Neuroscience Letters, 132 (1991) 191 194
191
(') 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 A D O N 1 S 030439409100639K NSL 08157
Isolation and sub-chromosomal localization of a D N A fragment of the human choline acetyltransferase gene Riccardo Cervini 1, M a r i a n o Rocchi 2, Stefano D i D o n a t o I a n d G a e t a n o F i n o c c h i a r o ~ ~lstituto Nacionale Neurologico, Divisione di Biochimica e Genetica, Milan (Italy) and ZDipartimento di Genetica, Llniversita' di Bari, Bari (Italy)
(Received 11 April 1991; Revised version received 30 July 199t: Accepted 31 July 1991) Key word~v
Choline acetyltransferase; Polymerase chain reaction; Gene mapping
A DNA fragment of 219 bp was obtained by polymerase chain reaction (PCR) on human genomic DNA using two oligonucleotide mixtures derived from peptide sequences of human placenta choline acetyltransferase (CHAT) and from partially conserved amino acid sequences of rat, porcine and Drosophila CHAT. Sequence homology with porcine ChAT demonstrated that this fragment is part of the human ChAT gene. This gene was assigned to chromosome 10 by hybridization of the 219 bp DNA probe with DNA from human-hamster somatic cell hybrids, and to region 10ql 1.2 10qter by PCR experiments.
The molecular biology of choline acetyltransferase (CHAT; EC 2.3.1.6.), the enzyme synthesizing acetylcholine in the nervous system and other organs, has been the subject of considerable work in the recent years [20] and the cDNAs encoding Drosophila, pig and rat CHAT, have been cloned and sequenced [1, 2, 9]. Deficiency of ChAT and cholinergic systems has been reported in several degenerative neurological disorders. Alterations of cholinergic pathways, altered affinity for choline and the presence of antibodies inhibiting ChAT activity have been reported in Alzheimer's disease [5, 7, 12]. Selective loss of cholinergic neurons has been described in the nucleus basalis of Meynert in Alzheimer's disease [25], Pick's disease [24], Parkinson's disease [17], Down's syndrome [3] and dominantly inherited olivopontocerebellar atrophy (OPCA) [23]. ChAT activity was also reduced in the cerebral cortex of OPCA patients [11] and in spinal motor neurons of patients with amyotrophic lateral sclerosis [10]. The identification and chromosomal localization of human ChAT gene could help the understanding of the molecular basis of these diseases. Here we describe the amplification of a region of such gene by polymerase chain reaction IPCR) [19] and its mapping on the long arm of chromosome 10. Human genomic DNA was extracted from lymphoid cells [16]. Primers for PCR were synthesized on a Gene Correspondence: G. Finocchiaro, Istituto Nazionale Neurologico C.
Besta, Divisione di Biochimica e Genetica, via Celoria 11, 20133 Milan~ Italy. Fax: (39) 011-392-2664236.
Assembler Plus (Pharmacia-LKB). Primer RG3 was a pool of 64 35mers containing 3 inosine residues (indicated as x), deduced from the amino acid sequence NGYGACYNPQPE, corresponding to amino acids 568 579 of pig ChAT [1]: AA(TC)GGCTA(TC)GGCGCXTG(TC)TA(TC)AA(TC)CCXCA(AG)CCXGA. Primer G F l l was reverse complementary to DNA sequences deduced from tryptic peptide ATRPSQGHQP, obtained from human placenta ChAT [8]. This peptide corresponds to the extreme C-terminal region of pig ChAT [1]. An EcoRI site preceded by bases AC was added on the 5' to facilitate subcloning procedures. GFI1 was a mix of 128 37mers and contained 5 inosine residues: ACGAATTCGGCTG(GA)TG(GX)CCCTGX(GC)(AT)GGGXC(GT)(GX)GT(CX)GC. PCR were performed in 100/zl mix using 150 pm of these two primers, 0.6/tg of genomic DNA, 0.15 mM dNTPs, 2.25 mM MgC12, 10 mM Tris-C1 pH 8.3, 50 mM KC1, 0.001% gelatin and 5 units of T. Aquaticus DNA polymerase (Perkin-Elmer Cetus). After 4 rain of denaturation at 94°C 40 cycles of amplifications were performed. In each cycle denaturation was at 9 4 C for 1 rain, annealing of primers at 53°C for 2 min and extension at 72'~C for 30 s. Primers R I and R2 correspond to nt 26-54 and 169 199 of the fragment amplified with GFI I and RG3. Two other primers amplified 128 bp of the gene for the human interstitial retinoid-binding protein (IRBP) [22]: A T G A G A G A A T G G G T T C T G C T C A T G T C C G T G and GGTTCTCCGGGAAGCAGTAGTTATCCAAGA. DNA sequences were determined by the Sanger meth-
