GENOMICS
6, 374-378
(1990)
SHORT COMMUNICATION Localization
of the Choline Acetyltransferase to Human Chromosome IO
(CHAT) Gene
ALEXIS BRICE, t SYLVIE BERRARD, t NGUYEN VAN CONG,* ODILE COHEN-HAGUENAUER,*,’ JACQUES MALLET,t AND JEAN FR~~ZAL* *Unite’ de Recherches de G&&ique Mt?dicale INSERM U 12, Clinique Maurice Lamy, H6ppital Necker Enfants-Malades, 749 rue de Se’vres 75743, Paris Cedex 15, and tD6partement de G&nCtique Molt?culaire, Laboratoire de Neurobiologie Cellulaireet Mol&ulaire, Centre National de la Recherche Scientifique, F9 7 190 Gif sur Yvette, France Received
October
Choline acetyltransferase (proposed HGM symbol CHAT), which catalyzes the biosynthesis of acetylcholine, constitutes the best marker for cholinergic neurons. Complementary DNA clones encoding the complete sequencesof porcine and, more recently murine, CHAT have been isolated (Berrard et al., 1987; Brice et al., 1989). The corresponding RNAs were shown to direct the synthesis of active enzymes both in Xenopus oocytes and in rabbit reticulocyte Ijlsate. Here, we have used the porcine probe to localize the human CHAT gene to chromosome 10, using a panel of human-rodent somatic cell hybrids. The porcine CHAT cDNA clone was isolated by Berrard et at. (1987) from a XgtlO cDNA library derived from the ventral spinal cord. The 2120-bp insert purified after EcoRI digestion includes the entire coding region flanked by 124 noncoding bp at the 5’ end and by 77 bp at the 3’ end. The CHAT insert was labeled to a specific activity of 1 X 10’ dpm/pg using random priming (Feinberg and Vogelstein, 1983,1984) and hybridized (see legend to Fig. 1) to TaqI DNA digests from a panel of 24 independent human-rodent somatic cell hybrids (Nguyen Van Cong et al., 1986) and from the parental human and rodent strains. In a first series of Southern blot experiments, we showed that the porcine probe recognizes both human and rodent CHAT genes and that the intensity of the signal is much stronger with the latter (data not shown). Therefore, the membranes were presaturated by hybridization with sonicated DNA from the parental rodent strains (Cohen-Haguenauer et aZ., 1987c). The human signal was easily distinguished (detailed hybridization conditions are given in the legend to Fig. l), permitting unambiguous analysis of its segregation among the hybrids.
A cDNA clone encoding the complete sequence of porcine choline acetyltransferase (CHAT) isolated by S. Berrard et al. (1987, Proc. Natl. Acad. Sci. USA 84: 9280-9284) was hybridized to TaqI digests of a panel of 26 human-rodent somatic cell hybrids and to a complementary panel of 10 human-rodent hybrids in order to determine the chromosomal localization of human CHAT. To enhance the detection of the human signal, hybridization and washings were performed under low stringency conditions on membranes presaturated with sonicated DNA from parental rodent strains. All informative human fragments had the same distribution among the hybrids, mapping CHAT to a single human chromosome. CHAT was assigned to chromosome 10 because all other chromosomes were eliminated by exclusion based on the analysis of the signal segregation. This result indicates that mutation of the CHAT gene cannot be responsible for the primary defect in familial Alzheimer’s disease. a 1990 Academic
Prew,
Inc.
Cholinergic systems are involved in numerous functions of the central nervous system. In particular, neurons of the septum, which project to the hippocampus, are thought to be implicated in learning and memory (Olton et al., 1979). An alteration of the corresponding neurons may account for some neuropsychological disorders in Alzheimer’s disease. ’ To whom reprint requests should be addressed at present address: INSERM 152, CNRS UA628, H5pitaI Cochin, 27 rue du faubourg Saint Jacques, 75014 Paris, France.
088%7543/90 $3.00 Copyright 0 1990 by Academic Press, AI1 rights of reproduction in any form
374 Inc. reserved.
23, 1989
SHORT
hamster kb
panel
1 2 3 4 5 6 7 8 9 1011 121314151617
ham~sier
man
COMMUNICATION
18192021
- man.
