| First Author | Reed V | Year | 1993 |
| Journal | Mouse Genome | Volume | 91 |
| Issue | 2 | Pages | 331-33 |
| Mgi Jnum | J:12800 | Mgi Id | MGI:61018 |
| Citation | Reed V, et al. (1993) Molecular analysis of a series of mutations at the mottled locus. Mouse Genome 91(2):331-33 |
| abstractText | Full text of Mouse Genome contribution: Molecular Analysis of a Series of Mutations at the Mottled Locus. Vivienne Reed, Andrew M. George, Carol Rasberry, Bruce M. Cattanach, Mary F. Lyon and Yvonne Boyd. Genetics Division, MRC Radiobiology Unit, Chilton, Didcot, Oxon OX11 ORD, England. Mottled mutants (Mo), which are presumed to be allelic mutations of a single X-linked locus, are reported frequently in the mouse. Affected mice can exhibit a range of phenotypic features such as defective pigmentation, wavy vibrissae and coat, tremor and incoordination, skeletal abnormalities and defective collagen and elastin (1). The phenotypic effects are due to abnormal activities of many copper dependent enzymes caused by an underlying defect in copper transport or copper binding (2, 3). Different Mo mutations have arisen both spontaneously and after mutagen treatment; some mutations cause death of male hemizygotes and female homozygotes whilst others permit viability and fertility in the hemizygous and homozygous states. Female heterozygotes are easy to identify as they have patches of light pigmentation on the coat, owing to the presence of cells which carry the mutation on an active X chromosome. Whilst the molecular lesions of the spontaneous mutation events are impossible to predict, mutations recovered after radiation treatment are frequently deletions (4, 5). Mo lies close to the locus for phosphoglycerate kinase (Pgk-1) as no recombination events have been reported between Mo and Pgk-1 (6, 7). In man, Menkes' syndrome (MNK) shares several characteristics with mottled and, as it maps to the equivalent conserved segment, is likely to be homologous to the murine defect. MNK and Mo lie in the conserved X chromosomal segment which runs from AR to COL4A5 in both man and mouse (8). MNK has recently been positioned immediately proximal to PGK1 on the human X chromosome (9, 10). If the local order of loci is the same in man and mouse, as current data suggest, Mo will lie in the Xist à Pgk-1 interval of the mouse X chromosome. We report here the analysis of six male mottled mutants for the presence of the three loci (Xist, DXNds3 and Pgk-1) closest to the mottled locus. Males were available from two radiation-induced and four spontaneous mutants (Moblo, Movbr, Mobr, Mo13H) with varying phenotypes and lethalities (Table 1). DNA was prepared from live-born males (around 10 days old) except for Modp, where males carrying the mutation die around 17 days in utero (11). A Modp male was identified at 17 days in utero by genital inspection and the presence of curly whiskers. In all cases sex was subsequently confirmed by demonstrating that the Y-linked gene (Ube1y1) was present using the polymerase chain reaction (PCR). A human cDNA probe for Xist, 14A3 (12), was labelled with 32P-dCTP by nick translation and hybridized to HindIII digests of male mottled DNA and suitable controls using methods described previously (13). PCR was performed on the same samples using primers for exon 1 of Pgk-1 (14) and for the DXNds3(Mintd) locus (15). Primers were used at a concentration of 0.5 uM in a 25 ul reaction in the presence of 0.25 uM dNTPs, 10 mM MgCl2 and 1 unit of Taq polymerase. The reactions were carried out in a Perkin-Elmer thermal cycler programmed for 35 cycles of (94 degrees c 1 min; 55 degrees c 30 secs) with an initial denaturation period of 10 min at 94 degrees C. The 6.5 kb Xist murine genomic fragment and the 170 bp and 111 bp amplification products for Pgk-1 and DXNds3 respectively were present in all six mutants (Fig. 1). Table 1 Details of mottled mutants used in this study Allele: dappled; Symbol: Modp; Origin: gamma low dose; Hemizygote phenotype: Die in utero, Skeletal