192
od [19] with the T7 Sequencing kit from Pharmacia after subcloning in plasmid pGEM7zf(Promega). Nucleotide and amino acid sequences were analyzed by MacMolly, a software from SoftGene, Berlin. Somatic cell hybrids were obtained as described [18]. Genomic DNA digests were transferred to Hybond-N membranes (Amersham) after agarose gel electrophoresis. PCR-amplified DNA was labeled with [c~-32P]dCTP by random primers [6]. In order to amplify a fragment of the ChAT gene from human genomic DNA we designed primers based on sequences of a tryptic peptide from human placental CHAT, corresponding to the extreme C-terminal region of porcine CHAT, and of upstream amino acids identical in rat and porcine ChAT and 67% homologous with those of Drosophila ChAT [see table in ref. 2]. The aminoacid sequence encompassed between these two primers in pig ChAT is of 73 residues, corresponding to 219 bp. PCR experiments on human genomic DNA yielded one single fragment of this size which was amplified and sequenced (Fig. IA). We compared both this nucleotide sequence and the deduced amino acid sequence with the European Molecular Biology Laboratory nucleic acid database (release 24) and the Swiss-Prot protein sequence database (release 15), respectively, and found significant homologies only with porcine ChAT cDNA and amino acid sequence. The degree of similarity at the pro-
tein level is of 76.7% (Fig. IB), clearly indicating that the PCR-amplified fragment is part of the human ChAT gene. The amino acid residues at positions 66 and 68 are serine and threonine, respectively. However, peptide 1t from human placental ChAT and porcine ChAT show in the same positions arginine and serine residues, respectively [8]. We used a mixture of degenerate oligonucleotide for PCR amplification: therefore the presence of codons AGC and ACC rather than CGC and TCC, respectively, might be the consequence of some misalignment to the original sequence and the presence of these two residues needs further control, Using the 219 bp band as a probe we also analyzed human-hamster somatic cell hybrids looking for the chromosomal localization of the ChAT gene. We analyzed a panel of HindIII digested DNA from 17 somatic cell hybrids. The results are shown in Table I and assign the gene encoding human ChAT to chromosome 10, in agreement with recent observations obtained using porcine ChAT as a probe [4]. To gain further insights on the location of the ChAT gene we also analyzed by PCR the DNA of hybrid HY.88E, which results from a translocation of 10ql 1.2 10qter on chromosome X. We observed that primers RG3 and GF 11 allowed the amplification from hamster DNA of a weak band similar to the one obtained from human DNA. Primers RI and R2 increased the specificity of the reaction, since the annealing temperature could be raised to 70°C and the expected product of 174
N AAC
G GGC
Y TAC
G GGC
A GCG
C TGC
Y TAT
N AAT
P CCG
Q CAG
P E CCG GAG
F TTT
H CAC
S AGC
C TGC
K AAA
E GAG
T ACT
S S TCT TCT
S AGC
K AAG
I AAT
D GAC
M ATG
R AGA
D GAC
L CTC
C TGC
S AGT
L CTG
L P CTG CCQ
K AAG
E GAA
K AAA
A GCC
T ACC
$ AGC
P CCC
T ACC
Q G CAG GGC
L CTT
F TTC
C TGC
I ATC
S TCT
S AGC
60
F A TTT GCA
K A AAA GCT
V GTG
E GAA
E GAA
$ AGC
L CTC
120
P T CCT ACT
E GAG
K AAG
P CCA
L TTG
A GCA
T ACA
180
H Q CAT CAG
T ACC
I ATC
$ AGC
P CCG
219
B o
hChAT pChAT
NG YGACYNPQPE II l l L l k l b l l l NG YGACYNPQPE
TILFCISSFH 11111qI[l SILFCISSFH
SCKETSSSKF AKAVEESLID ~lllql II llqLlll I GCKETSSTKF AKAVEESFIE
MRDLCSLLPP I Iii[ MKGLCSLSQS
TESKPLATKE klEIIll GMGKPLATKE
KATS PTQGHQ P l i I I II I KVTRPSQVHQ P
Fig. 1. A: DNA and deducedaminoacid sequenceof the 219 bp fragmentisolatedby polymerasechain reaction(PCR) on total humangenomic DNA with primers RG3 and GF11. B: comparisonof amino acid sequence deducedfrom the 219 bp product from PCR (hChAT) with amino acid sequenceof porcine ChAT(pChAT;aa 569~a41)[1].
