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Chromosomal assignment of the human immunophilin FKBP-12 gene
Vol. 179, No. 3, 1991 September 30, 1991
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1427-l 433
BIOCHEMICAL
CHROMOSOMAL
ASSIGNMENT
OF THE HUMAN IMMUNOPHILIN
FKBP-12 GENE
Anthony G. DiLella Department SmithKline
of Molecular Genetics
Beecham Pharmaceuticals
King of Prussia, PA 19406 Received
August
9, 1991
SUMMARY: FKBP-12 is the major T cell binding protein for the immunosuppressive drugs FK506 and rapamycin. It is a member of the immunophilin family of proteins which are believed to play a role in immunoregulation and basic cellular processes involving protein folding and trafficking. The chromosomal assignment of the human FKBP-12 gene was determined by using the polymerase chain reaction to amplify an intron-containing region of the gene in purified DNA isolated from 42 human-rodent somatic cell hybrids. The results of this analysis indicated that the FKBP-12 gene resides on human chromosome 20. 0 1991AcademicPress, Inc.
The immunophilins identified
(immunosuppressant
as a family of proteins that catalyze the interconversion
rotamers of the peptidyl-prolyl in ref. 1). lmmunophilins FK506,
binding proteins)
rapamycin,
proteins. cyclosporin
of the cis- and trans-
amide bond of peptide and protein substrates (reviewed
are the cytosolic receptors for the immunosuppressive
and cyclosporin
A, which inhibit the rotamase
The major FK506-binding A-binding
have recently been
T cell immunophilin
T cell immunophilin
is cyclophilin
activity of these
is FKBP-12
(2) and the
(3). Following binding to their
specific cytoplasmic
receptors, both FK506 and cyclosporin A can independently
the T cell receptor
signal transmission
expression
of the same set of lymphokine
related to FK506, also specifically
conserved
are also distributed
(6,7), suggesting
addition to immunoregulation.
of T cell activation
genes (4). Rapamycin,
binds to FKBP-12
cell activation by blocking the lymphokine lmmunophilins
pathway
in non-lymphoid
by inhibiting
a drug structurally
pathway (4,5).
cells and are phylogenetically
play a role in cellular
However, the endogenous
are not known; the biological significance
of immunophilin
processes
in
ligands of the immunophilins rotamase activity within cells 0006-291X/91
1427
block
(2) but inhibits a later stage of T
receptor signal transmission
that these proteins
drugs
$1.50
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 179, No. 3, 1991
BlOCHEMiCAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and the precise role of these proteins in immunoregulation immunophilin
FKBP-12
effort to understand (8). Further linkage FKBP-12
insight
chromosomal
the biological
into the physiological
gene to human chromosome
gene from human-rodent
The human
gene stucture was recently established
role and structure/function
with a human genetic disease.
is uncertain.
functions
relationship
of FKBP-12
of FKBP-12
could come from
In this paper, I report the localization 20 by using PCR to amplify
in an
of the
a region of the
somatic cell hybrids.
