). Bacterial de- bris were spun down at 9OOOg for 10 min. The supernatant was treated with DNase, RNase A, and RNase Tl, all at 1 pg/ml, for 15 min at 37°C. Phage were precipitated by the addition of 1 vol of 2 M NaCl in 20% polyethylene glycol (PEG 6000) and incubated for 15 min at O”C, followed by centrifugation at 9OOOg for 10 min. The phage pellets were thoroughly washed with cold 70% ethanol and dissolved in 500 ~1 of a solution containing 4.2 M guanidinium thiocyanate (Fluka), 10 mM Tris-HCl, pH 8.0,5 mM EDTA, and 0.2% Na-Sarcosyl. The samples were mixed vigorously and incubated for 20 min at 37°C. DNA was precipitated by the addition of 50 ~1 of 3 M NaAc, pH 5.2,l ml of 95% ethanol and centrifugation. The precipitate was dissolved in 400 al of 10 mM Tris-HCl, pH 8.0, 25 mM EDTA, and treated with RNase A and Tl, 50 pg/ml, for 15 min at 37°C. Remaining proteins were re- moved by the addition of 210 ~1 of 5 M NaCl and incubation for 15 min at 0°C followed by centrifugation for 15 min at 13,000g. The DNA was precipitated from the supernatant with 475 pl of 95% ethanol.

DNA fragments from XhBPl-2:l were isolated and subcloned in pGEM4Z. Plasmids phIGFBPl-E4.0 and phIGFBPl-B5.8 carry EcoRI and BamHI fragments isolated from the 5’ region of the IGBPl gene, and phIGFBPl-E5.2 and phIGFBPl-B3.4 carry fragments corresponding to the 3’ region of the gene, The DNA sequence of both strands was determined for selected parts of these clones by the di- deoxy method, using Sequenase (USB) and tailor-made sequencing primers (Pharmacia Gene Assembler).

Isolation of cosmid clones. A human cosmid (pWE15) library pre-

pared from placental DNA (Evans et al., 1989) was screened with EcoRI fragments from XhBPl-2:1, as well as with EcoRI to Sal1 frag- ments from p4.0hgBP3 and p6.2hgBP3. The identified cosmid clones were purified, and cosmid DNA was isolated as described (Berger and Kimmel, 1987). The human DNA inserts were isolated from the vector by Not1 cleavage and electrophoresis in low-melting-point agarose.

DNA and RNA analysis. Southern blotting and hybridization were performed as described (Berger and Kimmel, 1987). After hybrid- ization the filters were washed in 0.1X SSC, 0.1% SDS, at 65°C. T3 and T7 oligonucleotides were hybridized and washed in 6X SSC at 37°C. Complementary DNA was synthesized in 20 ~1 containing 50 mM Tris-HCl, pH 8.3, 10 mM MgC&, 10 mM DTT, 40 U RNasin (Promega), 200 pmol oligo(dT)n,-24 (Pharmacia), 1 mM dNTP, 10 I.rg total RNA, and 16 U of M-MuLV reverse transcriptase (BioLabs), for 1 h at 42°C. One microliter of each cDNA sample was PCR-amplified in a volume of 50 ~1 in the presence of 0.25 PM specific primer, 50 mM KCl, 1.5 mM MgCl,, 10 mMTris-HCl, pH 8.4, at 7O”C, 0.01% gelatin, 200 +M of each dNTP, and 1.25 U Tq polymerase (Perkin-Elmer Cetus). Two IGBPl primers were used (see Fig. 4): primer 51, 5’-CA- GAGAGCACGGAGATAACTGAG, located in exon 2 (2678-2700); and primer 53, 5’-ATTCCAAGGGTAGACGCACCAGC, located in exon 4 (5103-5125). Synthesis of single-stranded DNA for direct se- quencing of PCR products was performed as described (Wedell et al., 1990).

Pulsed-field gel electrophoresk. Preparation of DNA in agarose blocks and subsequent restriction enzyme cleavage were performed as described (Herrmann et al., 1987). Agarose gels (1%) were run in 0.5X

a b c

I 3.5 kb

FIG. 1. Southern analysis of TaqI-cleaved DNA from hybrid and human cells hybridized with IGBPB cDNA. Lane a, 10 pg DNA from the mouse-human hybrid cell line Cl21 containing chromosome 7 as

its only human chromosome; lanes b and c, 10 pg of human DNA. The size of the hybridizing fragment is indicated in kilobases.