193 TABLE I SYNTENY ANALYSIS FOR HUMAN ChAT Human chromosome present ( + ) or absent ( - ) . The percent concordance is calculated by dividing the number of concordant hybrids by the total number of hybrids. Hybrids
1
Positive HY.19.16T3D HY.95A1 Y.XY.8F6 Y.XY.8FT7 Negative HY.22AZA1 HY.60A HY.70B2 HY.75E1 HY.94A HY.95B HY.95S HY.137J HY. 166T4 RJ.387.51T5 RJ.387.58 Y.173.5CT3
3
4
5
4" 4-
-4"
+ 4-
4. 4.
6
7
8
4" +
+
4. +
-
9
4"
--
--
10
I1
4. 4" 4" +
4" + +
12
13
14
15
4.
+
4. +
+ . .
+
4. ~-
+
+ 4. 4.
4. 4.
4. +
4. + -
be obtained
65
from
. . 4"
+
.
+ -
+
+
-
+
71
65
59
47
.
human
+ 59
47
but not hamster
D N A . T h i s is s h o w n i n l a n e 3 a n d 7, r e s p e c t i v e l y , o f Fig.
.
X
4.
Jr + +
.
4.
4.
.
22
+
. 4.
+ 4.
21
4.
4.
.
20
+
+
4. 4.
+ +
.
+
59
19
+ .
4. +
18
4. 4.
4"
17
-
4.
+
4"
16
4.
t
59
-
Jr +
4.
%concordance
bp could
2
+
+
+ -
lOO 82
53
53
.
.
4" .
4.
+
59
53
.
.
.
.
.
.
1 234
65
47
65
65
+
+ +
--
+
4.
+
41
53
35
.
465
+
-
+ 44.
+ + + + +
+ q-
5 678
2. I n a g r e e m e n t w i t h d a t a o b t a i n e d b y S o u t h e r n h y b r i d i zation, this same product could be obtained from DNA of hybrids
HY.16T3D
and
HY.95A1
but
not
from
h y b r i d H Y . 6 0 A . I n t e r e s t i n g l y , t h e s a m e 174 b p f r a g m e n t was amplified from hybrid HY.88E thus assigning the human 10qll.2-10qter
ChAT
of chromosome
( l a n e 5 o f Fig. 2), gene to the region
10. A s i m i l a r p a t t e r n
was obtained using primers amplifying a fragment of the IRBP gene that has been localized to the proximal region of the long arm of chromosome
10 [14] ( d a t a n o t s h o w n ) .
O
I n c o n c l u s i o n , we h a v e i s o l a t e d a n d c l o n e d a D N A fragment of the human
ChAT gene using PCR on total
genomic DNA with degenerate primers. The availability of this DNA
fragment gives an important tool for clon-
ing the entire cDNA
of human
C H A T , a l l o w i n g t h e in-
v e s t i g a t i o n a t t h e m o l e c u l a r level o f c h o l i n e r g i c p a t h w a y a l t e r a t i o n s in d e g e n e r a t i v e d i s o r d e r s o f t h e n e r v o u s system and of mechanisms for the regulation of ChAT gene expression. Furthermore,
the subchromosomal
tion of the human
g e n e c a n b e o f u s e in l i n k a g e
ChAT
localiza-
studies of neurological diseases and indicates that mutations of such gene cannot cause hereditary forms of Alzheimer's disease linked with mutations on chromosome 19 o r 21 [15]. We
thank
Drs.
Nicoletta
Archidiacono,
Massimo
Fig. 2. Results of PCR with primers RI and R2 on hybrid HY.95 Al (lane 1), hybrid HY.16T3D (lane 2), human and hamster genomic DNA (lane 3 and 7, respectively), hybrid HY.88E (lane 5), and hybrid HY. 60A (lane 6). Lane 4: Mr markers (type V, Boheringer), lane 8, blank (no template), l0 20% of the reaction mixture were loaded on a 2% agarose gel and DNA stained by ethidium bromide.
