- Email: [email protected]
Assignment of the human slow skeletal troponin T gene to 19q13.4 using somatic cell hybrids and fluorescence in situ hybridization analysis
1374
BRIEF
Landegent, J. E., Jansen in de Wal, N., Dirks, R. W., Baas, F., and van der Ploeg, M. (1987). Use of whole cosmid cloned genomic sequences for chromosomal localization by non-radioactive in situ hybridization. Hum. Genet. 77: 366-370. McGarry, J. D., Woeltje, K. F., Kuwajima, M., and Foster, D. W. (1989). Regulation of ketogenesis and the reinassance of carnitine palmitoyltransferase. Diabetes Metab. Reu. 5: 271-284. Miyazawa, S., Ozasa, H., Osumi, T., and Hashimoto, T. (1983). Purification and properties of carnitine octanoyltransferase and carnitine palmitoyltransferase from rat liver. J. Biochem. 94: 529-542. Zabel, B. U., Naylor, S. L., Sakaguchi, A. Y., Bell, G. I., and Shows, T. B. (1983). High-resolution chromosomal localization of human genes for amylase, proopiomelanocortin, somatostatin, and a DNA fragment (D3Sl) by in situ hybridization. Proc. Natl. Ad. Sci. USA 80: 6932-6936.
Assignment of the Human Slow Skeletal Troponin T Gene to 19q13.4 Using Somatic Cell Hybrids and Fluorescence in Situ Hybridization Analysis Franqoise Samson,* Peter J. de Jong, t Barbara J. Trask, t Petra Koza-Taylor,* Marcy C. Speer,* Tamara Potter,* Allen D. Roses,* and John R. Gilbert* *Departments of Medicine and Neurobiology, Division of Neurology, Duke University Medical Center, Durham, North Carolina; and tHuman Genome Center, Biomedical Sciences Division, Lawrence Livermore National Laboratory, Livermore, California Received
December
2, 1991;
revised
April
REPORTS 42’C, in a 50% formamide, 5X SSPE, 2.5X Denhart’s, 0.5% SDS, 10% dextran sulfate solution containing 200 pg/ml of salmon sperm DNA. Washing conditions were I5 min at 55°C in 1X SSC, 0.1% SDS, followed by 15 min at 55’C in 0.1X SSC, 0.1% SDS. On Southern blot analysis of BglII-digested DNA, MSL-366 detects a 4.7-kb human fragment. Southern blotting demonstrated that MSL-366 did not hybridize to 2OXP35421-4, while a control CKMM clone gave a strong hybridization signal (data not shown). Combined with our previous localization of TNNTl distal to CKMM (5), these data place the slow troponin T gene telomeric to 2OXP3542-1-4 on the long arm of chromosome 19. This localization was confirmed by fluorescence in situ hybridization analysis. A TNNTl-positive cosmid, designated f16957, was isolated from a cosmid library prepared from flowsorted chromosome 19 DNA (2), by hybridizing high-density filters with the MSL-366 probe. Hybridization took place overnight at 75°C in 0.6 M NaCl, 0.02 M EDTA, 0.2 M TrisHCl, 0.5% SDS, 0.1% Na-pyrophosphate. The filters were washed at 75°C for 30 min in 0.1X SSC. Subsequent Southern blot hybridizations of the MSL-366 probe to EcoRI and PstI digests of cosmid F16957 revealed strong hybridization signals with characteristic bands at 4.5 and 9 kb in EcoRI digest and 1.8 kb in PstI digests (data not shown). Cosmid DNA was purified on Qiagen columns after RNase treatment. The DNA was nick translated to incorporate biotin-dUTP (Enzo) using a BRL kit supplemented with DNase (0.04 pg; BRL). .Hybridization, fluorescent detection, chromosome banding, and microscope analysis were as described (4, 6). Briefly, the biotinylated cosmid DNA was hybridized in the presence of unlabeled sonicated human genomic DNA to prometaphase spreads from peripheral blood lymphocyte cultures synchronized by methotrexate blocking and thymidine release. After hybridization, probe sites were labeled by sequen-
16, 1992
13.3