FIG. 1. Hybridization of the porcine choline acetyltransferase cDNA to the panel of human-hamster somatic cell hybrids. Prehybridization was performed in 45% formamide, 5X SSPE (1X SSPE is 0.15 M NaCl, 10 mM Na phosphate, 1 m&f EDTA, pH 7.4), 0.1% SDS, 0.04% Ficoll, 0.04% polyvinylpyrrolidone, 0.04% bovine serum albumin, 5% dextran sulfate with 100 rg/ml sonicated denatured rodent DNA and 250 rg/ml sonicated denatured salmon sperm DNA, overnight at 37°C. Then 1 X 10s cpm of the probe was added to 20 ml of prehybridization solution (0.5-10’ cpm/ml) for hybridization 24 h at 37°C. Blots were washed in 2X SSC, 0.1% SDS once for 10 min at room temperature and once for 2 min at 65°C. Filters were then exposed to Kodak KAR-5 films with intensifying screens at -70°C for 3 days. Lane 1, hamster DNA, lanes 2. 5, 13, and 16, human DNA, lanes 3 and 4, hybrids CHl and CH2; lanes 6 to 12, hybrids CH3 to CH9; lanes 14 and 15, hybrids CHlO and CHll; lanes 17 to 21, hybrids CH12 to CH16. Nine fragments were detected in the human lanes (2,5, 13, and 16); their molecular weights are indicated on the left. Three fragments were informative for the localization analysis (6.8, 4.3, and 1.85 kb) and correlated with the presence of chromosome 10 markers in the hybrids. The six additional human fragments comigrated with hamster bands (5.8 and 3.75 kb), were too faint to be followed in the hybrids (2.55,1.95, and 1.50 kb), or could be followed in some hybrids only (1.1 kb).
Nine TuqI fragments were detected in human DNA, 11 in hamster (Fig. l), and 5 in mouse. Only 3 human fragments (6.8, 4.3, and 1.85 kb) were informative for the localization analysis. Among the other 6 fragments, 2 (5.8 and 3.75 kb) comigrated with rodent bands, 3 (2.55, 1.95, and 1.50 kb) were too weak to be detected
375
in the hybrids where human DNA can be diluted up to l/15, and one could be followed only in somehybrids (1.1 kb) with a distribution similar to that of the 3 informative fragments. Analysis of the signal segregation among the 24 hybrids reveals that all informative fragments had the same distribution, indicating that they are localized to a single human chromosome. No gene dosage was observed in lymphoblastoid cell lines derived from XXY, XXX, XXXXY, and XYYYY individuals (kindly provided by Marc Fellous), thereby excluding mapping to either the X or the Y chromosomes. The analysis of the segregation of the CHAT signal among the 24 hybrids is summarized in Table 1. The absence of a detectable CHAT signal in cell hybrids where an intact human chromosome was present in more than 30% of mitoses excluded all but chromosome 10 as the site for the localization of the CHAT gene. In some hybrids (human-mouse M3, M5, and M6) where a human signal was detected even though cytogenetic analysis did not indicate the presence of an intact human chromosome 10, reference enzyme markers and reference DNA probes for chromosome 10 were all positive (Table 2) except for human-hamster hybrids 17 and 7 (CH17 and CH7). Chromosome breakage during the process of somatic cell hybridization could account for these discordant observations, since the translocation of a fragment from a human to a rodent chromosome would not be detected on a karyotype. In CH17, chromosome breakage might also have occurred, but neither the gene encoding GOT1 (glutamic-oxaloacetic transaminase 1, soluble) localized to lOq25.3 (Gerald and Grzeschik, 1984) nor the LAM1083 and LAM368 loci (see Table 2) would be involved. The latter have been shown to map to the long arm of chromosome 10 (Cohen-Haguenauer et al., 1987b, 1989; Donis-Keller et al., 1987). A striking discordance was observed in humanhamster hybrid 7 (CH7) as shown in Table 2. No positive signals were obtained for any of the markers of chromosome 10 tested, although the strongest hybridization signal with our CHAT probe was observed with these cells. This might indicate that CHAT maps to a portion of chromosome 10 that is not covered by any of the markers tested so far. According to this hypothesis, CHAT could not be located to a segment of chromosome 10 including the major part of the long arm up to lOq25.3. CHAT could map to lOq, distal to LAM1083, LAM368, and GOTl, i.e., distal to lOq25.3, which localizes the gene distal to FRAlOB; alternatively, CHAT might be localized to the proximal portion of the long arm or to the short arm of chromosome 10.