MATERIALS
AND METHODS
Oligonucleotides Oligonucleotides were synthesized on an Applied Biosystems DNA Synthesizer. The locations of the oligonucleotides used as PCR primers are shown in Figure 1. PCR primers I (3’-CCGACCCTTCTTCCCCAACGG-5’) and Ill (3’GATCCGTTCGTCCTCCACTAG-5’) are complementary to the sense-strand of exon 3. PCR primers II (5’-TTAGAACCATGGTGAGTGCCC-3’) and IV (5’CTTCCAGTAAAATCAGTFITG-3’) correspond to the sense-strand sequence of intron 2. Cell Lines and DNA Purified genomic DNA samples from a panel of 24 human-hamster cell lines were obtained from the BIOS Corp. (New Haven, CT). The cell lines were derived by fusing a Chinese hamster ovary cell line with peripheral blood leukocytes from human donors (9,lO) and were characterized by karyotype analysis. The chromosome content of the current passage of each somatic hybrid cell was determined by cytogenic analysis of Giemsa banded metaphase spreads and in situ chromosome “painting” using labeled human genomic DNA hybridized to metaphase spreads. Purified genomic DNA samples from a panel of 18 human-rodent somatic cell hybrids were obtained from NIGMS Human Genetic Mutant Cell Repository (Camden, N.J.). Cell lines NA09925 through NA09940 were derived from the fusion of fetal human male fibroblasts (IMR-91) with the thymidine kinase deficient mouse cell line B82 (11 ,12). The hybrid cell line NA10324 was derived in the laboratory of Dr. F. Ruddle (Yale University, New Haven, CT) by the fusion of the human fibroblast cell line GM001 44, with a 46,Xx,-6,+der(6)t(6;21) karyotype, with the HPRT and APRT deficient mouse cell line A9. The hybrid cell line NA10567, resulted from the fusion of the human fibroblast cell line GM02860, with a 46,XY,t(3;16)(ql3.2;ql3) karyotype, with the HPRT and APRT deficient mouse cell line A9 (13). Hybrid cell line NA10611 resulted from the microcell fusion of retroviral vector SP-1 infected human lymphoblast culture GM07890, with a 46,XX,del(13) karyoptype, with the Chinese hamster ovary line UV-135 (14). PCR, Electrophoresis, and Hybridization Analysis PCR was carried out using the nested primer approach as described (15). Reactions (100 pl) using the outside-set primers I and II were carried out in PCR mix (200 pM each dNTP, 10 mM Tris-HCI (pH 8.3), 50 mM KCI, 2 mM MgC12, 1 pM of each primer, and 2.5 units of Taq polymerase) containing loo-250 ng of genomic DNA. PCR amplification was carried out in a DNA thermal cycler (Per-kin-Elmer). The thermal profile consisted of 35 cycles of 95OC for 2 min., 50°C for 2 min., and 72OC for 2.5 min. Following amplification with the outer-set primers, a 2 pl aliquot from each 1428
Vol.
179,
No.
3, 1991
BIOCHEMICAL
AND
BiOPHYSICAL
RESEARCH
COMMUNICATIONS
reaction was then subjected to an additional 35 cycles of amplification in a final volume of lOOpI in PCR mix containing nested primers III and IV. Ten pl of nested primer reactions were electrophoresed on 1% agarose gels, stained with ethidium bromide, and processed for direct- el hybridization as described previously (16). Gel membranes were hybridized to a [32 PI-labeled oligonucleotide probe (3’-GTGTTATCCGACGTCACCGAA-5’) complementary to the sense-strand of intron 2 and specific for the PCR product (Fig. 1A). Gels were hybridized overnight at 37’C in (0.9 M NaCI, 6 mM EDTA, 0.5% SDS, and 0.9 M Tris-HCI, pH 7.5) containing 0.2 mg of salmon sperm DNA and 2x1 O6 cpm of probe per ml of hybridization solution. The gel membranes were then washed twice at O°C for 30 min in TMA (3 M tetramethylammonium chloride (Aldrich), 2 mM EDTA, and 50 mM Tris-HCI, pH 8) once each at 25OC (30 min) and 60°C (7 min) in TMA containing 0.2% SDS, and once at 25OC for 30 min in TMA as described (17). The gel strips were then autoradiographed at -8OOC. RESULTS I designed
PCR primers
of the human FKBP-12 pseudogenes
AND DISCUSSION
that would allow specific amplification
gene in the presence
(8) and the homologous
strategy.
Primers
were designed
12 gene.
The predicted
rodent
gene.
Figure
FKBP-12 1A shows
processed the PCR
from sequences
of intron 2 and exon 3 of the FKBP-
size of the PCR product
was about 1.5 kb. When PCR was
carried out using control human placental detected
of related human
of an intron region
DNA, only the expected
(Fig. 1 B, lane 1). To demonstrate
that the PCR product
1.5 kb fragment was specific
for the
IV-W II-,
H
I PCR product (1.5 kb)
Probe
B
Figure 1, PCR strategy for the amplification of the human FKBP-12 gene. A) The exon-intron organization of the FKBP-12 gene, as previously determined (8), is shown. Exons and introns are denoted by solid boxes and lines, respectively. An exploded view of a region containing the 3’-end of intron 2 and exon 3 is shown below the physical map of the gene; the PCR primer locationsare indicated. Restrictionenzyme abbreviations: B, E&JHI; E, mRI; X, ml; Sm, -1. B) Electrophoretic analysis of the PCR product derived from the intron2/exon3 region of the humanFKBP-12 gene is shown in lane 1. A ml digest of the PCR product is shown in lane 2. M, DNA size markers.