Troponin T is a subunit of a calcium-binding protein complex (troponin I, troponin C, troponin T) that plays a central role in the regulation of muscle contraction and relaxation. A cDNA probe corresponding to the human slow skeletal muscle troponin T gene (TNNTl), designated MSL-366, was previously isolated by subtractive hybridization of a cDNA muscle library (5). This probe was initially located to the q13.2q13.3 region of chromosome 19 distal to CKMM by somatic cell hybrid lines analysis. Refinement of regional assignment of TNNTl on chromosome 19 was achieved using the 2OXP3542-1-4 hybrid cell line and fluorescence in situ hybridization (FISH). 2OXP3542-1-4 contains an approximately lo- to 12-Mb fragment of human chromosome 19 that terminates in 19q13.3 approximatively 2 Mb distal to the CKMM gene (1, 3). The cDNA probe MSL366, cloned in Xgtll and consisting of 241 bp derived from the 3’ end of the human slow troponin T cDNA, was used for Southern blot analysis. Labeling reactions were performed by PCR amplification of the insert, using the Xgtll primers, purification of the amplified cDNA product with glassmilk (Geneclean Kit, Bio 101, La Jolla, CA), and labeling with [32P]dCTP (DuPont) by random priming to a specific activity of 10’ cpm/ Fg. Hybridization (25 X lo6 cpm per filter) was for 14 h at GENOMICS 13,1374-1375(1992) 0888.7543/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
13.2 P
13.1 12
12 13.1
q 13.2
moo
13.3
l O
13.4
0.00.. l ooooooooooooe
FIG. 1. Idiogram of a G-banded human chromosome 19 showing the location of cosmid clone F16957 identified by a TNNTl probe. The fluorescent signals seen matid (0) were scored for 10 pherical blood lymphocytes were produced by incubation followed by actinomycin. No chromosomes.
on both chromatids (0) or a single chrometaphase chromosome spreads of periof a normal donor. Fluorescent G bands of the slides before analysis in DAPI hybridization signals were seen on other
BRIEF tial incubation of slides in avidin-fluorescein (FITC), biotinylated goat anti-avidin antibody, and avidin-FITC, separated by washes. Bands were produced in chromosomes by incubation of slides in DAPI (4, 6’-diamidino-2-phenyindole) followed by actinomycin treatment. DAPI bands and FITC probe site signals were viewed through separate filter combinations on a Zeiss Axiophot microscope (excitation BP360-371, emission LP397 for DAPI, and excitation BP450-490, emission LP520 for FITC). The TNNTl-positive cosmid F16957 was localized relative to DAPI/actinomycin chromosome bands on metaphase spreads by fluorescence in situ hybridization as described previously (4, 6) (Fig. 1). Paired hybridization signals (signal on both chromatids) were observed at band q13.4 on all 20 chromosomes 19 scored in 10 metaphase spreads. This site accounted for 85% of all hybridization signals in these spreads. On 5 of the chromosomes 19, signals were also observed in the q13.2-q13.3 region on one or both chromatids. No signals were observed on any other chromosome. This localization by FISH to 19q13.4 is consistent with the results of somatic cell hybrid analysis and confirm the localization of TNNTl to the distal long arm of chromosome 19. ACKNOWLEDGMENTS We thank Dr. M. Sicilian0 and L. Bachinski for providing us with the ZOXP3542-1-4 hybrid cell line. We thank Suzanne Carter, Anne Fertitta, Jennifer Alleman, and Anne Bergman for technical assistance. The work at Duke was supported by the MDA and Philippe Foundation (F.S.), a Clinical Research Grant MOJ-RR-30, National Institute of Health Grant NS19999 (A.D.R.), the Leadership and Excellence in Alzeimer’s disease (LEAD) Award AG07922, and a MDA Clinical Research Grant (A.D.R.). The work at LLNL was performed under the auspices of the U.S. Department of Energy Office of Health and Environmental Research contract number W-7405ENG-48 with support from a U.S. Public Health Service Grant HG-00256-01.
REFERENCES 1.
2.
3. 4.
5.
6.