TBE using hexagonal field electronics (LKB Pulsaphor). Chromo- somes of Saccharomyces cerevkiae, strain YP 148, and Hansen& win- gei were used as size markers (Jones et al., 1989). DNA was transferred to a nylon filter in 0.5 M NaOH, 1.5 M NaCl, and hybridization was carried out in 6X SSC, 1% SDS, 5% dextran sulfate, 100 pg/ml salmon

sperm DNA at 65°C. Labeled probe was removed from the filter in 0.4 M NaOH for 20 min at room temperature, followed by neutralization in 0.2 M Tris-HCl, pH 7.5, 0.1X SSC.

RESULTS

The IGBPl and IGBP3 Genes Are Contiguous on Chromosome 7

We investigated the possible clustering of IGBP genes on human chromosome 7 by hybridization to DNA from the human-mouse hybrid cell line C121, which carries chromosome 7 as its only human chromosome. The cDNA probe specific for IGBP3 hybridized to DNA from the hybrid cell line (Fig. l), suggesting that the IGBP3 gene is located on human chromosome 7. In a parallel experiment no cross-hybridization was detected to mouse DNA under identical washing conditions.

Subsequently, pulsed-field gel electrophoresis was used to study whether the IGBPl and IGBP3 genes are closely linked. Human DNA samples digested with 11 different restriction enzymes were separated by pulsed- field gel electrophoresis, and the same filter was succes- sively hybridized with genomic probes specific for the two genes. Both genes hybridized to a 1.5Mh Not1 frag- ment (not shown) and to the same BssHII, iVae1, SacII, and SmaI fragments, all shorter than 70 kb in length (Fig. 2). This experiment demonstrates the close physi- cal linkage between the genes.

Cosmid clones containing the IGBPl and IGBP3 genes were isolated. One clone encompassing the IGBPl gene and two different clones encompassing the IGBP3 gene were isolated by screening of 6 X lo5 colonies with the corresponding genomic probes. One clone from each gene was cleaved with NotI, and the human DNA inserts were isolated. Not1 cleavage liberates the DNA inserted in the cosmid vector plus short flanking sequences en- coding the T3 and T7 promoters. The inserts were used

LOCALIZATION OF IGBPl AND IGBP3 GENES 499

90 kb

.90kb

FIG. 2. Pulsed-field gel electrophoresis of DNA isolated from pe- ripheral blood leukocytes. The filter was successively hybridized with genomic probes for IGBPl and IGBPB. DNA samples were cleaved with the indicated enzymes. M denotes size markers from Saccharo- myces cereuisiae strain YPl48. Two chromosomes, 1025 and 90 kb of this strain, cross-hybridize with pBR322.

in a cross-hybridization experiment to analyze whether the clones were overlapping. The IGBP3 insert hybrid- ized at high stringency with a 3%kb EcoRI fragment from the IGBPl cosmid, and the IGBPl insert hybrid- ized with an 11-kb EcoRI fragment from the IGBP3 cos- mid. These EcoRI fragments (3.8 and 11 kb) were also identified as end fragments of the two cosmids by hybrid- ization with the T3 primer. Thus, the two EcoRI frag- ments overlap and constitute the 12-kb genomic EcoRI fragment shown in the restriction map (Fig. 3). Orientation of the IGBPl and IGBP3 Genes

To determine the relative orientation of the two genes, a restriction map was established (Fig. 3). The cosmid

LhBPl-2:l chBPl-1:2

chBP3-3:12 IGBP3

-

clones were cleaved with iVot1, followed by partial as well as complete cleavage with BamHI or EcoRI or by only complete cleavage with BssHII, NaeI, Sad, or SmaI. The generated fragments were separated by electropho- resis, and after Southern blotting the DNA fragments were hybridized with the T3 and T7 primers. In addi- tion, isolated EcoRI and BamHI fragments from both cosmids were used as hybridization probes to confirm the restriction maps. The cosmids were shown to repre- sent authentic DNA from the IGBPl and IGBP3 gene regions, since the same length of BamHI and EcoRI fragments was detected in an experiment in which the whole cosmids were used as hybridization probes to hu- man DNA cleaved with BamHI or EcoRI.

