| First Author | Malas S | Year | 1995 |
| Journal | Mouse Genome | Volume | 93 |
| Issue | 2 | Pages | 433-5 |
| Mgi Jnum | J:26240 | Mgi Id | MGI:73919 |
| Citation | Malas S, et al. (1995) PCR Markers associated with the mouse nicotinic-acetylcholine-receptor-subunit genes alpha-4, alpha-7 and beta-4 on mouse Chromosomes 2, 7, and 9. Mouse Genome 93(2):433-5 |
| abstractText | Full text of Mouse Genome contribution: PCR MARKERS ASSOCIATED WITH THE MOUSE NICOTINIC- ACETYLCHOLINE-RECEPTOR-SUBUNIT GENES alpha4, alpha7 AND Beta4, ON MOUSE CHROMOSOMES 2, 7, AND 9. Stavros Malas and Olga Forero; University College London, Department of Genetics and Biometry, The Galton Laboratory, Wolfson House, 4, Stephenson Way, London, NW1 2HE, UK. INTRODUCTION Nicotinic acetylcholine receptors (nAchRs) are expressed both at the neuromuscular junction and in the central nervous system (reviewed in 1 & 2). The receptor from the neuromuscular junction consists of four homologous transmembrane subunits, designated alpha, Beta, gamma, (or Epsilon in the adult of some species) and delta, which assemble to form a pentamer of the type (alphal) 2Beta1gamma-delta (reviewed in 3). The neuronal receptor subunits share homology with the muscle ones and are of two types, alpha and Beta. It is also thought to be a pentamer of the type alpha2Beta3 (2). Eight alpha (alpha2-alpha9) and three beta (Beta2-Beta4) subunit cDNAs have been isolated in the rat (2, 4). Six of these genes (alpha2-alpha5, Beta2 and Beta4) have been assigned to mouse chromosomes 2. 3, 9 and 14 (5, 6). These genes have not as yet been associated with any neurological disorders in mice although in humans, a nonsense mutation in the alpha4 subunit gene has been associated with benign neonatal familial convulsions (7). The use of these genes as molecular genetic markers would be enhanced if specific PCR assays became available for mapping in crosses segregating for neurological mutations. Here we report the mapping of the alpha7 subunit gene to mouse chromosome 7 (MMU7) and three additional PCR markers, two of which are associated with the alpha4 and Beta4 subunit genes on mouse chromosomes 2 (MMU2) and 9 (MMU9), respectively. MATERIALS AND METHODS Backcross analysis and PCR conditions: DNAs from two interspecific backcrosses were obtained from the Genetic Mapping Resource of the Jackson Laboratory (Bar Harbor, ME). These were of the type: (C57BL/6J x Mus spretus)Fl x [C57BL/6J (BSB panel 1)] or [Mus spretus (BSS panel 2)] and each consisted of 94 N2 progeny. The typings for D2Mit25, D7Bir8, D7Bir15, D7Mit8, D9Mit2, Xmv16 and Xmv15 were obtained from the Jackson Laboratory. The primer sequences for the new loci are given in table 1; The PCR conditions are as for D2Ucl1 (9; see table 1 for annealing temp. and [Mg2+] ). The PCR conditions for D2Mit74 were as described (10).The gene order was determined by minimising the number of double recombination events. Table 1. Primer sequences and PCR conditions for loci mapped in this study. Locus: D2Uc129; Primer sequences: 5'-GACGAGAAGAACCAGATGAT-3Õ 5'-ATGTCAGGCCTCCAGATGAG-3'; PCR product size (bp)*: 214; Annealing Temp; [Mg2+]: 55 degrees C; 2 mM; Variation$: B6 > Sprp. Locus: Acra7; Primer sequences: 5'-ATGAAGAGGCCCGGAGAGGA-3Õ 5'-AGTTGGGGCACAGTGCATGC-3'; PCR product size (bp)*: 168; Annealing Temp; [Mg2+]: 57 degrees C; 1.5 mM; Variation$: B6 = Sprp. Locus: D7Uc11; Primer sequences: 5'-AGGTCTGTCATCTGAGGAAC-3Õ 5'-TGTAAACTGCCATGTGCTGG-3'; PCR product size (bp)*: 370; Annealing Temp; [Mg2+]: 59 degrees C; 2.5 mM; Variation$: B6 > Spra. Locus: D9Uc11; Primer sequences: 5'-CCTGAGCTGCTTCTTGTCAT-3Õ 5'-TCTGTTCTCGCTCATTCTGG-3'; PCR product size (bp)*: 203; Annealing Temp; [Mg2+]: 59 degrees C; 1.5 mM; Variation$: Spr > B6p. * determined through analysis in 8% native polyacrylamide gel and refer to C57BL/6J. $ allelic variants can be resolved by 2% agarose gels (a) or 8% polyacrylamide gels (p). Acra7 was typed by single strand conformation analysis as described (11). B6 - C57BL/6J, Spr - Mus spretus. The primers for D7Ucl1 were designed from MUS spretus sequence. Bacteriophage lambda library screening. The genomic bacteriophage lambda library was plated and screened as a primary library using bacterial strain LE392 (Promega). The transfer onto nylon membrane (Hybond N) was as described (12) and the membranes were processed according to the supplier's protocols (Amersham). The hybridisation and washes were as described (13). The probe was a rat cDNA corresponding to the alpha4 - 1 transcript of the rat nAchR (14), a gift from Dr. Jean-Louis Guenet. RESULTS PCR primers were designed to correspond to the 5' end of the rat alpha4 gene in an attempt to develop a specific assay for the corresponding mouse gene locus, Acra4, on distal MMU2. The expected PCR product size was 139 bp. The PCR product sizes of the C57BL/6J and Mus spretus parents were c. 218 and 214 bp, respectively. This locus was found to cosegregate in 94 animals with the Acra4 gene, previously mapped in a backcross segregating wasted (wst), by Southern blot analysis (see ref 9). It is also present in a 17 kb bacteriophage lambda clone which cross-hybridises with the alpha4 subunit cDNA. Therefore, it is possible that this locus defines the mouse alpha4 subunit gene and the amplified region could span a small intron of about 70-80 bp. Using the BSB backcross we mapped this locus to distal MMU2, denoted D2Ucl29 (fig la). A mouse bacteriophage lambda genomic library was partially screened using the rat cDNA of the alpha4 subunit gene, as part of a wider strategy to isolate genomic clones from the region of the mouse Acra4 gene, (SM; unpublished data). Eighteen clones (denoted CDA1-CDA18) were isolated. We anticipated that some of these clones would correspond to other related members of this gene family. Clone CDA6 did not contain D2Ucl29. A small region from this clone was sequenced, and a 65 bp domain showed 97% homology to exon 3 of the rat Beta4-subunit gene (fig 1b; 15). We postulate that this region could define part of the mouse Beta4 subunit gene. Primers were designed to amplify a 203 bp fragment. We followed the inheritance of this locus in the BSS backcross progeny (figure 1), and assigned it to MMU9, denoted as D9Ucl1 (fig 1a). The human homologue of alpha7, CHRNA7, was recently mapped to human chromosome 15q14 (16). We designed oligonucleotide primers to correspond to the 3' of the rat gene (17). A PCR product of the expected size, 168 bp, was obtained from both C57BL/6J, Mus spretus and rat DNA. We applied single-strand conformation analysis to follow the inheritance of this locus in the BSB backcross progeny which we assigned to MMU7, denoted as Acra7. D7Ucl1 is a microsatellite locus which defines a trinucleotide repeat of the type (GAG)2GAA(GAG)7 (SM; unpublished). DISCUSSION This study provides simple PCR assays associated with the mouse Acra4 (D2Ucl29) and Acrab4 (D9Ucl1) genes, on MMU2 and MMU9, and assigns the mouse homologue of the alpha7 nicotinic-acetylcholine- receptor-subunit gene to MMU7 (Acra7). The rat genes for the alpha3, alpha5 and