SHORT
376
COMMUNICATION
TABLE Comparison
of the CHAT
Signal
with
1
the Human
Chromosome Human
Chromosome hybridization
of the Hybrids
chromosome
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
X
Y
8 9
8 2
2 6
8 10
10 5
8 5
8 6
6 5
7 8
10 7
5 8
7 6
7 2
10 5
12 5
8 8
13 0
2 11
5
3
5
5
7
6
3
8
6 3
6 8
3 5
1 4
5 2
0 11
11 0
23
24
24
22
22
23
24
23
22
23
23
22
23
24
24
Correlation
+/+ -/-
4 7
5 9
8 5
7 4
7 6
9 4
Discordancy Exclusion
-/+ +/-
9 3
8 1
5 6
6 4
6 5
4 5 511 526406634242
23
23
24
21
24
Informative hybrids
Composition
22
24
21
23
Note. This table shows the correlation of the CHAT gene with the presence of each chromosome in the somatic cell hybrids. The number of hybrids that are concordant (+/+ or -/-), are discordant (-/+), or lead to exclusion of a chromosome for the localization of the CHAT gene (+/-) is given for each chromosome. Exclusion cases were taken into account only when a chromosome was observed in more than 30% of mitoses.
TABLE Comparison
of the CHAT
Somatic
Signal
Among the Hybrids with and Two Chromosome
cell hybrid
Mouse Ml M2 M3 M4 M5 M6 M7 Hamster CHl CH2 CH3 CH4 CH5 CH6 CH7 CH8 CH9 CHlO CHll CH12 CH13 CH14 CH15 CH16 CH17
CHAT
+ + + (weak) + + + + + + (intense) + + + +
Chr
10
-
2 the Presence of Chromosome 10 DNA Probes LAM1083 (DlOS7)
LAM368 (DlOS12)
-
-
+ + + (weak) + +
+ + + (weak) + +
+ -
+ -
+ -
+ + -
+ + + (weak) -
+ + + (weak) -
+ -
+ -
+ + -
+ + -
GOT1
-
+ -
+ + -
7
+ +
+ / + / 7 -
10, the Enzyme
+
+ -
7 -
+ -
GOTl,
Note. This table shows the segregation of the CHAT signal among the hybrids compared to the segregation of chromosome 10 (karyotypes); of a chromosome 10 enzyme marker, GOT1 (glutamic-oxaloacetic transaminase 1, soluble), localized to lOq25.3 (10); of two anonymous DNA probes (13) localized to chromosome 10, LAM1083 (DlOS7) and LAM368 (DlOS12) (4,6). Note the presence of an intense CHAT signal in hybrid CH7 negative for chromosome 10 and chromosome 10 markers. +, positive signal; -, negative signal; /, presence of chromosome 10 in less than 30% of mitoses.
SHORT
TABLE Segregation
of CHAT
Gene
L53C8 LA56AI CH35J6 CH56C CH56G CH56L CH56W CH56YI CH34Z CH35N
1
2
3
4
+ +-+---+ _____ -
+
-++++--+-++-+-+-++----
-----+-+-+-+ + iv--++-+/
5
6
7
3
in a Complementary Human
Human-rodent hybrid
8
9
10
11
Panel
chromosome 12
13
,--,-+---------++-----.
-+-+,--+,+ -+---+ + + + + -
+ +,+--+----+--+-,--.
377
COMMUNICATION
Informative
for Chromosome
present 14
15
16
+
/++--+++-.
17
18
19
---+
-
+
+
------
21
22
X
Y
CHAT + (weak) + -
---,--, +
‘-
. /+-+--. +
-,--.
+ -
/+/.---------
+--+.-
of the CHAT signal in an additional panel for the localization of the CHAT gene.