Brook, J. D., Harley, H. G., Walsh, K. V., Rundle, S. A., Siciliano, M. J., Harper, P. S., and Shaw, D. J. (1991). Identification of new DNA markers close to the myotonic dystrophy locus. J. Med. Genet. 28: 84-88. de Jong, P. J., Yokabata, K., Chen, C., Lohman, F., Pederson, L., McNinch, J., and van Dilla, M. (1989). Human chromosomespecific partial digest libraries in lambda and cosmid vectors. Cytogenet. Cell Genet. 51: 985. Liu, P., Legerski, R., and Siciliano, M. J. (1989). Isolation of human transcribed sequences from human-rodent somatic cell hybrids. Science 246: 813-815. Mohrenweiser, H. W., Carrano, A. V., Fertitta, A., Perry, B., Thompson, L. H., Tucker, J. D., and Weber, C. A. (1989). Refined mapping of the three DNA repair genes, ERCCl, ERCC2, and XRCCl, on human chromosome 19. Cytogenet. Cell Genet. 52: 11-14. Samson, F., Lee, J. E., Hung, W. Y., Potter, T. G., Herbstreich, M., Roses, A. D., and Gilbert, J. R. (1990). Isolation and localization of slow troponin (TnT) gene on chromosome 19 by subtraction hybridization of a cDNA muscle library using myotonic dystrophy muscle cDNA. J. Neurosci. Res. 27: 441-451. Trask, B. J., Massa, H. F., Kenwrick, S., and Gitschier, J. (1991). Mapping of human chromosome Xq28 by a-color fluorescence in situ hybridization of DNA sequences of interphase cell nuclei. Am. J. Hum. Genet. 48: 1-15.
REPORTS
1375
Assignment of the Gene Encoding the CY,Subunit of the Neuroendocrine/ Brain-Type Calcium Channel (C’CNLIA2) to Human Chromosome 3, Band ~14.3 SUSUMU SEINO, YUICHIRO YAMADA, RAFAEL ESPINOSA III, MICHELLE M. LE BEAU, AND GRAEME 1. BELL Departments and Howard 5841 South Received
of Medicine, Biochemistry and Molecular Biology, Hughes Medical Institute, University of Chicago, Maryland Avenue, MC 1028, Chicago, ///inok 60637
December
16. 1991;
revised
May
11. 1992
role in Voltage-sensitive Ca2+ channels play an important regulating hormone and neurotransmitter release, muscle contraction, and a large number of other cellular functions (reviewed in Ref. (1)). The voltage-sensitive Ca2+ channels are multisubunit proteins, and in skeletal muscle the complex has been shown to consist of four distinct subunits, o1 (170 kDa), (~~/8 (175 kDa), @ (52 kDa), and y (32 kDa) (2). Heterologous expression studies have shown that the (Ye subunit alone is sufficient for generating voltage-sensitive Ca2+ channel activity (1,3). The 01~ and /3 subunits are members of gene families, and cDNAs encoding four structurally related o1 subunits and two /3 subunits have been reported (1, 4). The CQ subunits have been termed the skeletal muscle, heart, brain, and neuroendocrine/brain
isoforms.
Four
types
of Ca2+
currents
have
been
described on the basis of their kinetics and pharmacology (L, N, T, and P) (1). The Ca2+ channel activity associated with the skeletal muscle, heart, and neuroendocrine/brain CQ subunit isoforms these
is inhibited
and
may
by dihydropyridine
drugs,
indicating
that
represent L-type currents (1,5). By contrast, the activity of the brain isoform is not inhibited by dihydropyridine drugs thus
represent
We have recently that unit
encodes (6). This
sequences muscle, somal
a P-type
isolated
from pancreatic
the human neuroendocrine/brain-type protein shares 68,64, and 41%
of the a, subunits
and location
current.
a cDNA
of rabbit
cardiac
identity
muscle, skeletal
brain, respectively. To determine of the human neuroendocrine/brain
gene, PCRable DNA from a somatic cell hybrid New Haven, CT) was typed using the polymerase tion
(PCR)
and
the
primers
fi-cells (pi subwith the
the
chromocri subunit
panel (Bios; chain reac-
5’-TGACTGAAGCCCAGCTT-
GTG-3’ and 5’-GTCTAGCAGCATCGATTGCAGC-3’. These primers amplify a 279-bp fragment of the human neuroendocrine/hrain-type a1 subunit gene that cosegregated with chromosome 3 in this hybrid cell panel (data not shown). To confirm the assignment to chromosome 3 and to obtain a regional localization, we performed fluorescence in situ hybridization of a biotin-labeled probe to human metaphase chromosomes. The probe was a human genomic clone, XhCaCh@108, that was isolated from a human genomic library (Cat. No. 946203, Stratagene; La Jolla, CA) by hybridization with a human neuroendocrine/brain (Ye subunit cDNA clone; this human genomic clone contained an insert of 15.5 kb. Fluorescence in situ GENOMICS
All
13, 1375-1377 (19%) o&38-7543/92 $5.00
Copyright 0 1992 by Academic Press, Inc. rights of reproduction in any form reserved.
