Mapping the human acetylcholinesterase gene to chromosome 7q22 by fluorescent in situ hybridization coupled with selective PCR amplification from a somatic hybrid cell panel and chromosome-sorted DNA libraries

Mapping the Human Acetylcholinesterase Gene to Chromosome 7q22 by Fluorescent in Situ Hybridization Coupled with Selective PCRAmplification from a Somatic Hybrid Cell Panel and Chromosome-Sorted DNA Libraries GAL EHRLICH, * EVANI WEGAs-PEQuwoT, t DALIA GINZBERG,* LILIAN SINDEL, + HERMONA SOREQ, *A AND HAIM ZAKUT+’ *Department of Biological Chemistry, The Life Sciences Institute, The Hebrew University of Jerusalem, Israel; tLaboratoire de Cytogenetique Humaine et Comparee, Hopital Necker-Enfants-Malades, Paris 15eme, France; and *Department of Obstetrics and Gynecology, The Edith Wolfson Medical Center, The Sackler Faculty of Medicine, Tel Aviv University, Holon, Israel Received

November

1, 1991,

To establish the chromosomal location of the human ACHE gene encoding the acetylcholine hydrolyzing enzyme acetylcholinesterase (ACHE, acetylcholine acetylhydrolase, E.C. 3.1.1.7), a human-specific polymerase chain reaction (PCR) procedure that supports the selective amplification of ACHE DNA fragments from human genomic DNA was employed with 19 humanhamster somatic cell hybrids carrying one or more human chromosomes. Informative ACHE-specific PCR fragments were produced from two cell lines, both of which include human chromosome 7, but not with DNA from 17 cell hybrids carrying various combinations of all human chromosomes other than 7. Fluorescent in S~~ZJ hybridization of biotinylated ACHE DNA with metaphase chromosomes from human peripheral blood lymphocytes revealed prominent labeling on the 7q22 position. Therefore, further tests were performed to confirm the chromosome 7 location. DNA samples from the two cell lines including chromosome 7 and the ACHE gene were positive with PCR primers informative for the human cystic fibrosis CFTR gene, known to reside at the 7q31.1 position, but negative for the ACHE-related butyrylcholinesterase (BCHE, acylcholine acylhydrolase, E.C. 3.1.1.8) gene, mapped at the 3q26-ter position, confirming that these lines contain chromosome 7 but not chromosome 3. In contrast, three other cell lines including chromosome 3, but not 7, were BCHE-positive and ACHE-negative. In addition, genomic DNA from a sorted chromosome 7 library supported the production of ACHEbut not BCHE-specific PCR products, whereas with DNA from a sorted chromosome 3 library, the BCHE but not the ACHE fragment was amplified. These findings assign the human ACHE gene to a single locus on chromosome 7q22 and should assist in establishing linkage between the in vivo amplification of the ACHE gene in ovarian tumors and leukemias and the phenomenon of tumor-related breakage in the long arm of chromosome 7. @ ISS!J Academic

PT~SS, Inc.

osss-7543/92 $5.00 Copyright @ 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