In order to further confirm the localization of CHAT to chromosome 10, a panel of 10 additional independent human-rodent hybrids was tested (Cohen-Haguenauer et al., 1987a). The distribution of the CHAT signal among these hybrids, summarized in Table 3, indicates that the only candidate is chromosome 10, since a positive signal was detected in CH56C and CH56YI only. A single discrepancy was observed in CH34Z, where no signal was detected even though it contained chromosome 10 in mitosis. The weak intensity of the hybridization signal to the complementary panel together with the infrequent presence of chromosome 10 in CH34Z can account for this observation. Taken together, data collected from the analyses of both panels clearly indicate that the gene encoding CHAT maps to human chromosome 10. A more precise localization by in situ hybridization on metaphasic chromosomes could not be performed in this experiment because the porcine probe yielded a signal that was too weak. Alzheimer’s disease is characterized mainly by the degeneration of cholinergic neurons. Our data indicate that the CHAT gene is not responsible for this defect, since in several pedigrees, the gene responsible for this disorder has been mapped to chromosome 21 (St Georges-Hyslop et al., 1987).
from the CEPH (Centre the Institut National de la the Centre National de la fellowships from the Fonthe Association Archibald the Fondation pour la Re-
of 10 independent
somatic
cells hybrids.
In this
cherche Midicale and the Fonds d’Etude du Corps Medical itaux de Paris. We are grateful to Francois Dreyfus, M.D. helpful discussion.
panel,
des HGpPh.D., for
REFERENCES 1.
BERRARD, S., BRICE, A., LOTTSPEICH, F., BRAUN, A., BARDE, Y. A., AND MALLET, J. (1987). cDNA cloning and complete sequence of porcine choline acetyltransferase: In vitro translation of the corresponding RNA yields an active protein. Proc. Natl. Acad. Sci. USA 84: 9260-9264.
2.
BFUCE, A., BERFURD, S., RAYNAUD, B., ANSIEAU, S., COPPOLA, T., WEBER, M., AND MALLET, J. (1989). Complete sequence of a cDNA encoding an active rat choline acetyltransferase: A tool to investigate the plasticity of the cholinergic phenotype expression. J. Neurosci. Res. 23: 266-273.
3.
COHEN-HAGUENAUER, O., PICARD, J. Y., MA’ITEI, M. G., SERERO, S., NGWEN VAN CONG, DE TAND, M. F., GUERRIER, D., HORS-CAYLA, M. C., Josso, N., AND F&AL, J. (1987a). Mapping of the gene for anti-Mtilerian hormone to the short arm of human chromosome 19. Cytogemt. Cell Gem% 44: 2-6. COHEN-HAGIJENAUER, O., NGWEN VAN CONG, KNOWLTON, R. G., DE TAND, M. F., JEGOU, C., GROSS, M. S., BROWN, V. A., BRAMAN, J. C., SCHUMM, J. W., BARKER, D. F., DONISKELLER, H., AND FR&AL., J. (1987b). Chromosomal assignment of 15 highly polymorphic random genomic probes: Human Gene Mapping 9. Cytogenet. Cell Genet. 46: 596.
4.
5.
COHEN-HAGIJENAUER, O., NGUYEN VAN CONG, AND FR$ZAL, J. (1987c). Prehybridization with rodent DNA for the chromosomal assignment of human genes using mouse specific probes: Human Gene Mapping 9. Cytogenet. Cell Genet. 46: 596.
6.
COHEN-HAGUENAIJER, O., NGUYEN VAN CONG, KNOWLTON, R. G., DE TAND, M. F., JECOU, C., GROSS, M. S., BROWN, V. A., FF&.&, J., AND DONIS-KELLER, H. (1989). Chromosomal assignment of 14 genomic probes for highly polymorphic loci. Cytogenet. Cell Genet. 50: 76-63. DONIS-KELLER, H., GREEN, P., HELMS, C., CARTINHOUR, S., WEIFFENBACH, B., STEPHENS, K., KEITH, T. P., BOWDEN,
ACKNOWLEDGMENTS This work was supported by grants d’Etudes du Polymorphiime Humain), Sant.& et de la Recherche Medicale, and Recherche Scientifique. O.C.H. received dation pour la Recherche Medicale and Garrod. A.B. received fellowships from
20
,--++-----++-+. + -
++++-++/-++--+--+
Note. This table shows the distribution too, chromosome 10 is the only candidate
10
7.