The 3.8-kb end fragment of the IGBPl cosmid and the genomic probe corresponding to the 3’ end of the IGBPS gene (p6.2hgBP3) hybridized with the same ll-kb end fragment of the IGBP3 cosmid. This indicated that the IGBPl gene is located 3’ of the IGBP3 gene. The 11-kb end fragment of the IGBPS cosmid was isolated and shown to hybridize with a 22-kb end fragment of the IGBPl cosmid generated by SnaI and Not1 cleavage. The 22-kb fragment is part of the genomic 30-kb frag- ment shown in Fig. 3. A probe specific for the 3’ end of the IGBPl gene (phIGFBPl-B3.4) also hybridized to the same 22-kb SmaI fragment. These results, in con- junction with the restriction map, showed that the two genes are oriented tail-to-tail and consequently are transcribed in opposite directions.

Sequence Determination and Identification of

Polymorphisms

Genomic clones were isolated from a human genomic h library using the cDNA clone encoding IGBPl as a probe. The DNA sequence was determined for 6218 bp covering the IGBPl gene. IGBPl is encoded by four exons (Fig. 4, top) (Brinkman et al., 1988b; Cubbage et

IGBPl

-

>2.0 . 0.9 6 o 0.6

3.2 3.4 1.1 6.5 1.7 12 3.5 1.2

4.0 1.8 5.2 40 o.9 . 5.6 2.3 2.0 EcoRI I I II 1 I 1 I I I I I II I I I I I I

>2.4 11.2 1.2 1.5 18 3.4 5.8 >ll BamHl I I I I I I I

0.9O.20.2 0.3

>21 33 0.2 >12 BssHII I” II

3.9 1.8 28 Nael _ - - - _ _ _ _ - _ _ I I I I------

>21 1.40e32.9 30 >13 Sac11 III I 1

0.6 1.3 >20 1.0 1.7 30 >13

Smal III I I 1

FIG. 3. Restriction endonuclease map of the DNA region covering the human IGBPl and IGBPB genes. The extension of X and cosmid clones is shown at the top. Direction of transcription is indicated by arrows and the location of exons is marked by solid boxes, Dashed lines indicate unmapped regions. Restriction sites for BssHII, NC& So&, and &no1 within the genes were deduced from determined DNA se- quences. All sizes of DNA fragments are given in kb.

500 EHRENBORG ET AL.

Exon 1 Exon 2 Exon 3 Exon 4 (514) (170) (129) (701)

-1 v,,,~ (1546) /1 t- I- 51 53-1

4 \ GluPr~CysArgIleGluLeuTyrArgValValGluSer~

4021 ATGTCAAGTTATTCTCTTAGGAGCCCTGCCGAATAGAACTCTACAGAGTCGTAGAGAGTT euAlaLysAlaGlnGluThrSerGlyGluGluIleSerLysPheTyrLeuProAsnCysA

4081 TAGCCAAGGCACAGGAGACATCAGGAGAAGAAATTTCCAAATTTTACCTGCCAAACTGCA snLysAsnGlyPheTyrHisSerArgGln

4141 ACAAGAATGGATTTTATCACAGCAGACAGGTAGGTGGCCTTGCCAGTGTGCGTCGTCAGG 4201 GTGAAAGGGACTACTGCCCTACATTCCTGCCAAGCCACGGTCATTCATGTCAAAGAAGGT 4261 CCACTCCAAAGTAGACACCAGAAGTGGTTGTATTGAGCCAGATCCACCCCTCTGGGAACC 4321 TGGAACAGCTAGGTGAAGAAAGCCTATTGAACAGGTCAATATGTCACCACCAGAGGTGGA 4381 AAACCTGCGCTGGACCAGGGGCCCTGGGGCTAAGCCTGAGCTCCATCACTCAGCAAGCCA 4441 ATAGCTATGGAAGACTTTCCTAGGCCCCCTAACGCTTGGATCTTGGGTTCCTCACCTATA 4501 CAAGAGAGGAAGGGAACAAACTTAACTTAGAAAAGCAAGTATCTATTTGGACAGCTCTTA 4561 ACACTTTTTTCTCCTTGAACTGTCTMTAAAACACAACAACATTTTTGTGGCTTGCCAGA 4621 TAATAAATGAGATCAAACAGACCAAGATTTCATATTGAGGAGTGCTTTAGGTCTCAGTGA