revised

January

INTRODUCTION

The acetylcholine hydrolyzing enzyme acetylcholine&erase (acetylcholine acetylhydrolase, E.C. 3.1.1.7, ACHE) has attracted scientific attention because of its function in terminating neurotransmission at cholinergic synapses and neuromuscular junctions (Soreq and Zakut, 1990). This is the target protein for a variety of neurotoxic compounds, including natural poisons, common agricultural insecticides, pharmacotherapeutic compounds, and chemical warfare agents (Taylor, 1990). Defects in the expression of the ACHE gene were reported in several neurodegenerative disorders, including Alzheimer disease (Navaratnam et aZ., 1991). In addition to brain and muscle, ACHE is expressed in multiple germ, embryonic, and tumor cells (Rakonczay and Brimijoin, 1988), where its function remains to be elucidated, and is secreted in large quantities into the amniotic fluid in cases of neural tube closure defects (Bonham and Atack, 1983). Early genetic linkage studies suggested the existence of allelic variants at a single locus for the human ACHE gene (Coates and Simpson, 1972). Molecular cloning of human ACHE cDNA later revealed that it is highly enriched (64%) in G-C residues (Soreq et al., 1990). Thus ACHE differs from the BCHE gene encoding the related protein butyrylcholinesterase (acylcholine acylhydrolase, E.C. 3.1.1.8), which is rich in A-T residues and was mapped to the 3q26-ter position (Gnatt et al., 1990). Blot hybridizations demonstrated that the ACHE and the BCHE genes are coamplified in leukemias and platelet disorders (Lapidot-Lifson et aZ., 1989; Zakut et al., 1992) and in ovarian adenocarcinomas (Zakut et al., 1990). Yet, cosmid cloning studies indicated that the ACHE and BCHE genes are physically unliked to each

1 To whom 1192

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correspondence

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be addressed.

ACHE

GENE

MAPPING

other (Gnatt er ul., 1991). The establishment of a specific chromosomal locus for the ACHE gene should, therefore, assist in further studies on the functioning of this gene in normal and diseased tissues. To this end, we employed direct polymerase chain reaction (PCR) amplification from somatic hybrid cell lines and chromosome-sorted libraries to map the ACHE gene to chromosome 7. Refinement of this analysis by chromosomal in situ hybridization with biotinylated ACHE DNA provided a single assignment for the ACHE gene at the 7q22 position.

MATERIALS

AND

METHODS

Oligodeoxynucleotide primers. Human-specific oligodeoxynucleotide primer pairs from the ACHE and BCHE genes were computer designed to avoid secondary structure interactions (Rychlik and Rhoads, 1989). Primer pairs from the ACHE gene included (1) P340(+) [5’-GCTTTCCTGGGCATCCCCTTTGCGGAGCCA-3’1 and P790(-) [5’-CAGGGCCAGCCTCTGATCCAGGAGACCCAG3’1; (2) Pl241(+) [5’-TGAAGGATGAGGGCTCGTATTTTCTGGTTT-3’] and Pl695(-) [5’-GTTGGCCCAGTATCGCATCAGTCGC-3’1; (3) Pl522(+) [5’-CGGGTCTACGCCTACGTCTTTGAACACCGTGCTTC-3’1 and Pl695(-); (4) Pl712(+) [5’-ATCCCAATGAGCCCCGAGACCCCAA-3’1 and Pl869(-) [5’-GAGCAATTTGGGGAGGAAGCGGTTCCAG-3’1. For the BCHE gene, we used P271(+) [5’-CTTGGTAGACTTCGATTCAAAAAGCCACAGTCT-3’1 and Pl588(-) [5’-ATTTTGCAAAATTTGCCCACCGTTTCACTATGGA-3’1. Primers were marked as (+) (downstream) or (-) (upstream) according to their 5’to 3’orientations; numbers indicate the position of 5’-end residues in these primers within the relevant cDNA sequences (Soreq et al., 1990, for ACHE, Prody et ul., 1987, for BCHE). Distinct 451-, 455-, 174-, 158-, and 1318-bp fragments were derived by these primers from the ACHE and BCHE genes, respectively, using the PCR at differential annealing temperatures (72OC for ACHE and 65’C for BCHE) that were experimentally determined. PCR primers for the human cystic fibrosis CFTR gene (201-3 and 201~$ Kerem et al., 1990), kindly provided by Dr. B. Kerem, were used as described. Sekctive PCR amplifiation from somatic cell hybrid DNA. DNA samples (50-100 ng) from 19 human-hamster somatic cell hybrids (Bios. Corp., New Haven, CT, see Gnatt et al., 1991 for details) and total human or hamster genomic DNA were subjected to PCR amplification with the human-specific ACHE and BCHE primer pairs. Cycling conditions were denaturation at 94OC for 30 s (first cycle 2 min); annealing at 72°C (for ACHE) and 65OC (for BCHE), 1.5 min; elongation at 72”C, 1 min (last cycle 6.5 min). Fifty cycles were performed in the presence of 2.5 units of Taq DNA polymerase (Boehringer-Mannheim, Germany) added prior to the 1st cycle and following the 25th cycle, 50 pmol of each primer, 200 PM of each deoxynucleoside triphosphate (Pharmacia, Sweden), 1.5 mA4 MgCls, 50 n&f KCl, 10 n&f Tris-HCl (pH 8.3 at 23’C), 0.01% gelatin in a final volume of 100 ~1, and 100 ~1 paraffin oil to avoid evaporation, all in 0.6-ml microtubes using the Programmable Automated Thermal Controller (MJ Research, Inc., Boston, MA). PCR amplification products (10%) were analyzed by agarose gel electrophoresis and compared with known size markers, and their identities were verified by hybridization to the relevant probes and direct DNA sequencing. Use of chrowsome-sorted Genomic ments 57751, MD), BCHE quences enzymes

