| First Author | Dutton ER | Year | 1996 |
| Journal | Mouse Genome | Volume | 94 |
| Issue | 3 | Pages | 692-4 |
| Mgi Jnum | J:35607 | Mgi Id | MGI:83055 |
| Citation | Dutton ER, et al. (1996) The high affinity neurotensin receptor, Ntsr, is proximal to Acra4 in distal mouse Chromosome 2 and cannot be a candidate gene for Wasted. Mouse Genome 94(3):692-4 |
| abstractText | Full text of Mouse Genome contribution: THE HIGH AFFINITY NEUROTENSIN RECEPTOR, Ntsr, IS PROXIMAL TO Acra4 IN DISTAL MOUSE CHROMOSOME 2 AND CANNOT BE A CANDIDATE GENE FOR WASTED. Elizabeth R. Dutton*, Catalina Betancur+, Christine M. Williamson*, Simon T. Ball*, William Rostene and Josephine Peters*. * Mammalian Genetics Unit, Medical Research Council, Harwell, Didcot, Oxon, OX11 ORD, United Kingdom. + INSERM U. 339, Hopital Saint-Antoine, 184 Rue du Fg St. Antoine, 75012 Paris, France. Neurotensin (NT) is a tridecapeptide which acts as a neurotransmitter and neuromodulator in the central nervous system and gastrointestinal tissues (2). The pharmacological effects of NT are mediated by specific membrane receptors characterised by their binding affinity. In mouse brain two spatially distinct types have been identified, a high affinity receptor and a low affinity binding site (3). Both the rat and human high affinity NT receptor cDNAs have been cloned (11, 12) and encode proteins with seven putative transmembrane domains which share 84% identity. Recently the gene for the high affinity receptor, Ntsr, has been localised by in situ hybridisation to distal mouse Chromosome 2H (5). An autosomal recessive mutation with neurological and immunological effects, wasted, (wst) is also located in distal mouse Chr 2 closely linked, but distal, to the gene coding for the alpha subunit of the acetyl choline receptor, Acra4 (1). Changes in NT have been associated with neurodegenerative disorders linked to altered dopaminergic transmission such as Parkinson's disease (12) and Huntington's chorea (7). Moreover, neurotensin binding sites have been shown to be located primarily on cell bodies and processes of dopaminergic neurons (9). wst/wst mice develop tremors, uncoordinated body movements and paralysis similar to those observed in human motor neuron diseases. Thus, from the available mapping and phenotype data, Ntsr is a candidate gene for wst. In order to test the candidacy further, brain neurotensin binding was compared in wst/wst mice and controls, and Ntsr was mapped relative to Acra4 by linkage analysis. Brain 125I-NT binding was studied in 21 day old wst/wst, wst/+ and +/+ mice by in vitro autoradiography as previously described (3). By this age wst/wst mice could be classified with confidence. wst is very closely linked to the dominant coat texture mutant ragged, Ra, (8), and is maintained at the Mammalian Genetics Unit by intercrossing +wst/Ra+. Results (Fig. 1A) are shown for 21 day old +wst/+wst (n = 3), +wst/Ra+ (n = 3), and Ra+/Ra+ (n = 2) and ++/++ (n =2) mice of both sexes. 125I-NT binding did not differ significantly in control ++/++ an d Ra+/Ra+ (data not shown); thus the data for these two groups were pooled. As previously described in the brain of other species (3), high densities of 125I-NT binding sites were detected in the midbrain, in the ventral tegmental area and substantia nigra, pars compacta. An intense labelling was also observed in frontal and parietal cortices, as well as in the hippocampal formation. Low levels of NT receptors were present in the caudate-putamen, a region primarily involved in motor control. As shown in Fig. 1A, 125 I-NT binding did not differ significantly in wasted, heterozygotes and control mice in any of the brain regions studied. Finally, no 125I-NT labelling was observed in the cerebellum where degenerative changes have been reported in wst/wst mice (10). Ntsr was mapped by linkage analysis of an interspecific cross previously described (13). Homozygotes for the reciprocal translocation T(2;8)2Wa were crossed to Mus spretus males and Fl female progeny outcrossed to C3H/HeH males. Southern blots of parental Mus spretus and laboratory mouse genomic DNA restricted with various enzymes were probed (13) with radiolabelled rat Ntsr cDNA (prNTR2) (11). Variant restriction length fragments of 3.4 and 2.3kb in Mus spretus and 4.4. and 2.2kb in laboratory mice were detected on HincII digested DNA as well as a non variant 2.0kb fragment. The Mus spretus specific fragments were used to follow the segregation of Ntsr in F2 progeny (Fig. 1B). The F2 progeny were also typed for Acra4 and two simple sequence repeats, D2Mit73 and D2Mit74 as described in the legend to Figure 1. The most likely marker order, determined by minimising the number of crossover events required to explain the haplotypes seen, together with recombination frequencies (expressed as genetic distances in centimorgans (cM) +/- standard error) is D2Mit73 - 4.9 +/- 2.1 - Ntsr - 1.0 +/- 1.0 - D2Mit74 - 1.0 +/- 1.0 Ð Acra4. Thus, our data place Ntsr proximal to D2Mit74 and Acra4. Since wst is clearly distal to D2Mit74 and Acra4 (1), Ntsr and wst must be separate loci (Fig. 1C). This is in accord with the finding that wst is not associated with modifications in NT binding in the brain. We compared our linkage data with the composite genetic map in the Mouse Chromosome Atlas (4), but there are no other mutants in the region with a phenotype that might be predicted for a mutation in Ntsr. ACKNOWLEDGMENTS We thank Dr. S. Nakanishi (Institute of Immunology, Kyoto, Japan) and Dr. D. Caput (Sanofi Elf Biorecherches, Labege Cedex, France) for the prNTR2 cDNA, Dr. J.-L. Guenet (Institut Pasteur, Paris, France) for the pSP65 cDNA and Anne Southwell and Philip Staniland for looking after the mice. E.R.D. is the recipient of a Studentship from the MRC Human Genome Mapping Project and C.B. was supported by a fellowship from the Fondation pour la Recherche Medicale (France). REFERENCES 1. Abbott, C., Malas, S., Pilz, A., Pate, L., Ali, R. & Peters, J. (1994). Genomics 20: 94-98. 2. Kitabgi, P., Checler, F., Mazella, J. & Vincent, J.P. (1985). Rev. Basic Clin. Pharmacol. 5: 397-486, 3. Kitabgi, P., Rostene, W., Dussaillant, M., Schotte, A., Laduron, P.M. & Vincent J.P. (1987). Eur. J. Pharmacol. 140: 285-293. 4. Lyon, M.F., Cocking, Y. & Gao, X. (1996). Mouse Genome 94:29-73. 5. Laurent, P., Clerc, P., Mattei, M.G., Forgez, P., Dumont, X., Ferrara, P., Caput, D, & Rostene, W. (1994). Mamm. Genome 5: 303-306. 6. Moyse, E., Rostene, W., Vial, M., Leonard, K., Mazella, J., Kitabji, P., Vincent, J.P. & Beaudet, A. (1987). Neuroscience 22: 525-536. 7. Nemeroff, C.B., Youngblood, W.W., Manberg, P.J., Prange, A.J. & Kizer, J.S. (1983). Science 221: 972-975. 8. Peters, J., Beechey, C.V., Ball, S.T. & Evans, E.P. (1994). Genet. Res. 63: 169-174. 9. Rostene, W., Brouard, A,, Dana, C., Masou, Y,, Agid, F., Vial, M., Lhiaubet, A.M. & Pelaprat, D. (1992). Ann. NYAcad. Sci. 668: 217-223. 10. Shultz, L.D., Sweet, H.O., Davisson, M.T. & Coman, D.R. (1982). Nature 297: 402-404. 11. Tanaka, K., Masu, M, & Nakanishi, S. (1990). Neuron 4: 847-854. 12. Vita, N., Laurent, P., Lefort, S., Chalon, P., Dumont, X., Kaghad, M., Gully, D., Le Fur, G., Ferrara, P. & Caput, D. (1993). FEBS Lett 317: 139-142. 13. Williamson, C.M., Dutton, E.R., Beechey, C.V. & Peters, J. (1994). Genomics 22: 240-242. FIG. 1. (Legend) (A) Quantification of 125I-NT binding in different brain regions of wasted, heterozygous and control mice. Coronal sections (20 um thick) were incubated with 125I-NT and subjected to film auto- radiography as previously reported (6). Film autoradiograms were analysed using a computer-assisted imaging system (HISTO-RAG, Biocom, Les Ulis, France). The optical density (O.D.) was determined using 6 sections per brain level per mouse. Each bar represents mean O.D. +/- S.E.M. Fr, frontal cortex; Par, parietal cortex, Cpu, caudate-putamen: Hip, hippocampus; SN, substantia nigra; VTA, ventral tegmental area. (B) Haplotype data analysis of 101 F2 progeny from the T2Wa interspecific cross. Each column represents the chromosome inherited from the Fl parent. Black boxes represent a laboratory mouse allele, white boxes represent a Mus spretus allele. The number of F2 progeny with each haplotype is shown below the column. To type D2Mit73 and D2Mit74 100 ng DNA was amplified by PCR in a total volume of 10 ul reaction mix (13) using a Perkin Elmer GeneAmp 9600 machine. Cycling times were 3 min at 94 degrees C (1 cycle), 1 min at 94 degrees C, 1 min at 55 degrees C, 2 mins at 72 degrees C (30 cycles), 10 mins at 72 degrees C (1 cycle). Samples were separated through a 4% agarose gel in 1 x TAE and visualised using ethidium bromide (0.4 ug/ml). Acra4 was typed by Southern analysis as previously described (1). (C) Linkage map from the haplotype data above showing the position of Ntsr (left) compared with the distal portion of the map from (1) showing the position of wst in relation to the same markers (right). Distances are in centimorgans (cM). |