BRIEF
Landegent, J. E., Jansen in de Wal, N., Dirks, R. W., Baas, F., and van der Ploeg, M. (1987). Use of whole cosmid cloned genomic sequences for chromosomal localization by non-radioactive in situ hybridization. Hum. Genet. 77: 366-370. McGarry, J. D., Woeltje, K. F., Kuwajima, M., and Foster, D. W. (1989). Regulation of ketogenesis and the reinassance of carnitine palmitoyltransferase. Diabetes Metab. Reu. 5: 271-284. Miyazawa, S., Ozasa, H., Osumi, T., and Hashimoto, T. (1983). Purification and properties of carnitine octanoyltransferase and carnitine palmitoyltransferase from rat liver. J. Biochem. 94: 529-542. Zabel, B. U., Naylor, S. L., Sakaguchi, A. Y., Bell, G. I., and Shows, T. B. (1983). High-resolution chromosomal localization of human genes for amylase, proopiomelanocortin, somatostatin, and a DNA fragment (D3Sl) by in situ hybridization. Proc. Natl. Ad. Sci. USA 80: 6932-6936.
Assignment of the Human Slow Skeletal Troponin T Gene to 19q13.4 Using Somatic Cell Hybrids and Fluorescence in Situ Hybridization Analysis Franqoise Samson,* Peter J. de Jong, t Barbara J. Trask, t Petra Koza-Taylor,* Marcy C. Speer,* Tamara Potter,* Allen D. Roses,* and John R. Gilbert* *Departments of Medicine and Neurobiology, Division of Neurology, Duke University Medical Center, Durham, North Carolina; and tHuman Genome Center, Biomedical Sciences Division, Lawrence Livermore National Laboratory, Livermore, California Received
December
2, 1991;
revised
April
REPORTS 42’C, in a 50% formamide, 5X SSPE, 2.5X Denhart’s, 0.5% SDS, 10% dextran sulfate solution containing 200 pg/ml of salmon sperm DNA. Washing conditions were I5 min at 55°C in 1X SSC, 0.1% SDS, followed by 15 min at 55’C in 0.1X SSC, 0.1% SDS. On Southern blot analysis of BglII-digested DNA, MSL-366 detects a 4.7-kb human fragment. Southern blotting demonstrated that MSL-366 did not hybridize to 2OXP35421-4, while a control CKMM clone gave a strong hybridization signal (data not shown). Combined with our previous localization of TNNTl distal to CKMM (5), these data place the slow troponin T gene telomeric to 2OXP3542-1-4 on the long arm of chromosome 19. This localization was confirmed by fluorescence in situ hybridization analysis. A TNNTl-positive cosmid, designated f16957, was isolated from a cosmid library prepared from flowsorted chromosome 19 DNA (2), by hybridizing high-density filters with the MSL-366 probe. Hybridization took place overnight at 75°C in 0.6 M NaCl, 0.02 M EDTA, 0.2 M TrisHCl, 0.5% SDS, 0.1% Na-pyrophosphate. The filters were washed at 75°C for 30 min in 0.1X SSC. Subsequent Southern blot hybridizations of the MSL-366 probe to EcoRI and PstI digests of cosmid F16957 revealed strong hybridization signals with characteristic bands at 4.5 and 9 kb in EcoRI digest and 1.8 kb in PstI digests (data not shown). Cosmid DNA was purified on Qiagen columns after RNase treatment. The DNA was nick translated to incorporate biotin-dUTP (Enzo) using a BRL kit supplemented with DNase (0.04 pg; BRL). .Hybridization, fluorescent detection, chromosome banding, and microscope analysis were as described (4, 6). Briefly, the biotinylated cosmid DNA was hybridized in the presence of unlabeled sonicated human genomic DNA to prometaphase spreads from peripheral blood lymphocyte cultures synchronized by methotrexate blocking and thymidine release. After hybridization, probe sites were labeled by sequen-
16, 1992
13.3