libraries for direct PCR amplifiation.

DNA libraries (Deaven et ul., 1986), each composed of fragfrom sorted human chromosome 7 or 3 (Catalog Nos. 57722 and respectively, American Type Culture Collection, Rockville, were used for direct PCR amplification with the ACHE and primer pairs. In both cases, we ensured that the genomic seto be amplified would not include the restriction sites for the employed in the construction and cloning of these libraries

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(Hi&II and EcoRI for chromosomes 3 and 7, respectively). Phages (1 x I@ PFU in 1 ~1 SM (Sambrook et al., 1989)) were lysed in 50 ~1 double-distilled water by heating to 70°C for 5 min, followed by two treatments of freezing in liquid nitrogen and then thawing. The resultant lysates were directly employed for PCR amplification and analysis as detailed above. Hybridization probes and conditions. Following gel electrophoresis, amplification products were hybridized as detailed elsewhere (Soreq et al., 1990) in a hybridization oven (HyBaid, Teddington, Middlesex, England) with a 2.2-kb-long fragment of ACHE DNA (clone GNACHE Soreq et al., 1990) or a 2.4-kb-long BCHE cDNA (Gnatt et al., 1991).

Fluorescent in situ hybridizution. FISH to spread mitotic chromosomes was performed using cesium-purified biotinylated ACHE DNA probes and fluorescence-intensified digital imaging microscopy, as previously detailed (Viegas-Pequignot et al., 1989). The probes employed were electrophoretically separated and electroeluted 2.2- and 5.5-kblong ACHE DNA inserts (Soreq et al., 1990). RESULTS

Chromosome Mapping by Direct PCR Amplification Direct PCR amplification with somatic cell hybrids and chromosome-sorted libraries was performed under species-specific conditions, so that human but not hamster DNA gave rise to four different PCR fragments informative for the ACHE gene and another one for the BCHE gene. DNA blot hybridization was employed to exclude the possibility of weak or invisible fragments. Figure 1 presents an example of such an experiment. ACHE-specific PCR products appeared with DNA from 2 of the 19 cell lines employe& 1006 and 683, both of which contain human chromosome 7. In contrast, no signal was observed with DNA from any of the other lines containing any human chromosome but 7 (Fig. 2A). Table 1 summarizes the results of this search in the human-hamster somatic hybrid cell lines, presenting a discordance value for chromosome 7 equal to zero. The validity of the PCR chromosomal assignment was examined by reconfirming our previous mapping of the BCHE gene to chromosome 3, which was performed by DNA blot hybridization using the same somatic hybrid cell panel (Gnatt et al, 1991). Figure 2B presents an ethidium bromide-stained agarose gel in which the 13X3bp-long BCHE-specific PCR product was generated from DNA from cell lines 423 and 507, which contain human chromosome 3, and cell line 860, in which only 15% of the cells carry this chromosome. The identity of the PCR fragments was verified by hybridization using [32P]ACHE DNA and [32P]BCHE cDNA probes (not shown). Regional Mapping on Chromosomes 7 by in Situ Hybridization Regional mapping of the ACHE gene on chromosomes 7 was achieved by fluorescent chromosomal in situ hybridization using a biotinylated ACHE DNA fragment as a probe (Fig. 3). Fifteen metaphases were analyzed, all of which carried at least one fluorescent spot on 7q22 (five with one spot, seven with two spots and, three with three spots). Two of these cells were also labeled by one spot at the 11~11 position, one with one spot on 12ql3, and another with one on 13ql4. These minor labelings