Beta4 are clustered within a 68 kb segment (15) and their human homologues map to chromosome 15q24 (6). The mouse genes were mapped to MMU9 by somatic cell genetics and linkage analysis (Acrab4 was mapped only in somatic cell hybrids; 5, 6). D9Ucl1 maps 7.1 cM proximal to Xmv15. Acra5 also maps proximal to Xmv15 by about 5 cM (18). Therefore, it is likely that Acra5 and D9Ucl1 are closely linked. If Acra3, Acra5, and Acrab4 are physically linked as their rat counterparts are, D9Ucl1 could be a useful marker for all three genes. Acra7 has been assigned to MMU7, mapping distal to ldh3 (see ref 8) and close to D7H15S12 and D7H15S9h1, whose human homologous sequences map to chromosome 15q11-q12 (19). Therefore Acra7, whose human homologue maps to 15q14, and the latter two loci establish a new syntenic group between chromosome 15q11-q14 and MMU7. The orientation of this region on MMU7 is yet undefined until Acra7 is mapped relative to D7H15S12 and D7H15S9h1. The alpha7 subunits are thought to associate and form a homo-oligomeric functional receptor (17). The expression pattern of alpha7 in the adult rat brain correlates with the distribution of a-bungarotoxin-binding (a-BTX) sites (17). Alpha-BTX blocks oligomerisation of alpha7 subunits and is therefore thought to be a component of rat brain a-BTX-binding proteins (17). The regions which express the alpha7 gene include the organs of the limbic system, such as the hypothalamus and hippocampus (17). Strains of mice with high sensitivity to nicotine-induced seizures exhibit high densities of a-BTX-binding sites in the hippocampus (20). So far, no mouse mutant genes which are associated with epilepsy, or other neurological abnormalities, have been mapped to the region of the Acra7 gene. Figure 1. (a) Linkage maps for MMU2, MMU7, and MMU9 showing the positions of D2Ucl29, D7Ucl1, Acra7 and D9Ucl1 (bold type; maps not in scale). The backcross used is given in brackets. The distances are expressed in centiMorgans +/- standard error. The number of recombinant chromosomes for each pair of markers is: Chr 2: D2Mit25 Ð1/94 - D2Ucl1 - 2/94 - D2Mit29, D2Mit74; Chr 7: D7Bir8 - 1/84 - Acra7 Ð 20/84 - D7Bir15 - 5/84 - D7Ucl1- 8/93 - D7Mit8; Chr 9: D9Mit2 - 2/94 - Xmv16 - 3/94 - D9Ucl1 - 3/42 - Xmv15. This order postulates two double recombination events between D7Bir15 and D7Mit8. (b) The sequence which defines locus D9Ucl1. The primer sequences are underlined. The 65 bp sequence (given in bold type and defined by l), is 97% homologous to the rat Beta4 subunit gene (15). Within this domain the sequences from the two species differ at two bases (positions 253 and 267 in fig 1b). The bases from the rat sequence at these two positions is given below the corresponding mouse bases. Acknowledgments We are grateful to Lucy Rowe and Mary Barter for the analysis of the backcross phenotypings and to Drs John Gubbay and Robin Lovell-Badge for the mouse genomic library. We also thank Cathy Abbott for valuable comments on the manuscript, and Lois Maltais for advice on nomenclature. This work was supported by a Wellcome Trust Prize award to S.M. References 1. Claudio, T. (1989) in Molecular Neurobiology, ed. D.M. Glover, B.D. Hames, pp. 63-142. Oxford: Oxford Univ. Press. 2. Sargent, P.B. (1993) Annu. Rev. Neurosci 16: 403-444. 3. Galzi, J.-L. et al., (1991) Annu. Rev. Pharmacol 31: 37-72. 4. Elgoyhen, A.B. et al., (1994) Cell 79: 705-715. 5. Bessis, A. et al., (1990) FEBS 1: 48-52. 6. Eng C.M, et al., (1991) Genomics 9: 278-282. 7. 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