Troponin T is a subunit of a calcium-binding protein complex (troponin I, troponin C, troponin T) that plays a central role in the regulation of muscle contraction and relaxation. A cDNA probe corresponding to the human slow skeletal muscle troponin T gene (TNNTl), designated MSL-366, was previously isolated by subtractive hybridization of a cDNA muscle library (5). This probe was initially located to the q13.2q13.3 region of chromosome 19 distal to CKMM by somatic cell hybrid lines analysis. Refinement of regional assignment of TNNTl on chromosome 19 was achieved using the 2OXP3542-1-4 hybrid cell line and fluorescence in situ hybridization (FISH). 2OXP3542-1-4 contains an approximately lo- to 12-Mb fragment of human chromosome 19 that terminates in 19q13.3 approximatively 2 Mb distal to the CKMM gene (1, 3). The cDNA probe MSL366, cloned in Xgtll and consisting of 241 bp derived from the 3’ end of the human slow troponin T cDNA, was used for Southern blot analysis. Labeling reactions were performed by PCR amplification of the insert, using the Xgtll primers, purification of the amplified cDNA product with glassmilk (Geneclean Kit, Bio 101, La Jolla, CA), and labeling with [32P]dCTP (DuPont) by random priming to a specific activity of 10’ cpm/ Fg. Hybridization (25 X lo6 cpm per filter) was for 14 h at GENOMICS 13,1374-1375(1992) 0888.7543/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
13.2 P
13.1 12
12 13.1
q 13.2
moo
13.3
l O
13.4
0.00.. l ooooooooooooe
FIG. 1. Idiogram of a G-banded human chromosome 19 showing the location of cosmid clone F16957 identified by a TNNTl probe. The fluorescent signals seen matid (0) were scored for 10 pherical blood lymphocytes were produced by incubation followed by actinomycin. No chromosomes.
on both chromatids (0) or a single chrometaphase chromosome spreads of periof a normal donor. Fluorescent G bands of the slides before analysis in DAPI hybridization signals were seen on other
BRIEF tial incubation of slides in avidin-fluorescein (FITC), biotinylated goat anti-avidin antibody, and avidin-FITC, separated by washes. Bands were produced in chromosomes by incubation of slides in DAPI (4, 6’-diamidino-2-phenyindole) followed by actinomycin treatment. DAPI bands and FITC probe site signals were viewed through separate filter combinations on a Zeiss Axiophot microscope (excitation BP360-371, emission LP397 for DAPI, and excitation BP450-490, emission LP520 for FITC). The TNNTl-positive cosmid F16957 was localized relative to DAPI/actinomycin chromosome bands on metaphase spreads by fluorescence in situ hybridization as described previously (4, 6) (Fig. 1). Paired hybridization signals (signal on both chromatids) were observed at band q13.4 on all 20 chromosomes 19 scored in 10 metaphase spreads. This site accounted for 85% of all hybridization signals in these spreads. On 5 of the chromosomes 19, signals were also observed in the q13.2-q13.3 region on one or both chromatids. No signals were observed on any other chromosome. This localization by FISH to 19q13.4 is consistent with the results of somatic cell hybrid analysis and confirm the localization of TNNTl to the distal long arm of chromosome 19. ACKNOWLEDGMENTS We thank Dr. M. Sicilian0 and L. Bachinski for providing us with the ZOXP3542-1-4 hybrid cell line. We thank Suzanne Carter, Anne Fertitta, Jennifer Alleman, and Anne Bergman for technical assistance. The work at Duke was supported by the MDA and Philippe Foundation (F.S.), a Clinical Research Grant MOJ-RR-30, National Institute of Health Grant NS19999 (A.D.R.), the Leadership and Excellence in Alzeimer’s disease (LEAD) Award AG07922, and a MDA Clinical Research Grant (A.D.R.). The work at LLNL was performed under the auspices of the U.S. Department of Energy Office of Health and Environmental Research contract number W-7405ENG-48 with support from a U.S. Public Health Service Grant HG-00256-01.
REFERENCES 1.
2.
3. 4.
5.
6.