[94
Pandolfo and Irma Colombo for help and suggestions. This work has been partially supported by a grant from the Muscular Dystrophy Association. 13 1 Berrard, S., Brice, A., Lottspeich, F., Braun, A., Barde, Y. and Mallet, J., eDNA cloning and complete sequence of porcine choline acetyltransferase: in vitro translation of the corresponding RNA yields an active protein, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 9280~9284. 2 Brice, A., Berrard, S., Raynaud, B., Ansieau, S., Coppola, T., Weber, M. and Mallet, J., Complete sequence of a cDNA encoding an active rat choline acetyltransferase: a tool to investigate the plasticity of cholinergic phenotype expression, J. Neurosci. Res., 23 (1989) 266-273. 3 Casanova, M.F.. Walker, L.C,, Whitehouse, P.J. and Price, D.L., Abnormalities of the nucleus basalis in Down' syndrome, Ann. Neurol., 18 (1985) 310-313. 4 Cohen-Haguenauer, O., Brice, A., Berrard, S., Van Cong, N., Mallet, J. and Frezal, J., Localization of the choline acetyltransferase (CHAT) gene to human chromosome 10, Genomics, 6 (1990) 374 378. 5 Davies, P. and Maloney, A.J.F., Selective loss of central cholinergic neurons in Alzheimer's disease, Lancet, ii (1976) 1403. 6 Feinberg, P.A. and Vogelstein, B., Addendum: a technique for radiolabeling DNA restriction endonuclease fragments to high specific activity, Anal. Biochem., 137 (1984) 266-267. 7 Fillit, H., Luine, V.N., Reisberg, B., Amador, R., McEwen, B. and Zabriskie, J.B., Studies of the specificity of antibrain antibodies in Alzheimer's disease. In J.D. Hutton and A.D. Kenny (Eds.), Senile Dementia of the Alzheimer Type, A. Liss, New York, 1985, pp. 307--318. 8 Hersh, L.B., Takane, K., Gylys, K., Moomaw, C. and Slaughter. C.. Conservation of amino acid sequences between human and porcine choline acetyltransferasc, J. Neurochem., 51 (1988) 1843-1845, 9 ltoh. N., Slemmon, J.R., Hawke, D.H., Williamson, R., Morita. E.. Itakura, K., Roberts, E., Shively, J.E., Crawford, G.D. and Salvaterra, P.M.. Cloning of Drosophila choline acetyltransferase cDNA, Proc. Natl. Acad. Sci. U.S.A., 83 (1986)4081-4085. 10 Kato. K., Choline acetyltransferase activities in single spinal motor neurons from patients with amyotrophie lateral sclerosis, J. Neurochem., 52 (1989) 636--640. I 1 Kish. S.J., Robitaille, Y., E1-Awar, M., Dock. J.H.N., Simmons, J., Schut, L., Chang, L.J., DiStefano, L. and Freedman, M., Non-Alzheimer type pattern of brain cholineacetyltransferase reduction in dominantly inherited olivopontocerebellar atrophy. Ann. Neurol., 26 (1989) 362-367. 12 Koshimura, K., Nakamura, S.. Miwa, S., Fujiwara, M. and
14
15 16
17
18
19
20 21
22
23
24
25
Kameyama, M., Regional difference in the kinetics of choline ace tyltransferase in brains of neurologically normal elderly people and those with Atzheimer-type dementia, J. Neurot. Sci, 84 (1988) 141 146. Lathe, R., Synthetic oligonucleotide probes deduced l¥om amino acid sequence data --- Theoretical and practical considerations, J. Mol. Biol., 183(1985) 1 12. Liou, G.I., Fong, S.-L., Gosden, J.. Van Tuinen, P., Ledbetter, D.H., Christie, S., Rout, D., Bhattacharya, S., Cook, R.G,, Li, Y., Wang, C. and Bridges, C.D.B., Human interstitial retinol binding protein (IRBP): cloning, partial sequence, and chromosomal localization, Somat. Celt Mol. Genct., 13 (1987) 315 323. Martin, G.M., Schellenberg, GD., Wijsmam E.M. and Bird, T.D,, Dominant susceptibility genes, Nature, 347 (1990) 124. Miller, S.A., Dykes, D.D. and Polesky, H.F., A simple salting out procedure for extracting DNA from human nucleated cells, Nucl. Acids Res., 16 (1988) 1215. Nakano, i. and Hirano, A., Parkinson's disease: neuron loss in the nucleus basalis without concomitant Alzheimer's disease, Ann. Neurol., 15 (1984) 415-418. Rocchi, M., Roncuzzi, L., Santamaria, R., Archidiacono, N., Dente, L. and Romeo, G., Mapping through somatic cell hybrids and eDNA probes of protein C to chromosome 2, factor X to chromosome 13, and al-acid glycoprotein to chromosome 9, Hum. Genet., 74 (1986) 30--33. