Vol.
176,
May
15, 1991
No.
BIOCHEMICAL
3, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
STRUCTURE
AND LOCALIZATION GROWTH FACTOR-BINDING
1250-l
255
OF THE HUMAN INSULIN-LIKE PROTEIN 2 GENE
Ewa Ehrenborg, Susanne Vilhelmsdotter, Svetlana Bajalica, Catharina Larsson, Ingrid Stem, Jbm Koch*, Karen Brondum-Nielsen, and Holger Luthman# Department of Clinical Genetics, Karolinska Institute, S-104 01 Stockholm, Sweden *Institute for Human Genetics, Aarhus University, Denmark Received
February
25,
1991
Insulin-like growth factor binding proteins(IGFBPs) areextracellular proteinsthat specifically bind IGF and modulatetheir effects. The humanIGFBP2 genewasstudiedand shown to be localized to chromosome2 region q33-q34, by somaticcell hybrid analysisand in situ hybridization. Structural characterizationof the geneshowedthat it consistsof four exons with three introns of lengths27.0, 1.O,and 1.9 kilobase-pairs.Comparisonof the encodedprotein sequenceof eachexon in IGFBPl, 2, and 3 revealsthe highestamino acid identity, 28%, in exon 1, while the lowest was found in exon 2. However, pairwise sequencecomparisons demonstrate50% identity betweenthe protein sequences encodedby exon 4 in IGFBPI and2, while their respectiveidentitieswith IGFBP3 areonly 25 and 30%. a 1991Academic Press,1°C.
Insulin-like growth factors I and II are boundto specific extracellular binding proteins (IGFBPs) with capacity to influence the activity of thesegrowth factors (1). Presently, cDNA clonesfor four membersof this family of binding proteinshave beenisolated(2-5); at leastfive different IGFBPs have beencharacterizedto someextent at the protein level (6), revealing differencesin their affinity for IGFI and II (7). The translatedparts of the genesfor IGFBPl and 3 are divided into four exonsshowinghomology both in size and sequence(8). The human IGFBPl and 3 genesare located at chromosomalregion 7p12-p14 separatedby only 20 kb of DNA and orientatedin a tail to tail fashion(Ehrenborget al., submitted).To further investigate the structural relationshipswithin the IGFBP family we have studiedthe humanIGFBP2 gene. MATERIALS
AND METHODS
Screening of a genomic library and restriction enzyme mapping A humancosmid(pWE15) library preparedfrom placentalDNA (9), was screenedwith the rat prBP2-cDNA clone (10). The identified cosmidcloneswere purified and cosmidDNA isolated.HumanDNA insertsincluding T3 andT7 promoter sequences were excisedfrom the vector by Not1 cleavage.The sizesof the end-fragmentsgeneratedby cleavage with BssHII, EcoRI, NaeI, or SacII were determinedby blotting and hybridization with [32P]dCTP-labelled # To whom correspondenceandreprint requestsshouldbe addressed. AbbreW. IGF, insulin-like growth factors; IGFBP, insulin-like growth factor-binding protein; kb, kilobase pairs; QFQ, quinacrinefluorescenceby quinacrine. 0006-291X/91 $1.50 Copyright 0 1991 by Academic Press. Inc. All rights of reproduction in any fi)rm reserved.
1250
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No.
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RESEARCH
COMMUNICATIONS
primers, T3 (5’-ATTAACCCICACTAAAG) and ‘I7 (5’CIATAGTGAGTCGTA’I-T). The relative order of the EcoRI fragments were determined by partial cleavage and hybridization with the flanking T3 and T7 primers.
Subcloning
and DNA-sequencing
An XbaI to EcoRI fragment hybridizing with an exon 1 specific oligonucleotide, and an EcoRI fragment hybridizing with oligonucleotides from exons 2 to 4 (Fig. 2) were cloned in pGEM7z(+) to generate phIGFBP2-XE6.4 and phIGFBP2-E5.8 (Fig. 1). The DNA sequence was determined by the dideoxy method using ‘I7-polymerase (Pharmacia) and tailor-made sequencing primers (Pharmacia Gene Assembler).