1429
was
Vol.
179,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1.5-
Figure 2. PCR analysis of human-rodent hybrid panels. Genomic DNA from human, rodent, and the indicated numbered cell lines were amplified by PCR with human FKBP-12 specific oligonucleotide primers. A) Panel I hybrid cell lines from the BIOS Corp. B) Panel II hybrid cell lines from NIGMS. The PCR product was electrophoresed on agarose gels and stained with ethidium bromide (top window of panels I and II). The gels were then hybridized to an oligonucleotide probe specific for the FKBP-12 PCR product (bottom window of panels I and II). “Neg” refers to the negative control (PCR reaction without added DNA). intron2/exon3 restriction
region
enzyme
of the FKBP-12
digestion.
the exon 3 PCR primers ml,
the expected
data indicated
1 .l- and 0.4-kb
on purified
genomic
about 400 bp upstream
detected
of the FKBP-12
of the human FKBP-12 DNA samples
panel I (Fig. 2A).
agarose
gel electrophoresis.
unrelated
human genomic
756) containing
were
analyzed
was digested
by of with
(Fig. 1 B, lane 2). The
gene in the presence
of the
pseudogenes.
localization
cell lines, representing
site is located
fragments
amplification
related human processed
analysis
A unique j@l
was further
(Fig. 1 A). When the 1.5 kb PCR fragment
specific
Chromosomal
gene, the fragment
from 24 individual PCR reaction
The diagnostic DNA controls
human chromosome
gene was determined human-hamster
products
1.5 kb product
were
by PCR hybrid
analyzed
was obtained
by
with three
and only hybrid cell lines (lines 507, 940, and
20 (Fig. 2A, top window). 1430
Hamster
DNA and the
Vol.
BIOCHEMICAL
179, No. 3, 1991
remaining
AND BIOPHYSICAL
hybrid cell lines which do not contain human chromosome
any PCR product.
The data indicated
specific amplification
gene in the presence of rodent chromosomal product
was further
oligonucleotide product hybridized
RESEARCH COMMUNICATIONS
verified
by direct-gel
of the human FKBP-12
DNA. The identity of the FKBP-12 hybridization
DNA control
samples
gene
using a [32P]-labeled
probe specific for intron 2 (Fig. 2A, bottom window).
from the human
20 did not show
The 1.5 kb PCR
and cell lines 507, 940, and 756
to the probe, whereas no hybridization
signal was detected
for hamster
DNA or cell lines that lacked the 1.5 kb band detected on ethidium-stained
gels. The
data suggested
gene with
cosegregation
human chromosome
of the 1.5 kb PCR product of the FKBP-12
20.
TABLE 1 Correlation
of
Gene with &man Chromosomes Somatic Cell Hybrids
F-P-12
in Euman/Rodent
Gene/Chromosome Human
Chromosome
1 2 3 4 5 6 1 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y
+/+
+/-
-/+
-/-
4 5 8 9 11 10 11 9 0 6 6 11 8 13 9 2 11
10
3 2 6 4 20 6 3 9 3 6 4 4 6
25 26 22 24 8 22 25 19 25 22 24 24 22 21 21 24 22 23 20 20 20 24 24 25
8 14 8 9 3 6
9 6 5 3 4 3 5 14 8 8 3 6 1 5 12 3
I 4 6 5 8 0 8 4 4 3
6 0 6 5 11 8
%Discordance 31 26 28 21 55 24 14 33 40 33 28 17 28 19 28 38 21 28 33 0 33 21 36 26
Percent discordance demonstrates the cosegregation of the Note. human FKBP-12 gene with human chromosome 20 by PCR analysis of 42 hybrid cell lines. Percent discordance for each chromosome represents: the presence of the gene in the absence of the chromosome (+/-) plus the absence of the gene in the presence of the chromosome (-/+) divided by the total number of hybrids analyzed, multiplied by presence of the gene/presence of the chromosome. 100. +/+, -I-, absence of the gene/absence of the chromosome.