378
SHORT D. W., SMITH, D. R., LANDER, E. S., BOTSTEIN, D., AKOTS, REDIKER, K. S., GRAVIUS, T., BROWN, V. A., RISING, M. PARKER, C., POWERS, J. A., WATT, D. E., KAUFFMAN, E. BRICKER, A., PHIPPS, P., MULLER-KAHLE, H., FULTON, T. NG, S., SCHUMM, J. W., BRAMAN, J. C., KNOWLTON, R. BARKER, D. F., CROOKS, S. M., LINCOLN, S. E., DALY, M. AND ABRAHAMSON, J. (1987). A genetic linkage map of the man genome. Cell 51: 319-337.
8.
9.
COMMUNICATION G., B., R., R., G., J., hu-
FEINBERG, A. P., AND VOGELSTEIN, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13. FEINBERG, A. P., AND VOGELSTEIN, B. (1984). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity: Addendum. Anal. B&hem. 137: 266-267.
10.
GERALD, P. S., AND GRZESCHIK, K. H. (1984). Report of the Committee on the Genetic Constitution of Chromosomes 10, 11, and 12 (HGM7). Cytogenet. Cell Genet. 37: 1033126.
11.
NGUYEN VAN CONG, WEIL, D., FINAZ, C., COHEN-HAGUENAUER, O., GROSS, M. S., JEGOU-FOUBERT, C., DE TAND, M. F., COCHET, C., DE GROUCHY, J., AND FRBZAL, J. (1986). Panel of twenty-
five independent mapping. Ann.
man-rodent Genet. 29:
hybrids 20-26.
for human
genetic
marker
12.
OLTON, D. S., BECKER, J. T., AND HANDELMAN, Hippocampus, space and memory. Behau. Brain 365.
G. E. (1979). Sci. 2: 313-
13.
SCHUMM, J. W., KNOWLTON, R. G., BRAMAN, J. C., BARKER, D. F., BOTSTEIN, D., AKOTS, G., BROWN, V. A., GRAVIUS, T. C., HELMS, C., HSIAO, K., REDIKER, K., THURSTON, J. G., AND DONIS-KELLER, H. (1988). Identification of more than 500 RFLRs by screening random genomic clones. Am. J. Hum. Genet. 42: 143-159.
14.
ST. GEORGE-HYSLOP, P. H., TANZI, R. E., POLINSKY, R. J., HAINES, J. L., NEE, L., WATKINS, P. C., MYERS, R. H., FELDMAN, R. G., POLLEN, D., DRACHMAN, D., GROWDON, J., BRUNI, A., FONCIN, J. F., SALMON, D., FROMMELT, P., AMADUCCI, L., SORBI, S., PLACENTINI, S., STEWART, G. D., HOBBS, W. J., CONNEALLY, P. M., AND GUSELLA, J. F. (1987). The genetic defect causing familial Alzheimer’s disease maps on chromocome 21. Science 235: 665-669.
6, 374-378
(1990)
SHORT COMMUNICATION Localization
of the Choline Acetyltransferase to Human Chromosome IO
(CHAT) Gene
ALEXIS BRICE, t SYLVIE BERRARD, t NGUYEN VAN CONG,* ODILE COHEN-HAGUENAUER,*,’ JACQUES MALLET,t AND JEAN FR~~ZAL* *Unite’ de Recherches de G&&ique Mt?dicale INSERM U 12, Clinique Maurice Lamy, H6ppital Necker Enfants-Malades, 749 rue de Se’vres 75743, Paris Cedex 15, and tD6partement de G&nCtique Molt?culaire, Laboratoire de Neurobiologie Cellulaireet Mol&ulaire, Centre National de la Recherche Scientifique, F9 7 190 Gif sur Yvette, France Received
October
Choline acetyltransferase (proposed HGM symbol CHAT), which catalyzes the biosynthesis of acetylcholine, constitutes the best marker for cholinergic neurons. Complementary DNA clones encoding the complete sequencesof porcine and, more recently murine, CHAT have been isolated (Berrard et al., 1987; Brice et al., 1989). The corresponding RNAs were shown to direct the synthesis of active enzymes both in Xenopus oocytes and in rabbit reticulocyte Ijlsate. Here, we have used the porcine probe to localize the human CHAT gene to chromosome 10, using a panel of human-rodent somatic cell hybrids. The porcine CHAT cDNA clone was isolated by Berrard et at. (1987) from a XgtlO cDNA library derived from the ventral spinal cord. The 2120-bp insert purified after EcoRI digestion includes the entire coding region flanked by 124 noncoding bp at the 5’ end and by 77 bp at the 3’ end. The CHAT insert was labeled to a specific activity of 1 X 10’ dpm/pg using random priming (Feinberg and Vogelstein, 1983,1984) and hybridized (see legend to Fig. 1) to TaqI DNA digests from a panel of 24 independent human-rodent somatic cell hybrids (Nguyen Van Cong et al., 1986) and from the parental human and rodent strains. In a first series of Southern blot experiments, we showed that the porcine probe recognizes both human and rodent CHAT genes and that the intensity of the signal is much stronger with the latter (data not shown). Therefore, the membranes were presaturated by hybridization with sonicated DNA from the parental rodent strains (Cohen-Haguenauer et aZ., 1987c). The human signal was easily distinguished (detailed hybridization conditions are given in the legend to Fig. l), permitting unambiguous analysis of its segregation among the hybrids.