A 4681 AGTACAGGTTCTGTAGATTTTATTGGGAGAAACTGAGGAGGACTAGGCCCTGCTTCAC~GGCA

T 4741 ATGAACAGTGGGGCACACACGAGACATGTTCCCTCTGGG~TGGGCTCCCCTGACATCAGG

G ( ApaLI) 4801 CTATGAAGCAGACAGCTGT~CACACACTGTACTGTTTAACACACATGGGAAGTCATTATT

4861 GCACATGCCACTCGGTCACTGTCTCTATTTTATGATGAGACCAGGAGGGTGTGAGATTT c s

4921 GCCTGCATCATGGGCAGCTGGTTTCACAGCCGGGBCTCTT~rCTTGGCTCTGC~CTC AALKKGPAMALLSLSACHLL

4981 GGCTGCACTGAAAAAA GGGCCAGCTATGGCTCTACTTTCCCTGTCAGCTTGTCATCTGCT L L T L V L S L Q CysGluThrSerMetAspGlyGluAlaGlyLe

5041 GCTCTT A GfTGAGACATCCATGGATGGAGAGGCGGGACT uCysTrpCysValTyrProTrpAsnGlyLysArgIleProGlySerProGluIleArgGl

5101 CTGCTGGTGCGTCTACCCTTGGAATGGGG~GAGGATCCCTGGGTCTCCAGAGATCAGGGG yAspProAsnCysGlnMetTyrPheAsnValGlnAsn

5161 AGACCCCAACTGCCAGATGTATTTTAATGTAC~CTG~CCAGATGAAATAATGTTC

FIG. 4. Schematic map of the IGBPl gene with a 3’ segment of the DNA sequence shown below. Boxes indicate exons with the translated segments hatched. The lengths of exons and introns in bp are stated within parentheses, and the positions of primers 51 and 53 are shown. A segment of the genomic IGBPl DNA sequence is shown, with the parts encoding IGBPl denoted by amino acids in three-letter code given above the corresponding codon. The sequence of the putative insert of 31 amino acids is indicated in single-letter code. Single-underlined sequences show the two 20 of 24-bp repeats of the intron 3/exon 4 splice junction. At positions 4736,4780, and 4820 (double-underlined) the nucleotide substitutions are indicated for the allelic variant.

al., 1989) and spans 5173 bp. This genomic sequence matches that of the cDNA in all positions, except that it codes for Met-228, while the cDNA clone phIGFBPl- 103 codes for Ile-228. This difference represents an es- tablished protein polymorphism (Luthman et al., 1989).

Possible DNA sequence polymorphisms were found at three positions (Fig. 4), 4736 (G/A), 4780 (A/T), and 4820 (A/G), when the DNA sequence was compared to that reported by Cubbage et al. (1989). The two se- quences were verified as allelic variants by sequencing a PCR product from DNA made from an astrocytoma cell line that was identical to that of Cubbage et al. (1989). Furthermore, the frequency of the substitution of an A for a G at position 4820 was possible to measure, as it forms an ApaLI site (Fig. 4). ApuLI cleavage identifies a two-allelic polymorphism with one fragment of 8.6 kb, allele 1, and two fragments of approximately 4.3 kb, al- lele 2 (not shown). Allele 1 was detected in 4 of 18 chro- mosomes from unrelated Scandinavians. In the XhBPl- 2:l DNA sequence the ApaLI site is absent and Met-228 is present. However, the opposite situation was found in

the previously reported IGBPl gene sequence (Cubbage et al., 1989); the ApaLI site and Ile-228 are present.

A detailed analysis of the IGBPl DNA sequence re- vealed a direct repeat of 20 of 24 bp from the splice junc- tion between intron 3 and exon 4. The splice junction, including a branch point, a pyrimidine-rich tract, and the AG dinucleotide, is repeated 93 bp upstream of exon 4 (Fig. 4). The repeat is located in frame with the normal exon 4 sequence, such that an alternatively spliced RNA would code for a protein with an in-frame insertion of 31 amino acids compared to the normal IGBPl protein.