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A., Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase, Science, 239 (1988) 487-491. Salvaterra, P.M., Molecular biology and neurobiology of choline acetyltransferase, Mol. Neurobiol., 1 (1987) 247-280. Sanger, F., Nicklen, S. and Coulson, A.R., DNA sequencing with chain-terminating inhibitors, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 5463-5467. Si, J.S., Borst, D.E., Redmond, T.M. and Nickerson, J.M., Cloning of cDNAs encoding human interstitial photoreceptor retinoidbinding protein (IRBP) and comparison with bovine IRBP sequences, Gene, 80 (1989) 99 108, Tagliavini, F. and Pilleri, G., Neuronal loss in the basal nucleus of Meynert in a patient with olivopontocerebellar atrophy, Acta Neuropathol., 66 (1985) 127-133. Uhl, G.R., Hilt, D.C., Hedreen, J.C., Whitehouse, P.J. and Price, D.L., Pick's disease (lobar sclerosis): depletion of neurons in the nucleus basalis of Meynert, Neurology, 33 (1983) 147(~1473. Wbitehouse, P.J., Price, D.L., Clark, A.W., Coyle, J.T. and DeLong, M.R., Alzheimer's disease: evidence for selective loss of cholinergic neurons in the nucleus basalis, Ann. Neurol., 10 (1981) 122-126.
191
(') 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 A D O N 1 S 030439409100639K NSL 08157
Isolation and sub-chromosomal localization of a D N A fragment of the human choline acetyltransferase gene Riccardo Cervini 1, M a r i a n o Rocchi 2, Stefano D i D o n a t o I a n d G a e t a n o F i n o c c h i a r o ~ ~lstituto Nacionale Neurologico, Divisione di Biochimica e Genetica, Milan (Italy) and ZDipartimento di Genetica, Llniversita' di Bari, Bari (Italy)
(Received 11 April 1991; Revised version received 30 July 199t: Accepted 31 July 1991) Key word~v
Choline acetyltransferase; Polymerase chain reaction; Gene mapping
A DNA fragment of 219 bp was obtained by polymerase chain reaction (PCR) on human genomic DNA using two oligonucleotide mixtures derived from peptide sequences of human placenta choline acetyltransferase (CHAT) and from partially conserved amino acid sequences of rat, porcine and Drosophila CHAT. Sequence homology with porcine ChAT demonstrated that this fragment is part of the human ChAT gene. This gene was assigned to chromosome 10 by hybridization of the 219 bp DNA probe with DNA from human-hamster somatic cell hybrids, and to region 10ql 1.2 10qter by PCR experiments.
The molecular biology of choline acetyltransferase (CHAT; EC 2.3.1.6.), the enzyme synthesizing acetylcholine in the nervous system and other organs, has been the subject of considerable work in the recent years [20] and the cDNAs encoding Drosophila, pig and rat CHAT, have been cloned and sequenced [1, 2, 9]. Deficiency of ChAT and cholinergic systems has been reported in several degenerative neurological disorders. Alterations of cholinergic pathways, altered affinity for choline and the presence of antibodies inhibiting ChAT activity have been reported in Alzheimer's disease [5, 7, 12]. Selective loss of cholinergic neurons has been described in the nucleus basalis of Meynert in Alzheimer's disease [25], Pick's disease [24], Parkinson's disease [17], Down's syndrome [3] and dominantly inherited olivopontocerebellar atrophy (OPCA) [23]. ChAT activity was also reduced in the cerebral cortex of OPCA patients [11] and in spinal motor neurons of patients with amyotrophic lateral sclerosis [10]. The identification and chromosomal localization of human ChAT gene could help the understanding of the molecular basis of these diseases. Here we describe the amplification of a region of such gene by polymerase chain reaction IPCR) [19] and its mapping on the long arm of chromosome 10. Human genomic DNA was extracted from lymphoid cells [16]. Primers for PCR were synthesized on a Gene Correspondence: G. Finocchiaro, Istituto Nazionale Neurologico C.
Besta, Divisione di Biochimica e Genetica, via Celoria 11, 20133 Milan~ Italy. Fax: (39) 011-392-2664236.