A cDNA clone encoding the complete sequence of porcine choline acetyltransferase (CHAT) isolated by S. Berrard et al. (1987, Proc. Natl. Acad. Sci. USA 84: 9280-9284) was hybridized to TaqI digests of a panel of 26 human-rodent somatic cell hybrids and to a complementary panel of 10 human-rodent hybrids in order to determine the chromosomal localization of human CHAT. To enhance the detection of the human signal, hybridization and washings were performed under low stringency conditions on membranes presaturated with sonicated DNA from parental rodent strains. All informative human fragments had the same distribution among the hybrids, mapping CHAT to a single human chromosome. CHAT was assigned to chromosome 10 because all other chromosomes were eliminated by exclusion based on the analysis of the signal segregation. This result indicates that mutation of the CHAT gene cannot be responsible for the primary defect in familial Alzheimer’s disease. a 1990 Academic
Prew,
Inc.
Cholinergic systems are involved in numerous functions of the central nervous system. In particular, neurons of the septum, which project to the hippocampus, are thought to be implicated in learning and memory (Olton et al., 1979). An alteration of the corresponding neurons may account for some neuropsychological disorders in Alzheimer’s disease. ’ To whom reprint requests should be addressed at present address: INSERM 152, CNRS UA628, H5pitaI Cochin, 27 rue du faubourg Saint Jacques, 75014 Paris, France.
088%7543/90 $3.00 Copyright 0 1990 by Academic Press, AI1 rights of reproduction in any form
374 Inc. reserved.
23, 1989
SHORT
hamster kb
panel
1 2 3 4 5 6 7 8 9 1011 121314151617
ham~sier
man
COMMUNICATION
18192021
- man.
FIG. 1. Hybridization of the porcine choline acetyltransferase cDNA to the panel of human-hamster somatic cell hybrids. Prehybridization was performed in 45% formamide, 5X SSPE (1X SSPE is 0.15 M NaCl, 10 mM Na phosphate, 1 m&f EDTA, pH 7.4), 0.1% SDS, 0.04% Ficoll, 0.04% polyvinylpyrrolidone, 0.04% bovine serum albumin, 5% dextran sulfate with 100 rg/ml sonicated denatured rodent DNA and 250 rg/ml sonicated denatured salmon sperm DNA, overnight at 37°C. Then 1 X 10s cpm of the probe was added to 20 ml of prehybridization solution (0.5-10’ cpm/ml) for hybridization 24 h at 37°C. Blots were washed in 2X SSC, 0.1% SDS once for 10 min at room temperature and once for 2 min at 65°C. Filters were then exposed to Kodak KAR-5 films with intensifying screens at -70°C for 3 days. Lane 1, hamster DNA, lanes 2. 5, 13, and 16, human DNA, lanes 3 and 4, hybrids CHl and CH2; lanes 6 to 12, hybrids CH3 to CH9; lanes 14 and 15, hybrids CHlO and CHll; lanes 17 to 21, hybrids CH12 to CH16. Nine fragments were detected in the human lanes (2,5, 13, and 16); their molecular weights are indicated on the left. Three fragments were informative for the localization analysis (6.8, 4.3, and 1.85 kb) and correlated with the presence of chromosome 10 markers in the hybrids. The six additional human fragments comigrated with hamster bands (5.8 and 3.75 kb), were too faint to be followed in the hybrids (2.55,1.95, and 1.50 kb), or could be followed in some hybrids only (1.1 kb).