EHRLICH

ET

AL,

some 7, the CFTR gene, mapped to the chromosomal 7q31.1 position (Kerem et al, 1989), was employed as a positive control. Oligodeoxynucleotide primers for the human CFTR gene were employed in PCR reactions with human and hamster genomic DNA or with DNA A. ACHE, primers: 1241(+), 1695(-) I 5’ 3!* P340(+) I 03

I 0

P790(-) -3, 5~

I I.0 Length,

I 1.5 Kb

I 2.0

I 2.5

BCHE 455 bp

B. BCHE, primers: 271(+), 1588(-)

i - 13 1X bp

I s 3’ + I’27 I (+I

Pl588(-) -3, 5,

1318 bp

FIG. 1. Direct PCR amplification of human-specific ACHE and BCHE genomic DNA sequences. Amplified PCR products from the ACHE and BCHE genes, 451 and 1318 bp long, respectively, were obtained using the p340(+) and p790(-) and p271(+) and pl588(-) human-specific primer pairs for the ACHE and BCHE genes, respectively (see Materials and Methods). The location of each primer within the two human genes is schematically presented according to its exon-intron structure (see Soreq et al., 1990, and Taylor, 1991, for details of the ACHE gene, and Gnatt et ul., 1991, and Arpagaus et al., 1990, for details on the BCHE gene). P, primer; E, exon; numbers are noted from 5’ to 3’. Introns are presented as triangles. P(A), polyadenylation site; N and C denote the ends of the open reading frames encoding the amino and carboxy termini of the mature proteins. The active site serine is represented by a dot. The annealing temperature is noted for each gene. The length of exon-DNA regions in kilobases is shown in the calibration scale.

were considered insignificant in view of the negative PCR results with somatic cell hybrids carrying chromosomes 11, 12, and 13, using four different ACHE-specific primer pairs. Additional in situ hybridization experiments, using a 5.5kb genomic probe from the ACHE gene (Soreq et al, 1990, clone GNACHE), also revealed labeling on chromosome 7 (not shown). Verification

of the Chromosome 7’ Assignment

To ensure beyond doubt that both of the somatic cell lines that included ACHE DNA indeed carry chromo-

FIG. 2. Screening of human-hamster somatic hybrid cell lines for human ACHE and BCHE DNA sequences by direct PCR amplification. (A) Association of the ACHE gene with chromosomes 7. DNA samples from human-hamster somatic cell hybrids containing one or more human chromosomes on a background of hamster chromosomes, and DNA from human or hamster origins for control, were subjected to PCR amplification with the human ACHE-specific primers. DNA fragments of the expected lengths, which hybridized with human ACHE DNA, were observed only in lanes loaded with PCR products generated from human genomic DNA or cell hybrids containing human chromosomes 7 as confirmed by positive PCR amplification of CFTR fragments (lines 1006 and 683, dotted above), but not in lanes loaded with PCR products generated from hamster genomic DNA and/or DNA from cell lines that do not carry these human chromosomes (undotted lines: 803,734,909,854,811,967,507,423 and 860). For chromosomal content of cell lines see Table 1. C, PCR reaction with no template DNA, to test for lack of contaminating amplifiable DNA in other reaction components; M, DNA size marker (marker VI, Boehringer-Mannheim). (B) Reexamination of BCHE gene mapping onto chromosome 3. DNA from human-hamster cell hybrids was PCR-amplified using the human BCHE-specific primers. A 1318-bplong PCR product, stained with ethidium bromide, appeared where total human DNA or hybrid cell lines containing human chromosome 3 were examined (lines 423,507, and 860, dotted above), but not from hamster genomic DNA and/or lines without human chromosome 3 (undotted lines 734,967,811,909,803,1006,854, and867). Gel electrophoresis, DNA staining and photography were as detailed under Materials and Methods. For chromosomal content of cell lines see Table 1.