TaqI cleavage also identified an RFLP when the IGBPl cosmid clone was used as a probe. Allele 1 gave a fragment of 6.1 kb and allele 2 a fragment of 4.4 kb (not shown). Allele 1 was detected in 7 of 16 chromosomes from unrelated Scandinavians.

DISCUSSION

These studies demonstrate that the genes encoding IGBPl and IGBP3 are contiguous on human chromo- some 7, where IGBPl earlier had been localized to region

LOCALIZATION OF IGBPl AND IGBP3 GENES 501

7p14-p12 (Alitalo et aZ., 1989; Ekstrand et cd., 1990). The gene family encoding IGF binding proteins includes four cloned genes (Brewer et al., 1988; Brinkman et al., 1988a; Julkunen et al., 1988; Lee et al., 1988; Luthman et al., 1989; Binkert et al., 1989; Wood et al., 1988; Mohan et al., 1989; Shimasaki et al., 1990; LaTour et aZ., 1990) and most likely at least one additional member (Roghani et al., 1989; Martin et al., 1990; Zapf et al., 1990). The evo- lution of gene families commonly involves duplication of an ancestral gene, which makes the generation of func- tional diversity possible within the family. Head-to-tail duplicated genes undergo frequent unequal crossing- over, which conserves sequence similarity between the genes (Nathans et aZ., 1986). However, evolution of ma- jor sequence differences required for the development of diversified function and regulation within a gene family is possible only if direct sequence repetition is in- terrupted. This can be achieved, e.g., by insertion of new DNA sequences in noncoding parts, inversion, or trans- position to another chromosome of one of the genes in the gene family. Accordingly, evolution of the structural differences between IGBPl and IGBP3 may be partially explained by their tail-to-tail orientation, as this pre- vents homogenization of their sequences by simple un- equal crossing-over. The differences are typified by the limited overall amino acid identity-33% (Wood et al., 1988)-and the presence of an extra intron in the 3’un- translated region of IGBP3 (Cubbage et al, 1990), as well as dissimilarities of the hormonal, developmental, and tissue-specific control of their gene expression (Brinkman et al., 1988a; Clemmons et al., 1989; Conover, 1990; Hardouin et al., 1989; Julkunen et al., 1988; Liu et al., 1991; Margot et al., 1989; Ooi et al., 1990; Seneviratne et al., 1990; Shimasaki et al., 1989; Unterman et al., 1990). When the respective IGF binding proteins of the human and the rat are compared, at least 67% amino acid homology is found (Shimasaki et al., 1989, 1990; Murphy et al., 1990). This indicates that the rearrange- ments that led to the evolution of the different forms of IGBPs occurred before the mammalian radiation, and it suggests that the IGBPl and IGBP3 genes are also linked in other mammalian species.

The normal intron 3/exon 4 splice site of the IGBPl gene fulfills all known criteria for a functional 3’ accep- tor splice junction (Smith et al., 1989): the dinucleotide AG located as the last two bases of the intron; a stretch of pyrimidines typically located immediately upstream of the dinucleotide; and a branch point consensus se- quence usually found about 30 nucleotides upstream of the 3’ splice site. The repeated sequence 93 bp upstream in intron 3 includes all these elements, except for a com- plete consensus branch point sequence (5’-PyNPyT- PuAPy-3’). However, the most conserved base in the branch point sequence (A) is present. Whether this al- ternative 3’ splice junction is used at all, and its potential functional consequences, should be investigated by di- rect means.

ACKNOWLEDGMENTS

We thank Drs. V. Sara and N. Hastie for gifts of RNA and DNA samples. The cosmid library was kindly provided by Dr. G. Evans.

This work was supported by grants from the Swedish Medical Re- search Council, the King Gustaf V and Queen Victoria Foundation,

the Knut and Alice Wallenberg Foundation, the Swedish Cancer Soci- ety, the Gustaf V Jubilee Fund, NIH FIRST Award DK38773, and March of Dimes Basic Research Grant 1-1186.

REFERENCES

Alitalo, T., Kontula, K., Koistinen, R., Aalto-Set&la, K., Julkunen, M., Jlnne, 0. A., Sepplll, M., and de la Chapelle, A. (1989). The gene encoding human low-molecular weight insulin-like growth- factor binding protein (IGF-BP25): Regional localization to 7p12-

p13 and description of a DNA polymorphism. Hum. Genet. 83: 335- 338.