Brook, J. D., Harley, H. G., Walsh, K. V., Rundle, S. A., Siciliano, M. J., Harper, P. S., and Shaw, D. J. (1991). Identification of new DNA markers close to the myotonic dystrophy locus. J. Med. Genet. 28: 84-88. de Jong, P. J., Yokabata, K., Chen, C., Lohman, F., Pederson, L., McNinch, J., and van Dilla, M. (1989). Human chromosomespecific partial digest libraries in lambda and cosmid vectors. Cytogenet. Cell Genet. 51: 985. Liu, P., Legerski, R., and Siciliano, M. J. (1989). Isolation of human transcribed sequences from human-rodent somatic cell hybrids. Science 246: 813-815. Mohrenweiser, H. W., Carrano, A. V., Fertitta, A., Perry, B., Thompson, L. H., Tucker, J. D., and Weber, C. A. (1989). Refined mapping of the three DNA repair genes, ERCCl, ERCC2, and XRCCl, on human chromosome 19. Cytogenet. Cell Genet. 52: 11-14. Samson, F., Lee, J. E., Hung, W. Y., Potter, T. G., Herbstreich, M., Roses, A. D., and Gilbert, J. R. (1990). Isolation and localization of slow troponin (TnT) gene on chromosome 19 by subtraction hybridization of a cDNA muscle library using myotonic dystrophy muscle cDNA. J. Neurosci. Res. 27: 441-451. Trask, B. J., Massa, H. F., Kenwrick, S., and Gitschier, J. (1991). Mapping of human chromosome Xq28 by a-color fluorescence in situ hybridization of DNA sequences of interphase cell nuclei. Am. J. Hum. Genet. 48: 1-15.
REPORTS
1375
Assignment of the Gene Encoding the CY,Subunit of the Neuroendocrine/ Brain-Type Calcium Channel (C’CNLIA2) to Human Chromosome 3, Band ~14.3 SUSUMU SEINO, YUICHIRO YAMADA, RAFAEL ESPINOSA III, MICHELLE M. LE BEAU, AND GRAEME 1. BELL Departments and Howard 5841 South Received
of Medicine, Biochemistry and Molecular Biology, Hughes Medical Institute, University of Chicago, Maryland Avenue, MC 1028, Chicago, ///inok 60637
December
16. 1991;
revised
May
11. 1992
role in Voltage-sensitive Ca2+ channels play an important regulating hormone and neurotransmitter release, muscle contraction, and a large number of other cellular functions (reviewed in Ref. (1)). The voltage-sensitive Ca2+ channels are multisubunit proteins, and in skeletal muscle the complex has been shown to consist of four distinct subunits, o1 (170 kDa), (~~/8 (175 kDa), @ (52 kDa), and y (32 kDa) (2). Heterologous expression studies have shown that the (Ye subunit alone is sufficient for generating voltage-sensitive Ca2+ channel activity (1,3). The 01~ and /3 subunits are members of gene families, and cDNAs encoding four structurally related o1 subunits and two /3 subunits have been reported (1, 4). The CQ subunits have been termed the skeletal muscle, heart, brain, and neuroendocrine/brain
isoforms.
Four
types
of Ca2+
currents
have
been
described on the basis of their kinetics and pharmacology (L, N, T, and P) (1). The Ca2+ channel activity associated with the skeletal muscle, heart, and neuroendocrine/brain CQ subunit isoforms these
is inhibited
and
may
by dihydropyridine
drugs,
indicating
that
represent L-type currents (1,5). By contrast, the activity of the brain isoform is not inhibited by dihydropyridine drugs thus
represent
We have recently that unit
encodes (6). This
sequences muscle, somal
a P-type
isolated
from pancreatic
the human neuroendocrine/brain-type protein shares 68,64, and 41%
of the a, subunits
and location
current.
a cDNA
of rabbit
cardiac
identity
muscle, skeletal
brain, respectively. To determine of the human neuroendocrine/brain
gene, PCRable DNA from a somatic cell hybrid New Haven, CT) was typed using the polymerase tion
(PCR)
and
the
primers
fi-cells (pi subwith the
the
chromocri subunit
panel (Bios; chain reac-
5’-TGACTGAAGCCCAGCTT-
GTG-3’ and 5’-GTCTAGCAGCATCGATTGCAGC-3’. These primers amplify a 279-bp fragment of the human neuroendocrine/hrain-type a1 subunit gene that cosegregated with chromosome 3 in this hybrid cell panel (data not shown). To confirm the assignment to chromosome 3 and to obtain a regional localization, we performed fluorescence in situ hybridization of a biotin-labeled probe to human metaphase chromosomes. The probe was a human genomic clone, XhCaCh@108, that was isolated from a human genomic library (Cat. No. 946203, Stratagene; La Jolla, CA) by hybridization with a human neuroendocrine/brain (Ye subunit cDNA clone; this human genomic clone contained an insert of 15.5 kb. Fluorescence in situ GENOMICS
All
13, 1375-1377 (19%) o&38-7543/92 $5.00
Copyright 0 1992 by Academic Press, Inc. rights of reproduction in any form reserved.