PCR analysis One ng of cloned DNA was amplified in 50 u.l in the presence of 0.25 uM primer, 50 mM KCl, 1.5 mM MgC12, 10 mM Tris-HCl, pH 8.4 at 7o”C, 0.01% gelatin, 0.2 mM of each dNTP, and 1.25 U Taq-polymerase (Perkin-Elmer &us). The samples were incubated for 5 min at 94°C followed by 22 cycles at 94°C for 1 min, 50°C for 2 min, and 72°C for 3 min. The primers used were 2-6,2-8, 2-9, and 58 (Fig. 2). Fifty ng of human DNA from 11 unrelated individuals was amplified in 50 u.l in the presence of 0.2 uM primer 57 and 58 (Fig. 2), 50 mM KCI, 1.0 mM MgC12. 10 mM Tris-HCl, pH 8.4 at 7o’C, 0.1% Tween 20,0.2 mM of each dNTP, 1.O U Taq-polymerase (Perkin-Elmer Cetus). The samples were incubated for 5 min at 94’C, followed by 30 cycles at 94°C for 1 min, 58°C for 1 min, and 72’C for 3 min. Amplification of 100 ng DNA from somatic cell hybrids (BIOS Corp.) with primers 2-6 and 2-9 were performed in the same buffer as above, and incubated for 5 min at 94°C followed by 35 cycles at 94°C for 1 min, 62°C for 0.5 min, and 72°C for 4 min. PCR products were separated in 2% agarose gels.
In situ hybridization In situ hybridization was carried out with the isolated insert from chBP2-2:4 labelled with bio- 1l-dUTP by nick-translation. Prehybridization and hybridization was performed essentially as described (11). Fifty ng of the probe was annealed with 1.5 pg human DNA for 15 min at 37-C. Slides with metaphase chromosomes were prepared, treated with RNase, and post-fixated in formaldehyde. Hybridization was performed at 37°C for 60 h. The probe was made fluorescent by 3 successive treatments with biotinylated anti-avidin antibodies alternating with fluorescein isothiocyanate-avidin (12). The result was analyzed and photographed in a confocal laser scanning microscope (Leitz). After destaining the chromosomal localization was confirmed by quinacrine (QFQJ-banding.
RESULTS Isolation
and structure
AND
of the IGFBP2
DISCUSSION gene.
Two clones were isolated by screening of 8x105 colonies with a rat IGFBP2 cDNA-clone. restriction map was established using BssHII, EcoRI, NaeI, and SacII (Fig. 1). The
chBP2-2:4 chBP2-2:6 phIGFBPZ-XE6.4
I
1 I
I I
I I
phIGFBP2.ES.8 El I . i
NaeI BssHll >12.8 EcoRI
I EZ E3 * II
E4 I .
i
SaeIl
NaeI 1.2 I I
4.0
I
6.3
I
2.6
1.2 II
4.3
5.8 I
0.3 so.9 II
Figure 1. Restriction endonuclease map of the human IGFBF’2 gene. The sizes of the two cosmid clones and subclones are shown at the top. Exons are indicated by solid boxes. Cleavage sites for BssHII,EcoRI,NaeI,andSacII are indicated. Lengths are given in kb.
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BIOCHEMICAL
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authenticity of the fragments was confiied by hybridization of EcoRI-cleaved total human DNA with labelled cosmids. The fragments encompassing coding parts of the gene were subcloned (Fig. 1). The localization of the four exons and the lengths of the introns were determined by the restriction map and PCR amplifications with exon specific primers. The DNA sequences were determined of exon-inn-on borders and coding regions (Fig. 2). All splice junctions conform to the consensus sequences of other vertebrate genes (13). However, the
GAGGGAGGAGGAAGAAGCGGAGGAGGCGGCTCCCGCGCTCGCAGGGCCGTGCCACCTGCCCGCCCGCCCGCTCGCTCGCTCGCCCGCCGC -39 MetLeuProArgValGlyCysProAlaLeuProLeuProProProPraLeuLeuProLeuLeuProLeu GCCGCGCTGCCGACCGCCAGCATGCTGCCGAGAGTGGGCTGCCCCGCGCTGCCGCTGCC~CGCCGCCGCTGCTGCCGCTGCTGCCGCTG
176,
May
15, 1991
No.