Nine TuqI fragments were detected in human DNA, 11 in hamster (Fig. l), and 5 in mouse. Only 3 human fragments (6.8, 4.3, and 1.85 kb) were informative for the localization analysis. Among the other 6 fragments, 2 (5.8 and 3.75 kb) comigrated with rodent bands, 3 (2.55, 1.95, and 1.50 kb) were too weak to be detected
375
in the hybrids where human DNA can be diluted up to l/15, and one could be followed only in somehybrids (1.1 kb) with a distribution similar to that of the 3 informative fragments. Analysis of the signal segregation among the 24 hybrids reveals that all informative fragments had the same distribution, indicating that they are localized to a single human chromosome. No gene dosage was observed in lymphoblastoid cell lines derived from XXY, XXX, XXXXY, and XYYYY individuals (kindly provided by Marc Fellous), thereby excluding mapping to either the X or the Y chromosomes. The analysis of the segregation of the CHAT signal among the 24 hybrids is summarized in Table 1. The absence of a detectable CHAT signal in cell hybrids where an intact human chromosome was present in more than 30% of mitoses excluded all but chromosome 10 as the site for the localization of the CHAT gene. In some hybrids (human-mouse M3, M5, and M6) where a human signal was detected even though cytogenetic analysis did not indicate the presence of an intact human chromosome 10, reference enzyme markers and reference DNA probes for chromosome 10 were all positive (Table 2) except for human-hamster hybrids 17 and 7 (CH17 and CH7). Chromosome breakage during the process of somatic cell hybridization could account for these discordant observations, since the translocation of a fragment from a human to a rodent chromosome would not be detected on a karyotype. In CH17, chromosome breakage might also have occurred, but neither the gene encoding GOT1 (glutamic-oxaloacetic transaminase 1, soluble) localized to lOq25.3 (Gerald and Grzeschik, 1984) nor the LAM1083 and LAM368 loci (see Table 2) would be involved. The latter have been shown to map to the long arm of chromosome 10 (Cohen-Haguenauer et al., 1987b, 1989; Donis-Keller et al., 1987). A striking discordance was observed in humanhamster hybrid 7 (CH7) as shown in Table 2. No positive signals were obtained for any of the markers of chromosome 10 tested, although the strongest hybridization signal with our CHAT probe was observed with these cells. This might indicate that CHAT maps to a portion of chromosome 10 that is not covered by any of the markers tested so far. According to this hypothesis, CHAT could not be located to a segment of chromosome 10 including the major part of the long arm up to lOq25.3. CHAT could map to lOq, distal to LAM1083, LAM368, and GOTl, i.e., distal to lOq25.3, which localizes the gene distal to FRAlOB; alternatively, CHAT might be localized to the proximal portion of the long arm or to the short arm of chromosome 10.
SHORT
376
COMMUNICATION
TABLE Comparison
of the CHAT
Signal
with
1
the Human
Chromosome Human
Chromosome hybridization
of the Hybrids
chromosome
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
X
Y
8 9
8 2
2 6
8 10
10 5
8 5
8 6
6 5
7 8
10 7
5 8
7 6
7 2
10 5
12 5
8 8
13 0
2 11
5
3
5
5
7
6
3
8
6 3
6 8
3 5
1 4
5 2
0 11
11 0
23
24
24
22
22
23
24
23
22
23
23
22
23
24
24
Correlation
+/+ -/-
4 7
5 9
8 5
7 4
7 6
9 4
Discordancy Exclusion
-/+ +/-
9 3
8 1
5 6
6 4
6 5
4 5 511 526406634242
23
23
24
21
24
Informative hybrids
Composition
22
24
21
23
Note. This table shows the correlation of the CHAT gene with the presence of each chromosome in the somatic cell hybrids. The number of hybrids that are concordant (+/+ or -/-), are discordant (-/+), or lead to exclusion of a chromosome for the localization of the CHAT gene (+/-) is given for each chromosome. Exclusion cases were taken into account only when a chromosome was observed in more than 30% of mitoses.