1431
Vol.
179, No. 3, 1991
The assignment
BIOCHEMICAL
of the FKBP-12
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
gene to human chromosome
20 was further
verified by PCR analysis of DNA samples from a different hybrid panel derived from 18 human-rodent
hybrid cell lines. Figure 28 (panel II) shows detection
1.5 kb PCR product
by ethidium-staining
of the diagnostic
(top window) and hybridization
(bottom
window) for human genomic DNA controls and only hybrid cell lines containing chromosome
20. No PCR product was detected
in panel II for either rodent DNA
samples or hybrid cell lines that lacked human chromosome The results are summarized
20, and 11 to 67% discordance
demonstrated
that the FKBP-12
genetic
disorders,
chromosome
for all other chromosomes.
gene is on human chromosome
and polymorphic
20 (18). Regional
20.
in Table 1. There is 0% discordance
chromosome
DNA markers
mapping
human
The data
20. Several genes,
have been assigned
of the FKBP-12
for human
to human
gene on chromosome
20
may be useful in linkage analysis between FKBP-12 and other human genetic loci.
ACKNOWLEDGMENT
I thank Dr. G. Sathe for the synthesis of oligonucleotide
PCR primers.
REFERENCES 1.
Schreiber,
2.
Fretz, H., Albers, M.W., Galat, A., Standaert, R.F., Lane, W.S., Burakoff, S.J., Bierer, B.E., and Schreiber, S.L. (1991) J. Am. Chem. Sot. 113, 1409-1411. Handschumacher, R.E., Harding, M.W., Rice, J., Drugge, R.J., and Speicher, D.W. (1984) Science 226, 544547. Tocci, M.J., Matkovich, D.A., Collier, K.A., Kwok, P., Dumont, F., Lin, S., Degubicibus, S., Siekierka, J-J., Chin, J., and Hutchinson, N.I. (1989) J. Immunol. 143, 718-729. Dumont, F.J., Staruch, M.J., Koprak, S.L., Melino, M.R., and Sigal, N.H. (1990) J. lmmunol. 144, 251-258. Siekierka, J.J., Weiderrecht, G., Greulich, H., Boulton, D., Hung, H., Cryan, J., and Sigal, N. (1990) J. Biol. Chem. 265, 2101 l-21 015. Koletsky, A., Harding, M., Handschumacher, R. (1986) J. Immunol. 137, 10541059. DiLella, A.G. and Craig, R.J. (1991) Biochemistry 30, 8512-8517. Dana, S. and Wasmuth, J.J. (1982) Somat. Cell Genet. 8, 245-264. Carlock, L.R., Smith, D., and Wasmuth, J. (1986) Somat. Cell Mol. Genet. 12,
3. 4.
5. 6. 7. 8. Q V. 10.
163-l
11. 12. 13.
S.L. (1991) Science 251, 283-287.
74.
Taggart, R.T., Mohandas, T.K., Shows, T.B., and Bell, G.I. (1985) Proc. Natl. Acad. Sci. USA 82, 6240-6244. Mohandas, T., Heinzmann, C., Sparkes. R.S., Wasmuth, J., Edwards, P., and Lusis, A.J. (1986) Somat. Cell Mol. Genet. 12,89-94. Callen, D.F. (1986) Ann. Genet. 29, 235-239. 1432
Vol.
14. 15. 16. 17. 18.