BIOCHEMICAL
3, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
STRUCTURE
AND LOCALIZATION GROWTH FACTOR-BINDING
1250-l
255
OF THE HUMAN INSULIN-LIKE PROTEIN 2 GENE
Ewa Ehrenborg, Susanne Vilhelmsdotter, Svetlana Bajalica, Catharina Larsson, Ingrid Stem, Jbm Koch*, Karen Brondum-Nielsen, and Holger Luthman# Department of Clinical Genetics, Karolinska Institute, S-104 01 Stockholm, Sweden *Institute for Human Genetics, Aarhus University, Denmark Received
February
25,
1991
Insulin-like growth factor binding proteins(IGFBPs) areextracellular proteinsthat specifically bind IGF and modulatetheir effects. The humanIGFBP2 genewasstudiedand shown to be localized to chromosome2 region q33-q34, by somaticcell hybrid analysisand in situ hybridization. Structural characterizationof the geneshowedthat it consistsof four exons with three introns of lengths27.0, 1.O,and 1.9 kilobase-pairs.Comparisonof the encodedprotein sequenceof eachexon in IGFBPl, 2, and 3 revealsthe highestamino acid identity, 28%, in exon 1, while the lowest was found in exon 2. However, pairwise sequencecomparisons demonstrate50% identity betweenthe protein sequences encodedby exon 4 in IGFBPI and2, while their respectiveidentitieswith IGFBP3 areonly 25 and 30%. a 1991Academic Press,1°C.
Insulin-like growth factors I and II are boundto specific extracellular binding proteins (IGFBPs) with capacity to influence the activity of thesegrowth factors (1). Presently, cDNA clonesfor four membersof this family of binding proteinshave beenisolated(2-5); at leastfive different IGFBPs have beencharacterizedto someextent at the protein level (6), revealing differencesin their affinity for IGFI and II (7). The translatedparts of the genesfor IGFBPl and 3 are divided into four exonsshowinghomology both in size and sequence(8). The human IGFBPl and 3 genesare located at chromosomalregion 7p12-p14 separatedby only 20 kb of DNA and orientatedin a tail to tail fashion(Ehrenborget al., submitted).To further investigate the structural relationshipswithin the IGFBP family we have studiedthe humanIGFBP2 gene. MATERIALS
AND METHODS
Screening of a genomic library and restriction enzyme mapping A humancosmid(pWE15) library preparedfrom placentalDNA (9), was screenedwith the rat prBP2-cDNA clone (10). The identified cosmidcloneswere purified and cosmidDNA isolated.HumanDNA insertsincluding T3 andT7 promoter sequences were excisedfrom the vector by Not1 cleavage.The sizesof the end-fragmentsgeneratedby cleavage with BssHII, EcoRI, NaeI, or SacII were determinedby blotting and hybridization with [32P]dCTP-labelled # To whom correspondenceandreprint requestsshouldbe addressed. AbbreW. IGF, insulin-like growth factors; IGFBP, insulin-like growth factor-binding protein; kb, kilobase pairs; QFQ, quinacrinefluorescenceby quinacrine. 0006-291X/91 $1.50 Copyright 0 1991 by Academic Press. Inc. All rights of reproduction in any fi)rm reserved.
1250
Vol.
176,
No.
BIOCHEMICAL
3, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
primers, T3 (5’-ATTAACCCICACTAAAG) and ‘I7 (5’CIATAGTGAGTCGTA’I-T). The relative order of the EcoRI fragments were determined by partial cleavage and hybridization with the flanking T3 and T7 primers.
Subcloning
and DNA-sequencing
An XbaI to EcoRI fragment hybridizing with an exon 1 specific oligonucleotide, and an EcoRI fragment hybridizing with oligonucleotides from exons 2 to 4 (Fig. 2) were cloned in pGEM7z(+) to generate phIGFBP2-XE6.4 and phIGFBP2-E5.8 (Fig. 1). The DNA sequence was determined by the dideoxy method using ‘I7-polymerase (Pharmacia) and tailor-made sequencing primers (Pharmacia Gene Assembler).
