Characterization and chromosomal localization of the human
insulin-like growth factor-binding protein 6 gene
Susanne V. Allander,
Department of Molecular Medicine, Rolf Luft Center for Diabetes Research, Karolinska Hospital, SE-171 76 Stockholm, Sweden
Department of Molecular Medicine, Endocrine Tumor Unit, Karolinska Hospital, CMM L8, SE-171 76 Stockholm, Sweden
Received: 14 October 1998 / Accepted: 1 December 1998
Abstract. Insulin-like growth factor-binding protein 6 (IGFBP6),
an extracellular protein with preferential affinity for insulin-like
growth factor (IGF) II, belongs to a family of binding proteins with
at least six members. We have characterized the genomic structure
and the chromosomal location of the human IGFBP6, which is
present in the human genome as a single-copy gene spanning 4.7
kb. It consists of four exons, encoding the translated regions, with
sizes of 334, 146, 120, and 123 bp, while the intervening introns
are 2661, 182, and 844 bp. Three mRNA cap sites were localized
101, 100, and 96 bp upstream of the ATG translation start codon
as determined by S1 nuclease analysis. The proximal 5Ј-flanking
region does not have any TATA or CAAT consensus sequences.
The IGFBP6 was localized to Chr 12 by analysis of somatic cell
hybrids and regionalized to 12q13 by fluorescence DNA in situ
Insulin-like growth factors I and II are complexed with specific
binding proteins in extracellular fluids. These binding proteins
constitute a separate protein family with pronounced structural
homologies, mainly owing to conserved amino acid sequences in
the N-terminal and C-terminal regions. At least six distinct
IGFBPs (IGFBP1–6) have been characterized thus far, and their
cDNAs, and in some cases proteins, have been isolated and se-
quenced (Murphy 1998). Despite their structural and functional
similarities, the binding proteins display unique expression pat-
terns and bind IGF-I and -II with different affinities (Murphy
1998). The IGFBPs have been isolated and characterized from
several mammalian species, and at least one binding protein,
IGFBP5, has been identified in non-mammalian vertebrates (Up-
ton et al. 1993). This suggests that modulation of IGF action by
specific binding proteins is important in vivo (James et al. 1993).
The IGF binding protein 6 gene shows a tissue-specific expression
in human but is ubiquitously expressed in rat (Kiefer et al. 1991b;
Shimasaki et al. 1991). IGFBP-6 binds predominantly IGF-II, pre-
venting it from binding to specific cell-surface receptors (Murphy
1998). Up to date, the genomic sequences of human IGFBP1–5
have been characterized and their chromosomal localizations de-
termined (Allander et al. 1993, 1994; Zazzi et al. 1998). These
genes show a similar genomic organization, although their sizes
varies from 5.2 kb for IGFBP1 to 33 kb for IGFBP5, mainly owing
to the lengths of the first intron, which range from 1.5 kb to 27 kb.
All members of the IGFBP family consist of four exons with the
exception of IGFBP3, which has an additional untranslated fifth
exon in the 3Ј end. IGFBP1–5 have been regionally localized to
human chromosomes (Allander et al. 1993, 1994). The IGF-
binding protein 1 and 3 genes are located at chromosomal region
7p14-12 in a tail-to-tail fashion separated by 20 kb. Likewise, the
IGFBP2 and 5 genes at 2q33-34 are arranged in opposite tran-
scriptional directions and separated by 20–40 kb. IGFBP4 is lo-
cated at chromosomal region 17q12-21.1, and IGFBP6 has earlier
been mapped to Chr 12 by PCR analysis of somatic cell hybrids
(Shimasaki et al. 1991). In order to further explore the genomic
organization of the human IGFBP gene family, we have isolated
and characterized the structure of IGFBP6. In addition, we report
the regional localization of the gene.
Materials and methods
Six primers were generated: 5Ј-GGAA-
GCTGAGGGCTGTCTCA (primer 6-11), 5Ј-AGCGAGCAGCAGAGC-
TAGCA (primer 6-6) and 5Ј-CCGTCCTCTCCTTCCAGAG fluorescein-
labeled (primer 6-12fl.) located in exon 1; 5Ј-CTGTTGCAGAG-
GAGAATCCT (primer 6-7) from exon 1 and exon 2; 5Ј-
CTACGTGCCCAATTGTGACC (primer 6-10) from exon 3; and 5Ј-
CAGCGACCCCAAGCACAGC (primer 6-4) from exon 4.
Somatic cell hybrids.
DNA isolated from the somatic human–rodent cell
hybrids (NIGMS Coriell Cell Repositories), from Chinese hamster cells,
and from peripheral leukocytes from normal individuals, was cleaved with
HindIII and Southern blotted. The filter was hybridized to a 570-bp probe
derived by PCR amplification with primers 6-7 and 6-4. PCR amplifica-
tions of the DNA were performed with primers 6-10 and 6-4. Genomic
DNAs (50 ng) were amplified in 50-l reactions containing 0.3
primer, 50 m
KCl, 1.5 m
Tris-HCl, 0.1% Tween 20, 0.2
of each dNTP, and 1.0 U Taq-polymerase (Perkin-Elmer-Cetus). The
reactions were incubated for 5 min at 95°C, followed by 35 cycles at 96°C
for 1 min, 58°C for 0.5 min, and 72°C for 1.5 min.
Isolation of genomic clones.
The cosmid clone chBP6-6:1 was isolated
colonies with a
P-labeled human IGFBP6 cDNA
probe consisting of a PCR-amplified fragment with primers 6-11 and 6-4.
Cosmid DNA was isolated from positive clones, an EcoRI restriction map
was constructed, and the authenticity of the cosmid clone was confirmed by
Southern hybridization to EcoRI-cleaved total human DNA. Two adjacent
EcoRI fragments, which contained exon sequences, 6.3 and 5.8 kb in size,
were subcloned into pGEM-7Zf(+) to generate plasmids phIGFBP6-E6.3
and phIGFBP6-E5.8 that were used for DNA sequencing.
Genomic sequencing of IGFBP6.
Ten g DNA of chBP6-6:1,
phIGFBP6-E6.3, or phIGFBP6-E5.8 were randomly sheared by sonication
Present address: Atherosclerosis Research Unit, King Gustaf V Research
Institute, Karolinska Hospital, SE-171 76 Stockholm, Sweden.
Present address: Department of Clinical Genetics, Uppsala University,
Children’s Hospital, SE-751 85 Uppsala, Sweden.
The nucleotide sequence data reported in this paper have been submitted to
the GenBank and have been assigned the accession number: AJ006952.
Correspondence to: E. Ehrenborg
Mammalian Genome 10, 376–380 (1999).
© Springer-Verlag New York Inc. 1999