TABLE Comparison
of the CHAT
Somatic
Signal
Among the Hybrids with and Two Chromosome
cell hybrid
Mouse Ml M2 M3 M4 M5 M6 M7 Hamster CHl CH2 CH3 CH4 CH5 CH6 CH7 CH8 CH9 CHlO CHll CH12 CH13 CH14 CH15 CH16 CH17
CHAT
+ + + (weak) + + + + + + (intense) + + + +
Chr
10
-
2 the Presence of Chromosome 10 DNA Probes LAM1083 (DlOS7)
LAM368 (DlOS12)
-
-
+ + + (weak) + +
+ + + (weak) + +
+ -
+ -
+ -
+ + -
+ + + (weak) -
+ + + (weak) -
+ -
+ -
+ + -
+ + -
GOT1
-
+ -
+ + -
7
+ +
+ / + / 7 -
10, the Enzyme
+
+ -
7 -
+ -
GOTl,
Note. This table shows the segregation of the CHAT signal among the hybrids compared to the segregation of chromosome 10 (karyotypes); of a chromosome 10 enzyme marker, GOT1 (glutamic-oxaloacetic transaminase 1, soluble), localized to lOq25.3 (10); of two anonymous DNA probes (13) localized to chromosome 10, LAM1083 (DlOS7) and LAM368 (DlOS12) (4,6). Note the presence of an intense CHAT signal in hybrid CH7 negative for chromosome 10 and chromosome 10 markers. +, positive signal; -, negative signal; /, presence of chromosome 10 in less than 30% of mitoses.
SHORT
TABLE Segregation
of CHAT
Gene
L53C8 LA56AI CH35J6 CH56C CH56G CH56L CH56W CH56YI CH34Z CH35N
1
2
3
4
+ +-+---+ _____ -
+
-++++--+-++-+-+-++----
-----+-+-+-+ + iv--++-+/
5
6
7
3
in a Complementary Human
Human-rodent hybrid
8
9
10
11
Panel
chromosome 12
13
,--,-+---------++-----.
-+-+,--+,+ -+---+ + + + + -
+ +,+--+----+--+-,--.
377
COMMUNICATION
Informative
for Chromosome
present 14
15
16
+
/++--+++-.
17
18
19
---+
-
+
+
------
21
22
X
Y
CHAT + (weak) + -
---,--, +
‘-
. /+-+--. +
-,--.
+ -
/+/.---------
+--+.-
of the CHAT signal in an additional panel for the localization of the CHAT gene.
In order to further confirm the localization of CHAT to chromosome 10, a panel of 10 additional independent human-rodent hybrids was tested (Cohen-Haguenauer et al., 1987a). The distribution of the CHAT signal among these hybrids, summarized in Table 3, indicates that the only candidate is chromosome 10, since a positive signal was detected in CH56C and CH56YI only. A single discrepancy was observed in CH34Z, where no signal was detected even though it contained chromosome 10 in mitosis. The weak intensity of the hybridization signal to the complementary panel together with the infrequent presence of chromosome 10 in CH34Z can account for this observation. Taken together, data collected from the analyses of both panels clearly indicate that the gene encoding CHAT maps to human chromosome 10. A more precise localization by in situ hybridization on metaphasic chromosomes could not be performed in this experiment because the porcine probe yielded a signal that was too weak. Alzheimer’s disease is characterized mainly by the degeneration of cholinergic neurons. Our data indicate that the CHAT gene is not responsible for this defect, since in several pedigrees, the gene responsible for this disorder has been mapped to chromosome 21 (St Georges-Hyslop et al., 1987).
from the CEPH (Centre the Institut National de la the Centre National de la fellowships from the Fonthe Association Archibald the Fondation pour la Re-
of 10 independent
somatic
cells hybrids.
In this
cherche Midicale and the Fonds d’Etude du Corps Medical itaux de Paris. We are grateful to Francois Dreyfus, M.D. helpful discussion.
panel,
des HGpPh.D., for
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ACKNOWLEDGMENTS This work was supported by grants d’Etudes du Polymorphiime Humain), Sant.& et de la Recherche Medicale, and Recherche Scientifique. O.C.H. received dation pour la Recherche Medicale and Garrod. A.B. received fellowships from
20
,--++-----++-+. + -
++++-++/-++--+--+
Note. This table shows the distribution too, chromosome 10 is the only candidate
10
7.
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