ACHE

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TABLE Discordance

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ACHE

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7

Chromosome Cell line

ACHE gene

324 423 734 750 803 860 867 940 507 683 811 983 909 854 967 968 1006 1049 1099 %Discordance

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2

3

4

5

6

7

8

9

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18

19

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+ 15% 60% + @

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55% + 26

15%

+

+

@I

+

+ 45%

32

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+ + 40%

+ +

+ +

+

+

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+

+

+ +

+

+ i + + + 68 26

+

+

t

+ +

+ +

@+ 0

+ +

+

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+

D

21

+

+

15%

+

+ +

+

32 26

26

10

16

+

+

42 32 26 21 21 36

36

26

+

+

26

16

21 21

Note. DNA samples from 19 hamster-human hybrid cell lines containing various combinations of human chromosomes were examined by direct PCR amplification for their content of human ACHE-specific DNA sequences as detailed in Figure 2A. Chromosomes present in each hybrid cell line are noted by (+) symbols. Chromosome 7-associated signals are circled. D, deleted %, percentage of cells in the hybrid cell lines that contain the noted human chromosome. The nercentage of findings discordant with the association of human ACHE-specific PCR fragment with each chromosome is given below.

from the same somatic hybrid cell lines employed in Fig. 2. PCR reactions using the CFTR primers with DNA from lines 1006 and 683, which contain human chromosome 7, gave rise to the human-specific CFTR product, whereas DNA from line 860, which contains human chromosome 3, did not (data not shown), thus strengthening the assignment of the human ACHE gene to chromosome 7. An independent assignment of the ACHE gene to chromosome 7 was obtained by direct PCR amplification using DNA from chromosome-sorted libraries. Lysed lambda phages containing fragments from sorted chromosome 7 or chromosome 3 (Deaven et uZ., 1986) libraries supported the appearance of the ACHE-specific PCR product from the human chromosome 7 library, but not from the chromosome 3 library. In contrast, the human BCHE-specific fragment was produced from chromosome 3 library phages, but not from chromosome 7 phages. Both of these PCR products hybridized with the relevant probes (not shown), confirming their identity as human ACHE- and BCHE-derived sequences; the hamster DNA impurity in the chromosome 7 library did not interfere with the PCR reaction because of the species-specific conditions that were employed.

DISCUSSION

Mapping of the human ACHE gene to a defined chromosomal location was performed in three steps. The

first phase of this study consisted of direct PCR amplification of human ACHE-specific DNA fragments from somatic cell hybrid DNAs and chromosome-sorted libraries. Reexamination by this technique of the chromosomal location of the BCHE gene, which we previously mapped to chromosome 3q26-ter (Gnatt et aZ., 1990), demonstrated the reliability and sensitivity of direct PCR amplification compared with blot hybridizations of somatic cell hybrid DNAs. To circumvent technical difficulties resulting from the high G-C content in the ACHE gene (Soreq et oz., 1990), a particularly high annealing temperature (72’C) was employed in the PCR procedure. This prevented the formation of nonspecific PCR fragments, which at 65’C also occurred with hamster DNA, but displayed no hybridization with the human ACHE DNA probe (Ehrlich et al., unpublished observations). Using the ACHE-specific primers, DNA from two different cell lines and one chromosome-sorted library supported PCR amplification of the ACHE fragment, as tested by blot hybridization. The element common to these sources was that they all contained DNA from human chromosome 7. In contrast, 17 cell lines and one chromosome-sorted library devoid of chromosome 7 gave negative signals. Regional mapping of the ACHE gene within chromosomes 7 was thereafter achieved by fluorescent in situ hybridization with biotinylated ACHE DNA. Double fluorescent spots were observed at the 7q22 location. Moreover, the two cell lines positive for chromosome 7 also carried the CFTR gene, known to reside on chromsome 7q31.1. Together, these data rein-