Berger, S. L., and Kimmel, A. R., Eds. (1987). “Methods in Enzymol- ogy,” Vol. 152, “Guide to Molecular Cloning techniques,” Academic Press, San Diego.

Binkert, C., Landwehr, J., Mary, J.-L., Schwander, J., and Heinrich, G. (1989). Cloning, sequence analysis and expression of a cDNA encoding a novel insulin-like growth factor binding protein (IGFBP-2). EMBO J. 8: 2497-2502.

Brewer, M. T., Stetler, G. L., Squires, C. H., Thompson, R. C., Busby, W. H., and Clemmons, D. R. (1988). Cloning, characterization, and expression of a human insulin-like growth factor binding protein. Biochem. Biophys. Res. Commun. 152: 1289-1297.

Brinkman, A., Groffen, C., Kortleve, D. J., Geurts van Kessel, A., and Drop, S. L. S. (1988a). Isolation and characterization of a cDNA encoding the low molecular weight insulin-like growth factor bind- ing protein (IBP-1). EMBO J. 7: 2417-2423.

Brinkman, A., Groffen, C., Kortleve, D. J., andDrop, S. L. S. (1988b). Organization of the gene encoding the insulin-like growth factor binding protein IBP-1. Biochem. Biophys. Res. Commun. 157: 898- 907.

Burch, W. M., Correa, J., Shively, J. E., and Powell, D. R. (1990). The 25-kilodalton insulin-like growth factor (IGF)-binding protein in- hibits both basal and IGF-I-mediated growth of chick embryo pelvic cartilage in vitro. J. Clin. Endocrinol. Metab. 70: 173-180.

Cascieri, M. A., Saperstein, R., Hayes, N. S., Green, B. G., Chicchi G. G., Applebaum, J., and Bayne, M. L. (1988). Serum half-life and biological activity of mutants of human insulin-like growth factor I which do not bind to serum binding proteins. Endocrinology 123: 373-381.

Clemmons, D. R., Thissen, J. P., Maes, M., Ketelslegers, J. M., and Underwood, L. E. (1989). Insulin-like growth factor-I (IGF-I) infu- sion into hypophysectomized or protein-deprived rats induces spe- cific IGF-binding proteins in serum. Endocrinology 125: 2967-2972.

Collins, V. P. (1983). Cultured human glial and glioma cells. In “Inter- national Review of Experimental Pathology” (G. W. Richter and M. A. Epstein, Eds.), Vol. 24, pp. 135-202, Academic Press, New York.

Conover, C. A. (1990). Regulation of insulin-like growth factor (IGF)-binding protein synthesis by insulin and IGF-I in cultured bovine fibroblasts. Endocrinology 126: 3139-3145.

Conover, C. A., Ronk, M., Lombana, F., and Powell, D. R. (1990). Structural and biological characterization of bovine insulin-like growth factor binding protein-3. Endocrinology 127: 2795-2803.

Cubbage, M. L., Suwanichkul, A., and Powell, D. R. (1989). Structure of the human chromosomal gene for the 25 kilodalton insulin-like growth factor binding protein. Mol. Endocrinol. 3: 846-851.

Cubbage, M. L., Suwanichkul, A., and Powell, D. R. (1990). Insulin- like growth factor binding protein-3 (IGFBP-3): Organization of the human chromosomal gene and demonstration of promotor activ- ity. J. Biol. Chem. 265: 12,642-12,649.

De Mellow, J. S. M., and Baxter, R. C. (1988). Growth hormone-de- pendent insulin-like growth factor (IGF) binding protein both in- hibits and potentiates IGF-I-stimulated DNA synthesis in human skin fibroblasts. Biochem. Biophys. Res. Commun. 166: 199-204.

Ehrenborg, E., Vilhelmsdotter, S., Bajalica, S., Larsson, C., Stern, I.,

502 EHRENBORG ET AL.

Koch, J., Brondum-Nilsen, K., and Luthman, H. (1991). Structure and localization of the human insulin-like growth factor-binding protein 2 gene. Biochem. Biophys. Res. Commun. 176: 1250-1255.

Ekstrand, J., Ehrenborg, E., Stern, I., Stellan, B., Zech, L., and Luth- man, H. (1990). The gene for insulin-like growth factor-binding protein-l is localized to human chromosomal region 7~14-12. Germ- mics. 6: 413-418.