179,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Warburton, D., Gersen, S., Yti, M.-T., Jackson, C., Handelin, B., and Housman, D. (1990) Genomics 6, 358-366. Mullis, K.B., and Faloona, F.A. (1987) Meth. Enzymol. 155, 335-350. DiLella, A.G., Marvit, J., Lidsky, A.S., Guttler, F., and Woo, S.L.C. (1986) Nature 322, 799-803. DiLella, A.G., and Woo, S.L.C. (1987) Meth. Enzymol. 152, 447-451. McKusick, V.A. (1990) In Genetic Maps (S.J. O’brien, Ed.), Book 5: Human Maps, pp. 5.47-5.114. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
1433
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1427-l 433
BIOCHEMICAL
CHROMOSOMAL
ASSIGNMENT
OF THE HUMAN IMMUNOPHILIN
FKBP-12 GENE
Anthony G. DiLella Department SmithKline
of Molecular Genetics
Beecham Pharmaceuticals
King of Prussia, PA 19406 Received
August
9, 1991
SUMMARY: FKBP-12 is the major T cell binding protein for the immunosuppressive drugs FK506 and rapamycin. It is a member of the immunophilin family of proteins which are believed to play a role in immunoregulation and basic cellular processes involving protein folding and trafficking. The chromosomal assignment of the human FKBP-12 gene was determined by using the polymerase chain reaction to amplify an intron-containing region of the gene in purified DNA isolated from 42 human-rodent somatic cell hybrids. The results of this analysis indicated that the FKBP-12 gene resides on human chromosome 20. 0 1991AcademicPress, Inc.
The immunophilins identified
(immunosuppressant
as a family of proteins that catalyze the interconversion
rotamers of the peptidyl-prolyl in ref. 1). lmmunophilins FK506,
binding proteins)
rapamycin,
proteins. cyclosporin
of the cis- and trans-
amide bond of peptide and protein substrates (reviewed
are the cytosolic receptors for the immunosuppressive
and cyclosporin
A, which inhibit the rotamase
The major FK506-binding A-binding
have recently been
T cell immunophilin
T cell immunophilin
is cyclophilin
activity of these
is FKBP-12
(2) and the
(3). Following binding to their
specific cytoplasmic
receptors, both FK506 and cyclosporin A can independently
the T cell receptor
signal transmission
expression
of the same set of lymphokine
related to FK506, also specifically
conserved
are also distributed
(6,7), suggesting
addition to immunoregulation.
of T cell activation
genes (4). Rapamycin,
binds to FKBP-12
cell activation by blocking the lymphokine lmmunophilins
pathway
in non-lymphoid
by inhibiting
a drug structurally
pathway (4,5).
cells and are phylogenetically
play a role in cellular
However, the endogenous
are not known; the biological significance
of immunophilin
processes
in
ligands of the immunophilins rotamase activity within cells 0006-291X/91
1427
block
(2) but inhibits a later stage of T
receptor signal transmission
that these proteins
drugs
$1.50
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 179, No. 3, 1991
BlOCHEMiCAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and the precise role of these proteins in immunoregulation immunophilin
FKBP-12
effort to understand (8). Further linkage FKBP-12
insight
chromosomal
the biological
into the physiological
gene to human chromosome
gene from human-rodent
The human
gene stucture was recently established
role and structure/function
with a human genetic disease.
is uncertain.
functions
relationship
of FKBP-12
of FKBP-12
could come from
In this paper, I report the localization 20 by using PCR to amplify
in an
of the
a region of the
somatic cell hybrids.