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mammalian species and that the gene duplication event that led to their appearance might have occurred before the evolution of mammals (Taylor, 1991), The different composition of nucleotide sequences in the BCHE compared with those in the ACHE gene (Soreq et aZ., 1990) further indicates that these two genes are evolutionarily relatively distant from each other, and the mapping results presented in this paper showing that the ACHE and BCHE genes are located on separate chromosomes strengthen this assumption. The ACHE gene is amplified in leukemias (LapidotLifson et aZ., 1989), ovarian carcinomas (Zakut et r~l., 1990), and platelet disorders (Zakut et aZ., 1992). The long arm of chromosome 7 tends to break in cancerous cells (Mitelman, 1988). Further studies will be required to determine any putative correlation(s) between these events and the ACHE gene. It is interesting to note that the AT-rich BCHE gene, which presents almost a mirror image of the properties of the ACHE gene, is likewise subject to frequent amplifications and resides on the 3q26-ter chromosomal fragment, breakable in thrombopoietic disorders (Pedersen, 1990), a phenomenon that awaits explanation. FIG. 3. Refinement of ACHE gene mapping to 7q22 by fluorescent in situ hyhridization with biotinylated ACHE cDNA. In s&u hybridization was performed in metaphase cells obtained from lymphocyte cultures of normal donors. The probe employed was a 2.2-kh-long recombinant ACHE DNA (Soreq et ul., 1990) nick-translated by biotiny1 dUTP (Bio-Rad Laboratories) according to Boehringer-Mannheim protocols and as previously described (Viegas-Pequignot et al., 1989). Detection was by indirect immunofluorescence. The first antibody was an anti-biotin and the second a fluorescein conjugate. Chromosomes were counterstained by propidium iodide. Arrows indicate fluorescent spots located on 7q22 over the R-banded chromosomes.

ACKNOWLEDGMENTS We are grateful to Dr. B. Kerem, Jerusalem, for the CFTR primers. This research was supported by the U.S. Army Medical Research and Development Command (Grant DAMDl7-90-Z-0038), the U.S. Israel Binational Science Foundation (Grant 89-0205), the Association Francaise Contre le Myopathies (AFM) (to H.S. and H.Z.), and the E.W.H. British Research Trust, London (to H.Z.). G.E. is a GoldaMeir Predoctoral Fellow.

REFERENCES

forced the assignment of the ACHE gene to chromosome 7q22. Our findings imply that a single chromosomal site harbors ACHE coding sequences in the human genome, corroborating previous genetic predictions (Coates and Simpson, 1972). In addition, the data presented in this report confirm and extend our previous observations that the ACHE and BCHE genes, which encode two closely related cholinesterase proteins, are not genetically linked to each other in the human genome (Gnatt et ul., 1991). The similar but not identical exon-intron organization in these two human genes (Soreq and Zakut, 1990; Taylor, 1991) implies that they arose during evolution by gene duplication. This is evident from the presence of both ACHE and BCHE in early species such as Torpedo murmoruta (Toutant and Massoulie, 1985), although in insects, a single gene encodes one cholinesterase protein with mixed ACHE/BCHE properties (see Hall and Malcolm, 1991, for a recent example). All mammals studied so far have distinct ACHE and BCHE genes, with each of the homologous amino acid sequences within these cholinesterases being highly conserved (Soreq and Zakut, 1990). This suggests that the two cholinesterases are both physiologically required in

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