Assembler Plus (Pharmacia-LKB). Primer RG3 was a pool of 64 35mers containing 3 inosine residues (indicated as x), deduced from the amino acid sequence NGYGACYNPQPE, corresponding to amino acids 568 579 of pig ChAT [1]: AA(TC)GGCTA(TC)GGCGCXTG(TC)TA(TC)AA(TC)CCXCA(AG)CCXGA. Primer G F l l was reverse complementary to DNA sequences deduced from tryptic peptide ATRPSQGHQP, obtained from human placenta ChAT [8]. This peptide corresponds to the extreme C-terminal region of pig ChAT [1]. An EcoRI site preceded by bases AC was added on the 5' to facilitate subcloning procedures. GFI1 was a mix of 128 37mers and contained 5 inosine residues: ACGAATTCGGCTG(GA)TG(GX)CCCTGX(GC)(AT)GGGXC(GT)(GX)GT(CX)GC. PCR were performed in 100/zl mix using 150 pm of these two primers, 0.6/tg of genomic DNA, 0.15 mM dNTPs, 2.25 mM MgC12, 10 mM Tris-C1 pH 8.3, 50 mM KC1, 0.001% gelatin and 5 units of T. Aquaticus DNA polymerase (Perkin-Elmer Cetus). After 4 rain of denaturation at 94°C 40 cycles of amplifications were performed. In each cycle denaturation was at 9 4 C for 1 rain, annealing of primers at 53°C for 2 min and extension at 72'~C for 30 s. Primers R I and R2 correspond to nt 26-54 and 169 199 of the fragment amplified with GFI I and RG3. Two other primers amplified 128 bp of the gene for the human interstitial retinoid-binding protein (IRBP) [22]: A T G A G A G A A T G G G T T C T G C T C A T G T C C G T G and GGTTCTCCGGGAAGCAGTAGTTATCCAAGA. DNA sequences were determined by the Sanger meth-
192
od [19] with the T7 Sequencing kit from Pharmacia after subcloning in plasmid pGEM7zf(Promega). Nucleotide and amino acid sequences were analyzed by MacMolly, a software from SoftGene, Berlin. Somatic cell hybrids were obtained as described [18]. Genomic DNA digests were transferred to Hybond-N membranes (Amersham) after agarose gel electrophoresis. PCR-amplified DNA was labeled with [c~-32P]dCTP by random primers [6]. In order to amplify a fragment of the ChAT gene from human genomic DNA we designed primers based on sequences of a tryptic peptide from human placental CHAT, corresponding to the extreme C-terminal region of porcine CHAT, and of upstream amino acids identical in rat and porcine ChAT and 67% homologous with those of Drosophila ChAT [see table in ref. 2]. The aminoacid sequence encompassed between these two primers in pig ChAT is of 73 residues, corresponding to 219 bp. PCR experiments on human genomic DNA yielded one single fragment of this size which was amplified and sequenced (Fig. IA). We compared both this nucleotide sequence and the deduced amino acid sequence with the European Molecular Biology Laboratory nucleic acid database (release 24) and the Swiss-Prot protein sequence database (release 15), respectively, and found significant homologies only with porcine ChAT cDNA and amino acid sequence. The degree of similarity at the pro-
tein level is of 76.7% (Fig. IB), clearly indicating that the PCR-amplified fragment is part of the human ChAT gene. The amino acid residues at positions 66 and 68 are serine and threonine, respectively. However, peptide 1t from human placental ChAT and porcine ChAT show in the same positions arginine and serine residues, respectively [8]. We used a mixture of degenerate oligonucleotide for PCR amplification: therefore the presence of codons AGC and ACC rather than CGC and TCC, respectively, might be the consequence of some misalignment to the original sequence and the presence of these two residues needs further