Somatic Cell and Molecular Genetics, Vol. 21, No. 2, 1995, pp. 147-150

Brief Communication

Identification of the Human Neuronal Nicotinic Cholinergic oz2 Receptor Locus, (CHRNA2), within an 8p21 Mapped Locus, by Sequence Homology with Rat DNA

Stephen Wood, I Michae l Schertzer , a and M. Lisa Y a r e m k o 2

IDepartment of Medical Genetics, University of British Columbia, Vancouver, Canada; and 2Department of Pathology, University of Chicago, Chicago, Illinois

Received 25 January 1995--Final 7 April 1995

Abstrac t - -We have identified a cosmid, at the D8S131 locus, that shows sequence homology with exon 2 of the rat gene for the neuronal nicotinic acetylcholine receptor a2 subunit. A 357-bp sequence surrounding a rare cutter AscI site contains a 152-bp region of homology. The human CHRNA2 gene is therefore positioned at the D8S131 locus, which has been mapped to 8p21.

INTRODUCTION MATERIALS A N D M E T H O D S

Neuronal nicotinic acetylcholine recep- tors are a family of acetylcholine responsive cation channels that are thought to consist of pentamers of two acetylcholine binding cx subunits and three structural 13 subunits (1). The genes for the a2 (CHRNA2) and 133 (CHRNB3) subunits have been assigned to human chromosome 8 using rat cDNAs to probe a cell hybrid panel (2).

We have found that sequence flanking a rare cutter AscI (GGCGCGCC) site in a cosmid from the flow-sorted chromosome 8 cosmid library, LA08NC01 (3), is homolo- gous with exon 2 of the rat et2 gene. This identification of the human homolog posi- tions the CHRNA2 gene at the D8S131 locus, which has been mapped to 8p using cell hybrids and linkage analysis (4) and to 8p21 using FISH (5).

Cosmid Identification and Analysis. The flow-sorted chromosome 8 library, LA08NC01, has been described (3). Cosmid 132C2 was identified by hybridizing the Y19-1D probe, which detects a BamHI polymorphism at the D8S131 locus (6), against the arrayed cosmid library. Two dinucleotide repeat SSRs (simple sequence repeats) were identified within cosmid 132C2 by hybridization with poly-GT. PCR primers were developed for both of these polymor- phic loci. Cosmid 132C2 was found to contain a single rare cutter AscI (GGCGCGCC) site by restriction digestion. The sequence adja- cent to the single AscI site was obtained by subcloning EcoRI-AscI fragments in pNEB193 (New England Biolabs). Dideoxy- nucleotide sequencing reactions on double- stranded templates were carried out using

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148 Wood et al.

vector primers and a Sequenase protocol (7). The GenBank data base was searched for these cosmid sequences using the BLAST algorithm (8). Fluorescence in situ hybridiza- tion was performed, using cosmid 132C2 DNA, as previously described (9).

RESULTS

Sequence Analysis of Cosmid 132C2. Sequencing through the AscI site of both EcoRI-AscI subclones produced 357 bp of sequence surrounding the AscI site (Gen- Bank Ul1384). The BLAST search detected

homology with a rat gene for the nicotinic, cholinergic or2 receptor locus. Figure I shows a comparison of the D8S131 sequence with that for exon 2 of the rat locus (10) and the putative translation of these sequences. From a comparison of 49 amino acids, there were six differences observed. This corre- sponds to 87.8% sequence homology for this region. In addition, 16 silent nucleotide replacements at codon position 3 were noted. This degree of divergence is typical between rodent and human gene loci. The invariant ag dinucleotide corresponding to the rat acceptor splice junction is not conserved at

D8S131:

Rat:

D8S131:

Rat:

L P Q G G S H agtcccacgg CA TTG CCG CAG GGA GGC TCG CAT 188

llll I l l Ill i 111 11 II tatcccacag TG TTG ACC CAG CAA GGC TCA CAC 15

L T Q Q G S H

T E T E D R L F K H ACC GAG ACT GAG GAC CGG CTC TTC AAA CAC 218

L1 II I III I[ II I11 Ill III ACC CAT GCT GAG GAC CGC CTG TTC AAA CAC 45 T H A E D R L F K H

D8S131:

Rat:

L F R G Y R W A R CTC TTC CGG GGA TAC AAN CGC TGG GCG CGC 248

I I [ I LII II 111 III il II CTG TTT GGA GGC TAC AAT CGC TGG GCA CGG 75 L F G G Y N R W A R

D8S131:

Rat:

P V P N T S D V V I CCG GTG CCC AAC ACT TCA GAC GTG GTG ATT 278

I I I III I [II I[ II III II II CCA GTG CCC AAC ACT TCT GAT GTG GTC ATC 105 P V P N T S D V V I

D8S131:

Rat:

V R F G L S I A Q L GTG CGC TTT GGA CTG.TCC ATC GCT CAG CTC 308

II [ I III I I Ill 11 Ill Ill Ill GTG CGC TTT GGA TTA TCC ATT GCT CAG CTC 135 V R F G L S I A Q L

S D V D8S131: AGC GAT GTG gt 319

I [ Ill Rat: ATA GAT GTG gt 146

I D V

Fig. 1. Comparison of DSS131 sequence with rat neuronal nicotinic acetylcholine receptor a2 gene, exon 2. D8S131 sequence was obtained from the region flanking the sole Asc l site in cosmid LA08NC01-132C2. The rat sequence is numbered starting with the first nucleotide in the codon corresponding to the proposed amino terminus of the mature protein (10). The Ascl site in the human sequence is at nucleotides 242-249 (shown in bold).