Evans, G. A., Lewis, K., and Rothenberg, B. E. (1989). High efficiency vectors for cosmid microcloning and genomic analysis. Gene 79:

9-20.

Froesch, E. R., Schmid, C., Schwander, J., and Zapf, J. A. (1985). Actions of insulin-like growth factors. Annu. Rev. Physiol. 47: 443- 467.

Grossberger, D. (1987). Minipreps of DNA from bacteriophage lambda. Nucleic Acids Res. 15: 6737.

Hardouin, S., Gourmelen, M., Noguiez, P., Seurin, D., Roghani, M., Lebouc, Y., Povoa, G., Merimee, T. J., Hossenlopp, P., and Binoux, M. (1989). Molecular forms of serum insulin-like growth factor

(IGF)-binding proteins in man: Relationships with growth hormone and IGFs and physiological significance. J. Clin. Endocrinol. Metab. 69: 1291-1301.

Herrmann, B. G., Barlow, D. P., and Lehrach, H. (1987). A large in- verted duplication allows homologous recombination between chro- mosomes heterozygous for the proximal T-complex inversion. CeI1 48: 813-825.3

Jones, C. P., Janson, M., and Nordenskjold, M. (1989). Separation of yeast chromosomes in the megabase range suitable as size markers for pulse-field gel electrophoresis. Technique 1: 90-95.

Julkunen, M., Koisinen, R., Aalto-Set&i, K., Seppiila, M., Janne, 0. A., and Kontula, K. (1988). Primary structure of human insulin- like growth factor-binding protein/placental protein 12 and tissue- specific expression of its mRNA. FEBS Lett. 236: 295-302.

LaTour, D., Mohan, S., Linkhart, T. A., Baylink, D. J., and Strong, D. D. (1990). Inhibitory insulin-like growth factor-binding protein: Cloning, complete sequence, and physiological regulation. Mol. En- docrinol. 4: 1806-1814.

Lee, Y.-L., Hintz, R. L., James, P. M., Lee, P. D. K., Shively, J. E., and Powell, D. R. (1988). Insulin-like growth factor (IGF) binding pro- tein complementary deoxyribonucleic acid from human HepG2 hep- atoma cells: Predicted protein sequence suggests an IGF binding domain different from those of the IGF-I and IGF-II receptors. Mol. Endocrinol. 2: 404-411.

Liu, F., Powell, D. R., Styne, D. M., and Hintz, R. L. (1991). Insulin- like growth factors and insulin-like growth factor binding proteins in the developing Rhesus monkey. J. Clin. Endocrinol. Metab. 72: 905-911.

Lund, E., Bostock, C., Robertson, M., Christie, S., Mitchen, J. L., and Dahlberg, J. E. (1983). Ul small nuclear RNA genes are located on

human chromosome 1 and are expressed in mouse-human hybrid cells. Mol. Cell. Biol. 3: 2211-2220.

Luthman, H., Soderling-Barros, J., Persson, B., Engberg, C., Stern, I., Lake, M., Fran&n, S.-A., Israelsson, M., R&den, B., Lindgren, B., Hjelmqvist, L., Enerbtick, S., Carlsson, P., Bjursell, G., Povoa, G., Hall, K., and Jiirnvall, H. (1989). Human insulin-like growth-fac- tor-binding protein. Low-molecular-mass form: Protein sequence and cDNA cloning. Eur. J. &o&em. 180: 259-265.

Margot, J. B., Binkert, C., Mary, J.-L., Landwehr, J., Heinrich, G., and Schwander, J. (1989). A low molecular weight insulin-like growth factor binding protein from rat: cDNA cloning and tissue distribution of its messenger RNA. Mol. Endocrinol. 3: 1053-1060.

Martin, J. L., Willetts, K. E., and Baxter, R. C. (1990). Purification and properties of a novel insulin-like growth factor-II binding pro- tein from transformed human fibroblasts. J. Biol. Chem. 265: 4124- 4130.

Mohan, S., Bautista, C. M., Wergedal, J., and Baylink, D. J. (1989). Isolation of an inhibitory insulin-like growth factor (IGF) binding

protein from bone cell-conditioned medium: A potential local regula- tor of IGF action. Proc. Natl. Acad. Sci. USA 86: 8338-8342.

