| First Author | Dutton ER Williamson P | Year | 1993 |
| Journal | Mouse Genome | Volume | 91 |
| Issue | 1 | Pages | 121-23 |
| Mgi Jnum | J:4221 | Mgi Id | MGI:52717 |
| Citation | Dutton ER Williamson P, et al. (1993) D2Har1 detected by clone pG3.1 maps between Acra and Il-1b on chromosome 2. Mouse Genome 91(1):121-23 |
| abstractText | Full text of Mouse Genome contribution: D2Har1 DETECTED BY CLONE pG3.1 MAPS BETWEEN Acra AND Il-1b ON CHROMOSOME 2 E R Dutton1, P Williamsonl, A Pilz2, C M Abbott2, H Moseley1, Y Boyd1, J Peters1; 1MRC Radiobiology Unit, Chilton, Didcot, Oxon OX11 ORD; 2Department of Genetics & Biometry, University College London, Wolfson House, 4, Stephenson Way, London NW1 2HE. Introduction The clone pG3.1 is a 2.6kb murine genomic fragment cloned into the EcoRI site of the vector pAT153. The genomic insert was derived from a cosmid isolated from a library constructed from a somatic cell hybrid which retained four human Y chromosomes on a RAG (BALB/c) mouse background (1, 2), Routine analysis of this clone revealed that it detected restriction length variants (RFLVs) between different mouse strains, which would be useful in linkage analysis if the position of the locus detected by pG3.1 was established. This was investigated by hybridization to a panel of somatic cell hybrids to determine its chromosomal origin, and then by linkage analysis to map it to a specific region within that chromosome. Methods and Results 1) Somatic cell hybrid panel analysis A chromosomal assignment of the locus detected by pG3.1 was obtained through the analysis of a mouse (CBA) x Chinese hamster (V79) somatic cell hybrid panel (3). The chromosome content of the hybrid mapping panel was defined using chromosome-specific PCR reactions supplemented by cytogenetic studies and hybridization to probes of defined locations (4). Purified insert DNA was labelled by nick translation and used to probe EcoRI digests of hybrid DNA displayed on a nylon membrane (Hybond N+). Hybridization was carried out overnight at 63 degrees C and the filters were washed at 63 degrees C (0.5xSSC, 0.1 %SDS). No signal was observed in Chinese hamster control tracks, whereas a 2.6kb band was detected in the mouse control track. Hybrid DNAs were scored for the presence or absence of the 2.6kb fragment. The locus detected by pG3.1 segregated with mouse chromosome 2 and no other mouse chromosome (Table 1). It was therefore assigned to Chr 2 and given the locus symbol D2Har1. Table 1. (Legend). Assignment of D2Har1 to mouse chromosome 2. Figure 1. (Legend). Autoradiograph of RFLVs detected by D2Harl on BamHI digested DNA samples from Lane 1) heterozygous and Lane 2) homozygous backcross progeny. Figure 2. (Legend). Haplotype data for 14 loci, including D2Harl, for 126 backcross progeny. Black squares represent Mus spretus haplotypes and white squares represent AN haplotypes. a) Backcross progeny showing parental haplotype or single recombination event. b) Backcross progeny showing double recombination events. 2) Linkage analysis In order to position D2Har1 on chromosome 2 linkage analysis was performed on an interspecific backcross between laboratory mice and Mus spretus described previously (5). Briefly, a Mus spretus male was crossed to a female of the laboratory stock AN, maintained on a C3H background, and resulting Fl females were backcrossed to AN males. The D2Harl probe was hybridised to Southern blots of BamHI digested DNA samples from 126 backcross progeny previously characterised for 14 markers on Chr 2 (5, 6, 7). Standard methods and procedures were used throughout the investigation (5, 8). Hybridization was carried out overnight at 65 degrees C and then the filters were washed at high stringency (0. lxSSC, 0.1 %SDS, 65 degrees C). The D2Harl probe detected RFLVs of 9.7kb and 5.4kb in AN, and 9.1kb and 5.0kb in Mus spretus (Fig. 1). Figure 2 shows the haplotype data for the segregation of these RFLVs in the backcross progeny. Twelve recombinants were found between Acra and D2Har1, and eleven recombinants were found between D2Harl and Il-1b in 126 backcross progeny. For the locus order Acra - D2Harl - Il-1b all the offspring can be explained by single crossovers. The recombination frequencies obtained +/-standard error (%) are Acra - 9.5 +/- 2.6 - D2Har1 - 8.7 +/-2.5 - Il-1b. Comments D2Har1 will be a useful marker for the central region of Chr 2. This region is of considerable interest as it contains several genes which are important in development including Pax-6 and Wt-1. Acknowledgements Jonathan Wolfe and Peter Goodfellow for supplying the original plasmid clone; Adrian Edwards for assistance with probe characterisation; HGMP for support. References 1. Wolfe, J. et al. (1984) EMBO J. 3: 1997-2003. 2. Klebe, R.J., Chen T.R. and Ruddle F.H. (1970) J. Cell Biol. 45: 74. 3. Williamson, P. et al. (1991) Somat. Cell Mol. Genet. 17: 609-615. 4. Williamson, P. et al. manuscript in preparation. 5. Pilz, A., Moseley H., Peters J. and Abbott C. (1992a) Genomics 12: 715-719. 6. Abbott, C., Pilz A., Moseley H. and Peters J. (1992) Mammalian Genome 3(5): 286-289. 7. Pilz, A., Moseley H., Peters J. and Abbott C. (1992b) Mammalian Genome, in press. 8. Maniatis, T., Fritsch E. and Sambrook J. (1982) "Molecular Cloning: A Laboratory Manual" Cold Spring Harbour Laboratory, Cold Spring Harbour, N.Y. |