MATERIALS
AND METHODS
Oligonucleotides Oligonucleotides were synthesized on an Applied Biosystems DNA Synthesizer. The locations of the oligonucleotides used as PCR primers are shown in Figure 1. PCR primers I (3’-CCGACCCTTCTTCCCCAACGG-5’) and Ill (3’GATCCGTTCGTCCTCCACTAG-5’) are complementary to the sense-strand of exon 3. PCR primers II (5’-TTAGAACCATGGTGAGTGCCC-3’) and IV (5’CTTCCAGTAAAATCAGTFITG-3’) correspond to the sense-strand sequence of intron 2. Cell Lines and DNA Purified genomic DNA samples from a panel of 24 human-hamster cell lines were obtained from the BIOS Corp. (New Haven, CT). The cell lines were derived by fusing a Chinese hamster ovary cell line with peripheral blood leukocytes from human donors (9,lO) and were characterized by karyotype analysis. The chromosome content of the current passage of each somatic hybrid cell was determined by cytogenic analysis of Giemsa banded metaphase spreads and in situ chromosome “painting” using labeled human genomic DNA hybridized to metaphase spreads. Purified genomic DNA samples from a panel of 18 human-rodent somatic cell hybrids were obtained from NIGMS Human Genetic Mutant Cell Repository (Camden, N.J.). Cell lines NA09925 through NA09940 were derived from the fusion of fetal human male fibroblasts (IMR-91) with the thymidine kinase deficient mouse cell line B82 (11 ,12). The hybrid cell line NA10324 was derived in the laboratory of Dr. F. Ruddle (Yale University, New Haven, CT) by the fusion of the human fibroblast cell line GM001 44, with a 46,Xx,-6,+der(6)t(6;21) karyotype, with the HPRT and APRT deficient mouse cell line A9. The hybrid cell line NA10567, resulted from the fusion of the human fibroblast cell line GM02860, with a 46,XY,t(3;16)(ql3.2;ql3) karyotype, with the HPRT and APRT deficient mouse cell line A9 (13). Hybrid cell line NA10611 resulted from the microcell fusion of retroviral vector SP-1 infected human lymphoblast culture GM07890, with a 46,XX,del(13) karyoptype, with the Chinese hamster ovary line UV-135 (14). PCR, Electrophoresis, and Hybridization Analysis PCR was carried out using the nested primer approach as described (15). Reactions (100 pl) using the outside-set primers I and II were carried out in PCR mix (200 pM each dNTP, 10 mM Tris-HCI (pH 8.3), 50 mM KCI, 2 mM MgC12, 1 pM of each primer, and 2.5 units of Taq polymerase) containing loo-250 ng of genomic DNA. PCR amplification was carried out in a DNA thermal cycler (Per-kin-Elmer). The thermal profile consisted of 35 cycles of 95OC for 2 min., 50°C for 2 min., and 72OC for 2.5 min. Following amplification with the outer-set primers, a 2 pl aliquot from each 1428
Vol.
179,
No.
3, 1991
BIOCHEMICAL
AND
BiOPHYSICAL
RESEARCH
COMMUNICATIONS
reaction was then subjected to an additional 35 cycles of amplification in a final volume of lOOpI in PCR mix containing nested primers III and IV. Ten pl of nested primer reactions were electrophoresed on 1% agarose gels, stained with ethidium bromide, and processed for direct- el hybridization as described previously (16). Gel membranes were hybridized to a [32 PI-labeled oligonucleotide probe (3’-GTGTTATCCGACGTCACCGAA-5’) complementary to the sense-strand of intron 2 and specific for the PCR product (Fig. 1A). Gels were hybridized overnight at 37’C in (0.9 M NaCI, 6 mM EDTA, 0.5% SDS, and 0.9 M Tris-HCI, pH 7.5) containing 0.2 mg of salmon sperm DNA and 2x1 O6 cpm of probe per ml of hybridization solution. The gel membranes were then washed twice at O°C for 30 min in TMA (3 M tetramethylammonium chloride (Aldrich), 2 mM EDTA, and 50 mM Tris-HCI, pH 8) once each at 25OC (30 min) and 60°C (7 min) in TMA containing 0.2% SDS, and once at 25OC for 30 min in TMA as described (17). The gel strips were then autoradiographed at -8OOC. RESULTS I designed
PCR primers
of the human FKBP-12 pseudogenes
AND DISCUSSION
that would allow specific amplification
gene in the presence
(8) and the homologous
strategy.
Primers
were designed
12 gene.
The predicted
rodent
gene.
Figure
FKBP-12 1A shows
processed the PCR
from sequences
of intron 2 and exon 3 of the FKBP-
size of the PCR product
was about 1.5 kb. When PCR was
carried out using control human placental detected
of related human
of an intron region
DNA, only the expected
(Fig. 1 B, lane 1). To demonstrate
that the PCR product
1.5 kb fragment was specific
for the
IV-W II-,
H
I PCR product (1.5 kb)
Probe
B
Figure 1, PCR strategy for the amplification of the human FKBP-12 gene. A) The exon-intron organization of the FKBP-12 gene, as previously determined (8), is shown. Exons and introns are denoted by solid boxes and lines, respectively. An exploded view of a region containing the 3’-end of intron 2 and exon 3 is shown below the physical map of the gene; the PCR primer locationsare indicated. Restrictionenzyme abbreviations: B, E&JHI; E, mRI; X, ml; Sm, -1. B) Electrophoretic analysis of the PCR product derived from the intron2/exon3 region of the humanFKBP-12 gene is shown in lane 1. A ml digest of the PCR product is shown in lane 2. M, DNA size markers.