PCR analysis One ng of cloned DNA was amplified in 50 u.l in the presence of 0.25 uM primer, 50 mM KCl, 1.5 mM MgC12, 10 mM Tris-HCl, pH 8.4 at 7o”C, 0.01% gelatin, 0.2 mM of each dNTP, and 1.25 U Taq-polymerase (Perkin-Elmer &us). The samples were incubated for 5 min at 94°C followed by 22 cycles at 94°C for 1 min, 50°C for 2 min, and 72°C for 3 min. The primers used were 2-6,2-8, 2-9, and 58 (Fig. 2). Fifty ng of human DNA from 11 unrelated individuals was amplified in 50 u.l in the presence of 0.2 uM primer 57 and 58 (Fig. 2), 50 mM KCI, 1.0 mM MgC12. 10 mM Tris-HCl, pH 8.4 at 7o’C, 0.1% Tween 20,0.2 mM of each dNTP, 1.O U Taq-polymerase (Perkin-Elmer Cetus). The samples were incubated for 5 min at 94’C, followed by 30 cycles at 94°C for 1 min, 58°C for 1 min, and 72’C for 3 min. Amplification of 100 ng DNA from somatic cell hybrids (BIOS Corp.) with primers 2-6 and 2-9 were performed in the same buffer as above, and incubated for 5 min at 94°C followed by 35 cycles at 94°C for 1 min, 62°C for 0.5 min, and 72°C for 4 min. PCR products were separated in 2% agarose gels.
In situ hybridization In situ hybridization was carried out with the isolated insert from chBP2-2:4 labelled with bio- 1l-dUTP by nick-translation. Prehybridization and hybridization was performed essentially as described (11). Fifty ng of the probe was annealed with 1.5 pg human DNA for 15 min at 37-C. Slides with metaphase chromosomes were prepared, treated with RNase, and post-fixated in formaldehyde. Hybridization was performed at 37°C for 60 h. The probe was made fluorescent by 3 successive treatments with biotinylated anti-avidin antibodies alternating with fluorescein isothiocyanate-avidin (12). The result was analyzed and photographed in a confocal laser scanning microscope (Leitz). After destaining the chromosomal localization was confirmed by quinacrine (QFQJ-banding.
RESULTS Isolation
and structure
AND
of the IGFBP2
DISCUSSION gene.
Two clones were isolated by screening of 8x105 colonies with a rat IGFBP2 cDNA-clone. restriction map was established using BssHII, EcoRI, NaeI, and SacII (Fig. 1). The
chBP2-2:4 chBP2-2:6 phIGFBPZ-XE6.4
I
1 I
I I
I I
phIGFBP2.ES.8 El I . i
NaeI BssHll >12.8 EcoRI
I EZ E3 * II
E4 I .
i
SaeIl
NaeI 1.2 I I
4.0
I
6.3
I
2.6
1.2 II
4.3
5.8 I
0.3 so.9 II
Figure 1. Restriction endonuclease map of the human IGFBF’2 gene. The sizes of the two cosmid clones and subclones are shown at the top. Exons are indicated by solid boxes. Cleavage sites for BssHII,EcoRI,NaeI,andSacII are indicated. Lengths are given in kb.
1251
A
Vol.
176,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
authenticity of the fragments was confiied by hybridization of EcoRI-cleaved total human DNA with labelled cosmids. The fragments encompassing coding parts of the gene were subcloned (Fig. 1). The localization of the four exons and the lengths of the introns were determined by the restriction map and PCR amplifications with exon specific primers. The DNA sequences were determined of exon-inn-on borders and coding regions (Fig. 2). All splice junctions conform to the consensus sequences of other vertebrate genes (13). However, the
GAGGGAGGAGGAAGAAGCGGAGGAGGCGGCTCCCGCGCTCGCAGGGCCGTGCCACCTGCCCGCCCGCCCGCTCGCTCGCTCGCCCGCCGC -39 MetLeuProArgValGlyCysProAlaLeuProLeuProProProPraLeuLeuProLeuLeuProLeu GCCGCGCTGCCGACCGCCAGCATGCTGCCGAGAGTGGGCTGCCCCGCGCTGCCGCTGCC~CGCCGCCGCTGCTGCCGCTGCTGCCGCTG