defects; Reference: 11. Allele: brindled; Symbol: Mobr; Origin: spontaneous; Hemizygote phenotype: Die at 14 days; Reference: 16. Allele: 10H; Symbol: Mo10H; Origin: 6Gy X-ray; Hemizygote phenotype: Die at 14 days; Reference: 17. Allele: 13H; Symbol: Mo13H; Origin: spontaneous; Hemizygote phenotype: Die at 14 days; Reference: Lyon, unpublished. Allele: viable brindled; Symbol: Movbr; Origin: spontaneous; Hemizygote phenotype: Reduced viability, sterile; Reference: 18. Allele: blotchy; Symbol: Moblo; Origin: spontaneous; Hemizygote phenotype: Reduced viability, often fertile; Reference: 19. Figure 1. (Legend). Xist, DXNds3 and Pgk-1 are present in six mottled mutants. (top) Hybridization of Xist to HindIII digested DNA of mottled mutants and controls, the mouse and human restriction fragments detected by the probe are arrowed and the approximate sizes indicated (middle) PCR amplification of genomic DNA using primers which flank the first exon of Pgk-1, the 111 bp amplification product is arrowed. (bottom) PCR amplification of genomic DNA using primers which amplify the DXNds3 locus, the 170 bp amplification product is arrowed. Track 1, Moblo; 2, Mobr; 3, Modp; 4, Movbr; 5, Mo10H; 6, Mo13H; 7, Ta26H; 8, 3H1; 9, clone 8.0; 10, H2O replacement for template DNA as a control for PCR reactions. Details of mutants and loci are given in Table 1 and in the text respectively. Ta25H and 3H1 are control murine DNAs (20), clone 8.0 is a somatic cell hybrid which retains a portion of the X chromosome (21, 22). We have shown that three loci thought to lie close to Mo have not been deleted in two radiation- induced and 4 spontaneous mouse mutants, although it is not known whether any of these represent the null phenotype. Assuming that the relative order of loci around Pgk-1 is the same in man and mouse, if any deletions are present they must be contained within the interval which separates Xist from Pgk-1, for which the accepted genetic distance is ~2cM (8). The estimated distance between PGKI and the chromosomal breakpoints associated with MNK in man is around 200kb. This separation distance is consistent with the observation that Mo and Pgk-1 have never been reported to recombine in the mouse. The absence of large deletions (>200kb - 2Mb) in this region may be because (a) these are not compatible with survival in heterozygous females due to lethality caused by loss of the mottled locus itself or other loci in the Xist-Pgk-1 interval (b) large deletions are not a common class of mutation at Mo and examination of additional male lethal and radiation-induced mutations is required (c) the mottled phenotype is caused by a mutation in a different locus from that implicated in Menkes' syndrome and lies on the distal side of Pgk-1 (d) the local order of loci is different in man and mouse. The recent cloning of the human Menkes' locus (23-25) will enable us to examine the last two possibilities and to search for the presence of small deletions within this locus. In conclusion, an initial molecular genetic analysis of a graded series of mottled mutations has shown that three loci known to map within ~2cM of the Mo locus are present in six mutants, including two radiation-induced mutations (Mo10H and Modp) one of which is associated with male lethality (Modp). Acknowledgements We thank H. Brown for invaluable assistance in the maintenance of mouse stocks, P.H. Glenister for recovering frozen mutant stocks; A. Ballabio for supplying human XIST probes; E. Evans and K. Glover for photographic assistance. References 1. Lyon, M.F. and Searle, A.G,: GSVLM, Oxford University Press, 1989. 2. Hunt, D.M.: Nature 249: 852-854, 1974. 3, Danks, D.M. in Scriver (ed.); Metabolic Basis of Disease, 1251-1267, McGraw-Hill. 4. Rinchik, E.M. and Russell, L.B. in Davies (ed.); Genome Analysis 1, 121-158, IRL press. 5. Cattanach, B.M. et al.: Nature Ganetics, in press. 6. Nielsen, J.T. and Chapman, V.M.: Genetics 87:319-325, 1977. 7. 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