1429
was
Vol.
179,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1.5-
Figure 2. PCR analysis of human-rodent hybrid panels. Genomic DNA from human, rodent, and the indicated numbered cell lines were amplified by PCR with human FKBP-12 specific oligonucleotide primers. A) Panel I hybrid cell lines from the BIOS Corp. B) Panel II hybrid cell lines from NIGMS. The PCR product was electrophoresed on agarose gels and stained with ethidium bromide (top window of panels I and II). The gels were then hybridized to an oligonucleotide probe specific for the FKBP-12 PCR product (bottom window of panels I and II). “Neg” refers to the negative control (PCR reaction without added DNA). intron2/exon3 restriction
region
enzyme
of the FKBP-12
digestion.
the exon 3 PCR primers ml,
the expected
data indicated
1 .l- and 0.4-kb
on purified
genomic
about 400 bp upstream
detected
of the FKBP-12
of the human FKBP-12 DNA samples
panel I (Fig. 2A).
agarose
gel electrophoresis.
unrelated
human genomic
756) containing
were
analyzed
was digested
by of with
(Fig. 1 B, lane 2). The
gene in the presence
of the
pseudogenes.
localization
cell lines, representing
site is located
fragments
amplification
related human processed
analysis
A unique j@l
was further
(Fig. 1 A). When the 1.5 kb PCR fragment
specific
Chromosomal
gene, the fragment
from 24 individual PCR reaction
The diagnostic DNA controls
human chromosome
gene was determined human-hamster
products
1.5 kb product
were
by PCR hybrid
analyzed
was obtained
by
with three
and only hybrid cell lines (lines 507, 940, and
20 (Fig. 2A, top window). 1430
Hamster
DNA and the
Vol.
BIOCHEMICAL
179, No. 3, 1991
remaining
AND BIOPHYSICAL
hybrid cell lines which do not contain human chromosome
any PCR product.
The data indicated
specific amplification
gene in the presence of rodent chromosomal product
was further
oligonucleotide product hybridized
RESEARCH COMMUNICATIONS
verified
by direct-gel
of the human FKBP-12
DNA. The identity of the FKBP-12 hybridization
DNA control
samples
gene
using a [32P]-labeled
probe specific for intron 2 (Fig. 2A, bottom window).
from the human
20 did not show
The 1.5 kb PCR
and cell lines 507, 940, and 756
to the probe, whereas no hybridization
signal was detected
for hamster
DNA or cell lines that lacked the 1.5 kb band detected on ethidium-stained
gels. The
data suggested
gene with
cosegregation
human chromosome
of the 1.5 kb PCR product of the FKBP-12
20.
TABLE 1 Correlation
of
Gene with &man Chromosomes Somatic Cell Hybrids
F-P-12
in Euman/Rodent
Gene/Chromosome Human
Chromosome
1 2 3 4 5 6 1 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y
+/+
+/-
-/+
-/-
4 5 8 9 11 10 11 9 0 6 6 11 8 13 9 2 11
10
3 2 6 4 20 6 3 9 3 6 4 4 6
25 26 22 24 8 22 25 19 25 22 24 24 22 21 21 24 22 23 20 20 20 24 24 25
8 14 8 9 3 6
9 6 5 3 4 3 5 14 8 8 3 6 1 5 12 3
I 4 6 5 8 0 8 4 4 3
6 0 6 5 11 8
%Discordance 31 26 28 21 55 24 14 33 40 33 28 17 28 19 28 38 21 28 33 0 33 21 36 26
Percent discordance demonstrates the cosegregation of the Note. human FKBP-12 gene with human chromosome 20 by PCR analysis of 42 hybrid cell lines. Percent discordance for each chromosome represents: the presence of the gene in the absence of the chromosome (+/-) plus the absence of the gene in the presence of the chromosome (-/+) divided by the total number of hybrids analyzed, multiplied by presence of the gene/presence of the chromosome. 100. +/+, -I-, absence of the gene/absence of the chromosome.