Murphy, L. J., Seneviratne, C., Ballejo, G., Croze, F., and Kennedy, T. G. (1990). Identification and characterization of a rat decidual

insulin-like growth factor-binding protein complementary DNA. Mol. Endocrinol. 4: 329-336.

Nathans, J., Piantanida, T. P., Eddy, R. L., Shows, T. B., and Hog- ness, D. S. (1986). Molecular genetics of inherited variation in hu- man color vision. Science 232: 203-210.

Ooi, G. T., Orlowski, C. C., Brown, A. L., Becker, R. E., Unterman, T. G., and Rechler, M. M. (1990). Different tissue distribution and

hormonal regulation of messenger RNAs encoding rat insulin-like growth factor-binding proteins-l and -2. Mol. Endocrinol. 4: 321- 328.

Ritvos, O., Ranta, T., Jalkanen, J., Suikkari, A.-M., Voutilainen, R., Bohn, H., and Rutanen, E.-M. (1988). Insulin-like growth factor (IGF) binding protein from human decidua inhibits the binding and biological action of IGF-I in cultured choriocarcinoma cells. Endo- crinology 122: 2150-2157.

Roghani, M., Hossenlopp, P., Lepage, P., Balland, A., and Binoux, M. (1989). Isolation from human cerebrospinal fluid of a new insulin- like growth factor-binding protein with a selective affinity for IGF- II. FEBS Lett. 255: 253-258.

Seneviratne, C., Luo, J., and Murphy, L. J. (1990). Transcriptional regulation of rat insulin-like growth factor-binding protein-l ex- pression by growth hormone. Mol. Endocrinol. 4: 1199-1204.

Shimasaki, S., Koba, A., Mercado, M., Shimonaka, M., and Ling, N. (1989). Complementary DNA structure of the high molecular weight rat insulin-like growth factor binding protein (IGF-BP3) and tissue distribution of its mRNA. B&hem. Biophys. Res. Com- mun. 165: 907-912.

Shimasaki, S., Uchiyama, F., Shimonaka, M., and Ling, N. (1990). Molecular cloning of the cDNAs encoding a novel insulin-like growth factor-binding protein from rat and human. Mol. Endo- crinol. 4: 1451-1458.

Smith, C. W. J., Patton, J. G. and Nadal-Ginard, B. (1989) Alternative splicing in the control of gene expression. Annu. Rev Genet. 23: 527-577.

Ui, M., Shimonaka, M., Shimasaki, S., and Ling, N. (1989). An insu- lin-like growth factor-binding protein in ovarian follicular fluid blocks follicle-stimulating hormone-stimulated steroid production by ovarian granulosa cells. Endocrinology 125: 912-916.

Unterman, T. G., Patel, K., Mahathre, V. K., Rajamohan, G., Oehler, D. T., and Becker, R. E. (1990). Regulation of low molecular weight insulin-like growth factor binding proteins in experimental diabetes mellitus. Endocrinology 126: 2614-2624.

Wedell, A., Datta, S., Andersson, B., and Luthman, H. (1990). A modi- fied procedure for polymerase chain reaction amplification of single stranded DNA improves direct sequencing. Technique 2: 23-26.

White, R. M., Nissley, S. P., Moses, A. C., Rechler, M. M., and John- sonbaugh, R. E. (1981). The growth hormone dependence of a soma- tomedin-binding protein in human serum. J. Clin. Endocrinol. Me- tab. 53: 49-57.

Wood, W. I., Cachianes, G., Henzel, W. J., Winslow, G. A., Spencer, S. A., Hellmiss, R., Martin, J. L., and Baxter, R. C. (1988). Cloning and expression of the growth hormone-dependent insulin-like growth factor-binding protein, Mol. Endocrinol. 2: 1176-1185.

Zapf, J., Kiefer, M., Merryweather, J., Masiarz, F., Bauer, D., Born, W., Fischer, J. A., and Froesch, E. R. (1990). Isolation from adult human serum of four insulin-like growth factor (IGF) binding pro- teins and molecular cloning of one of them that is increased by IGF I administration and in extrapancreatic tumor hypoglycemia. J. Biol. Chem. 265: 14,89214,898.