CHRNA2 Maps to 8p21 149

the human locus. It is replaced by gg, which raises the possibility that a pseudogene may have been detected. However, the upstream human ag dinucleotide is preceded by 12 pyrimidines, raising the possibility that the human and rat loci have diverged by exon sliding.

Mapping of D8S131 (CHRNA2). The D8S131 locus has previously been mapped to 8p21 using the Y19 probe for fluorescent in situ hybridization (FISH) (5). Cosmid 132C2, isolated using the Y19 probe, was verified to map to this location using FISH (Fig. 2).

DISCUSSION

The AscI restriction endonuclease has a recognition sequence of GGCGCGCC, mak- ing it a rare cutter that is expected to fre- quently lie in GC-rich HTF islands, character- istic of the upstream region of many genes

(11). Screening cosmids that are already mapped for rare cutter sites can therefore provide an effective method for mapping genes. In common with most human chromo- somes, the number of anonymous DNA markers mapped to chromosome 8 far exceeds the number of mapped genes, while it is the genes that are the targets for in- herited disease. The few hundred base pairs of sequence flanking a rare cutter site that are easily obtained far exceed the amount of sequence required to obtain a "hi t" in sequence data bases. As more expressed sequences (ESTs) become available for data- base searching, we anticipate this approach will yield an increasing frequency of hits. The end result of this strategy is the combination of gene locus and map position that is the critical information required for evaluating candidate genes for inherited disease or candidate diseases for identified genes.

Fig. 2. Chromosomal localization of the human CHRNA2 gene by fluorescence in situ hybridization. A metaphase spread shows hybridization with cosmid LA08NC01-132C2 DNA to 8p21 and a chromosome 8 a-satellite probe.

150 Wood et al.

The nicotinic, cholinergic a2 receptor (CHRNA2) has previously been assigned to chromosome 8 by hybridization of a rat cDNA to human-hamster somatic cell hy- brids (2). Our detection of this locus by sequence homology has refined this place- ment to 8p21. The nicotinic, cholinergic B3 receptor (CHRNB3) has also been assigned to chromosome 8 using somatic cell hybrids (2). Recently using an exon trapping strategy to identify expressed sequences from cos- mids that had been mapped by FISH, the CHRNB3 locus has been localized to 8p12 (12). These two loci thus have different loca- tions on chromosome 8. Do the CHRNA2 and CHRNB3 loci have any likely disease candi- dates? Considering the function of the pro- ducts of these loci, it would seem that genetic defects might Iead to neurological disease.

ACKNOWLEDGMENTS

This work was supported by the Cana- dian Genome Analysis and Technology Pro- gram (CGAT) grant GO-12753.

LITERATURE CITED

1. Cooper, E., Couturier, S., and Ballivet, M. (1991). Nature 350:235-238.

2. Anand, R., and Lindstrom, J. (1992). Genomics 13:962-967.

3. Wood, S., Schertzer, M., Drabkin, H., Patterson, D., Longmire, J.L., and Deaven, L i . (1992). Cytogenet. Cell Genet. $9:243-247.

4. Tomfohrde, J., Wood, S., Schertzer, M., Wagner, M.J., Wells, D., Parrish, J., Sadler, L.A., Blanton. S.H., Daiger, S.P., Wang, Z., Wilkie, P.J., and Weber, J.L. (1992). Genomics 14:144-152.

5. Weber, B., Allen, L., Magenis, R.E'., Goodfellow, P.J., Smith, L., and Hayden, M.R. (1991). Mam- mal. Gen. 1:211-216.

6. Weber, B., Reiss, O., Kreklywich, C.N., Wood, S., and Hayden, M.R. (1991). Nucleic Acids Res. 19:1725.

7. Seto, D. (1990). Nucleic Acids Res~ 18:5905-5906. 8. Altschul, S.F., Gish, W., Miller, W., Myers, E.W.,

and Lipman, D.J. (1990). J. Mol. Biol. 215:403410. 9. Wood, S., Yarerako, M.L., Schertzer, M., Kele-

men, P.R., Minna, J., and Westbrook, C.A. (1994). Genomics 24:597-600.

10. Wada, K., Ballivet, M., Boulter, J., Connofly, J., Wada, E., Deneris, E.S., Swanson, L.W., Heine- mann, S., and Patrick, J. (1988). Science 240:330- 334.

11. Lindsay, S., and Bird, A.P. (1987). Nature 327:336- 338.

12. Koyama, K., Sudo, K., and Nakamura, Y. (1994). Genomics 21:460~61.