1431
Vol.
179, No. 3, 1991
The assignment
BIOCHEMICAL
of the FKBP-12
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
gene to human chromosome
20 was further
verified by PCR analysis of DNA samples from a different hybrid panel derived from 18 human-rodent
hybrid cell lines. Figure 28 (panel II) shows detection
1.5 kb PCR product
by ethidium-staining
of the diagnostic
(top window) and hybridization
(bottom
window) for human genomic DNA controls and only hybrid cell lines containing chromosome
20. No PCR product was detected
in panel II for either rodent DNA
samples or hybrid cell lines that lacked human chromosome The results are summarized
20, and 11 to 67% discordance
demonstrated
that the FKBP-12
genetic
disorders,
chromosome
for all other chromosomes.
gene is on human chromosome
and polymorphic
20 (18). Regional
20.
in Table 1. There is 0% discordance
chromosome
DNA markers
mapping
human
The data
20. Several genes,
have been assigned
of the FKBP-12
for human
to human
gene on chromosome
20
may be useful in linkage analysis between FKBP-12 and other human genetic loci.
ACKNOWLEDGMENT
I thank Dr. G. Sathe for the synthesis of oligonucleotide
PCR primers.
REFERENCES 1.
Schreiber,
2.
Fretz, H., Albers, M.W., Galat, A., Standaert, R.F., Lane, W.S., Burakoff, S.J., Bierer, B.E., and Schreiber, S.L. (1991) J. Am. Chem. Sot. 113, 1409-1411. Handschumacher, R.E., Harding, M.W., Rice, J., Drugge, R.J., and Speicher, D.W. (1984) Science 226, 544547. Tocci, M.J., Matkovich, D.A., Collier, K.A., Kwok, P., Dumont, F., Lin, S., Degubicibus, S., Siekierka, J-J., Chin, J., and Hutchinson, N.I. (1989) J. Immunol. 143, 718-729. Dumont, F.J., Staruch, M.J., Koprak, S.L., Melino, M.R., and Sigal, N.H. (1990) J. lmmunol. 144, 251-258. Siekierka, J.J., Weiderrecht, G., Greulich, H., Boulton, D., Hung, H., Cryan, J., and Sigal, N. (1990) J. Biol. Chem. 265, 2101 l-21 015. Koletsky, A., Harding, M., Handschumacher, R. (1986) J. Immunol. 137, 10541059. DiLella, A.G. and Craig, R.J. (1991) Biochemistry 30, 8512-8517. Dana, S. and Wasmuth, J.J. (1982) Somat. Cell Genet. 8, 245-264. Carlock, L.R., Smith, D., and Wasmuth, J. (1986) Somat. Cell Mol. Genet. 12,
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Taggart, R.T., Mohandas, T.K., Shows, T.B., and Bell, G.I. (1985) Proc. Natl. Acad. Sci. USA 82, 6240-6244. Mohandas, T., Heinzmann, C., Sparkes. R.S., Wasmuth, J., Edwards, P., and Lusis, A.J. (1986) Somat. Cell Mol. Genet. 12,89-94. Callen, D.F. (1986) Ann. Genet. 29, 235-239. 1432
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COMMUNICATIONS
Warburton, D., Gersen, S., Yti, M.-T., Jackson, C., Handelin, B., and Housman, D. (1990) Genomics 6, 358-366. Mullis, K.B., and Faloona, F.A. (1987) Meth. Enzymol. 155, 335-350. DiLella, A.G., Marvit, J., Lidsky, A.S., Guttler, F., and Woo, S.L.C. (1986) Nature 322, 799-803. DiLella, A.G., and Woo, S.L.C. (1987) Meth. Enzymol. 152, 447-451. McKusick, V.A. (1990) In Genetic Maps (S.J. O’brien, Ed.), Book 5: Human Maps, pp. 5.47-5.114. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
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