control, Using the 219 bp band as a probe we also analyzed human-hamster somatic cell hybrids looking for the chromosomal localization of the ChAT gene. We analyzed a panel of HindIII digested DNA from 17 somatic cell hybrids. The results are shown in Table I and assign the gene encoding human ChAT to chromosome 10, in agreement with recent observations obtained using porcine ChAT as a probe [4]. To gain further insights on the location of the ChAT gene we also analyzed by PCR the DNA of hybrid HY.88E, which results from a translocation of 10ql 1.2 10qter on chromosome X. We observed that primers RG3 and GF 11 allowed the amplification from hamster DNA of a weak band similar to the one obtained from human DNA. Primers RI and R2 increased the specificity of the reaction, since the annealing temperature could be raised to 70°C and the expected product of 174
N AAC
G GGC
Y TAC
G GGC
A GCG
C TGC
Y TAT
N AAT
P CCG
Q CAG
P E CCG GAG
F TTT
H CAC
S AGC
C TGC
K AAA
E GAG
T ACT
S S TCT TCT
S AGC
K AAG
I AAT
D GAC
M ATG
R AGA
D GAC
L CTC
C TGC
S AGT
L CTG
L P CTG CCQ
K AAG
E GAA
K AAA
A GCC
T ACC
$ AGC
P CCC
T ACC
Q G CAG GGC
L CTT
F TTC
C TGC
I ATC
S TCT
S AGC
60
F A TTT GCA
K A AAA GCT
V GTG
E GAA
E GAA
$ AGC
L CTC
120
P T CCT ACT
E GAG
K AAG
P CCA
L TTG
A GCA
T ACA
180
H Q CAT CAG
T ACC
I ATC
$ AGC
P CCG
219
B o
hChAT pChAT
NG YGACYNPQPE II l l L l k l b l l l NG YGACYNPQPE
TILFCISSFH 11111qI[l SILFCISSFH
SCKETSSSKF AKAVEESLID ~lllql II llqLlll I GCKETSSTKF AKAVEESFIE
MRDLCSLLPP I Iii[ MKGLCSLSQS
TESKPLATKE klEIIll GMGKPLATKE
KATS PTQGHQ P l i I I II I KVTRPSQVHQ P
Fig. 1. A: DNA and deducedaminoacid sequenceof the 219 bp fragmentisolatedby polymerasechain reaction(PCR) on total humangenomic DNA with primers RG3 and GF11. B: comparisonof amino acid sequence deducedfrom the 219 bp product from PCR (hChAT) with amino acid sequenceof porcine ChAT(pChAT;aa 569~a41)[1].
193 TABLE I SYNTENY ANALYSIS FOR HUMAN ChAT Human chromosome present ( + ) or absent ( - ) . The percent concordance is calculated by dividing the number of concordant hybrids by the total number of hybrids. Hybrids
1
Positive HY.19.16T3D HY.95A1 Y.XY.8F6 Y.XY.8FT7 Negative HY.22AZA1 HY.60A HY.70B2 HY.75E1 HY.94A HY.95B HY.95S HY.137J HY. 166T4 RJ.387.51T5 RJ.387.58 Y.173.5CT3
3
4
5
4" 4-
-4"
+ 4-
4. 4.
6
7
8
4" +
+
4. +
-
9
4"
--
--
10
I1
4. 4" 4" +
4" + +
12
13
14
15
4.
+
4. +
+ . .
+
4. ~-
+
+ 4. 4.
4. 4.
4. +
4. + -
be obtained
65
from
. . 4"
+
.
+ -
+
+
-
+
71
65
59
47
.
human
+ 59
47
but not hamster
D N A . T h i s is s h o w n i n l a n e 3 a n d 7, r e s p e c t i v e l y , o f Fig.
.
X
4.
Jr + +
.
4.
4.
.
22
+
. 4.
+ 4.
21
4.
4.
.
20
+
+
4. 4.
+ +
.
+
59
19
+ .
4. +
18
4. 4.
4"
17
-
4.
+
4"
16
4.
t
59
-
Jr +
4.
%concordance
bp could
2
+
+
+ -
lOO 82
53
53
.
.
4" .
4.
+
59
53
.
.
.
.
.
.
1 234
65
47
65
65
+
+ +
--
+
4.
+
41
53
35
.
465
+
-
+ 44.
+ + + + +
+ q-
5 678
2. I n a g r e e m e n t w i t h d a t a o b t a i n e d b y S o u t h e r n h y b r i d i zation, this same product could be obtained from DNA of hybrids
HY.16T3D
and
HY.95A1
but
not
from
h y b r i d H Y . 6 0 A . I n t e r e s t i n g l y , t h e s a m e 174 b p f r a g m e n t was amplified from hybrid HY.88E thus assigning the human 10qll.2-10qter
ChAT
of chromosome
( l a n e 5 o f Fig. 2), gene to the region
10. A s i m i l a r p a t t e r n
was obtained using primers amplifying a fragment of the IRBP gene that has been localized to the proximal region of the long arm of chromosome
10 [14] ( d a t a n o t s h o w n ) .
O
I n c o n c l u s i o n , we h a v e i s o l a t e d a n d c l o n e d a D N A fragment of the human
ChAT gene using PCR on total
genomic DNA with degenerate primers. The availability of this DNA
fragment gives an important tool for clon-
ing the entire cDNA
of human
C H A T , a l l o w i n g t h e in-
v e s t i g a t i o n a t t h e m o l e c u l a r level o f c h o l i n e r g i c p a t h w a y a l t e r a t i o n s in d e g e n e r a t i v e d i s o r d e r s o f t h e n e r v o u s system and of mechanisms for the regulation of ChAT gene expression. Furthermore,
the subchromosomal
tion of the human
g e n e c a n b e o f u s e in l i n k a g e
ChAT
localiza-
studies of neurological diseases and indicates that mutations of such gene cannot cause hereditary forms of Alzheimer's disease linked with mutations on chromosome 19 o r 21 [15]. We
thank
Drs.
Nicoletta
Archidiacono,
Massimo
Fig. 2. Results of PCR with primers RI and R2 on hybrid HY.95 Al (lane 1), hybrid HY.16T3D (lane 2), human and hamster genomic DNA (lane 3 and 7, respectively), hybrid HY.88E (lane 5), and hybrid HY. 60A (lane 6). Lane 4: Mr markers (type V, Boheringer), lane 8, blank (no template), l0 20% of the reaction mixture were loaded on a 2% agarose gel and DNA stained by ethidium bromide.
[94
Pandolfo and Irma Colombo for help and suggestions. This work has been partially supported by a grant from the Muscular Dystrophy Association. 13 1 Berrard, S., Brice, A., Lottspeich, F., Braun, A., Barde, Y. and Mallet, J., eDNA cloning and complete sequence of porcine choline acetyltransferase: in vitro translation of the corresponding RNA yields an active protein, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 9280~9284. 2 Brice, A., Berrard, S., Raynaud, B., Ansieau, S., Coppola, T., Weber, M. and Mallet, J., Complete sequence of a cDNA encoding an active rat choline acetyltransferase: a tool to investigate the plasticity of cholinergic phenotype expression, J. Neurosci. Res., 23 (1989) 266-273. 3 Casanova, M.F.. Walker, L.C,, Whitehouse, P.J. and Price, D.L., Abnormalities of the nucleus basalis in Down' syndrome, Ann. Neurol., 18 (1985) 310-313. 4 Cohen-Haguenauer, O., Brice, A., Berrard, S., Van Cong, N., Mallet, J. and Frezal, J., Localization of the choline acetyltransferase (CHAT) gene to human chromosome 10, Genomics, 6 (1990) 374 378. 5 Davies, P. and Maloney, A.J.F., Selective loss of central cholinergic neurons in Alzheimer's disease, Lancet, ii (1976) 1403. 6 Feinberg, P.A. and Vogelstein, B., Addendum: a technique for radiolabeling DNA restriction endonuclease fragments to high specific activity, Anal. Biochem., 137 (1984) 266-267. 7 Fillit, H., Luine, V.N., Reisberg, B., Amador, R., McEwen, B. and Zabriskie, J.B., Studies of the specificity of antibrain antibodies in Alzheimer's disease. In J.D. Hutton and A.D. Kenny (Eds.), Senile Dementia of the Alzheimer Type, A. Liss, New York, 1985, pp. 307--318. 8 Hersh, L.B., Takane, K., Gylys, K., Moomaw, C. and Slaughter. C.. Conservation of amino acid sequences between human and porcine choline acetyltransferasc, J. Neurochem., 51 (1988) 1843-1845, 9 ltoh. N., Slemmon, J.R., Hawke, D.H., Williamson, R., Morita. E.. Itakura, K., Roberts, E., Shively, J.E., Crawford, G.D. and Salvaterra, P.M.. Cloning of Drosophila choline acetyltransferase cDNA, Proc. Natl. Acad. Sci. U.S.A., 83 (1986)4081-4085. 10 Kato. K., Choline acetyltransferase activities in single spinal motor neurons from patients with amyotrophie lateral sclerosis, J. Neurochem., 52 (1989) 636--640. I 1 Kish. S.J., Robitaille, Y., E1-Awar, M., Dock. J.H.N., Simmons, J., Schut, L., Chang, L.J., DiStefano, L. and Freedman, M., Non-Alzheimer type pattern of brain cholineacetyltransferase reduction in dominantly inherited olivopontocerebellar atrophy. Ann. Neurol., 26 (1989) 362-367. 12 Koshimura, K., Nakamura, S.. Miwa, S., Fujiwara, M. and
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