| First Author | Blair H | Year | 1992 |
| Journal | Mouse Genome | Volume | 90 |
| Issue | 4 | Pages | 672-74 |
| Mgi Jnum | J:3461 | Mgi Id | MGI:51974 |
| Citation | Blair H, et al. (1992) Detailed characterization of the X chromosome content of the somatic cell hybrid, "Clone 8.0". Mouse Genome 90(4):672-74 |
| abstractText | Full text of Mouse Genome contribution: DETAILED CHARACTERIZATION OF THE X CHROMOSOME CONTENT OF THE SOMATIC CELL HYBRID, "CLONE 8.0" Helen Blair, Vivienne Reed, Andrew George, Steven Laval, Sheila Holt, Yvonne Boyd*. Genetics Division, MRC Radiobiology Unit, Chilton, Didcot, Oxon OX11 ORD. * Author to whom correspondance should be addressed Introduction The somatic cell hybrid, clone 8.0 (previously known as clone 8 e.p.), was recovered after a fusion between the murine L-cell derived line, clone 1D, and a derivative of the human cell line D98 (Herman et al. 1991). Clone 8.0 retains material from only the mouse X chromosome on a human background. This has made it valuable as a mapping reagent for the assignment of loci to the mouse X chromosome (Amar et al. 1985) and as reagent for the generation of X-specific probes (Brockdorff et al. 1990; Herman et al. 1991). Seven loci from the mouse X chromosome have been shown to be present in clone 8.0; however, several pseudoautosomal sequences from the telomeric end of the mouse X chromosome along with two juxtacentromeric loci appear to be deleted (Amar et al. 1985; Herman et al, 1991). Thus, clone 8.0 appears to contain a large portion of the mouse X chromosome with the proximal and distal ends of the chromosome missing; a conclusion which is supported by the ease at which probes for the murine X chromosome have been isolated from the hybrid (Brockdorff et al. 1990; Herman et al. 1991 ). However, cytogenetic analysis revealed that the mouse X chromosome present in clone 8.0 did not possess a standard banding pattern which suggested that the chromosome had been rearranged during the generation of the hybrid (Herman et at. 1991). Therefore the possibility remains that sizeable interstitial deletions could have occurred which would not have been detected by previous analyses. In order to further characterize the X chromosome content of this hybrid, we have analyzed for the presence or absence of an additional sixteen X chromosome loci of defined location, either by Southern blotting, or by amplification of locus-specific products using the polymerase chain reaction (PCR). Table 1. Analysis of the mouse X chromosome content of clone 8.0. Locus: DXF34h*; +/-: -; Probe name/PCR: pH3-7; Reference: Level & Boyd. 1992. Locus: Otc; +/-: -; Probe name/PCR: (a); Reference: Herman et al. 1991. Locus: Maoa; +/-: -; Probe name/PCR: 2.2a; Reference: Laval et al. 1991. Locus: Timp; +/-: +; Probe name/PCR: Timp; Reference: Jackson et al. 1987. Locus: DXPas3; +/-: +; Probe name/PCR: (a); Reference: Amar et al. 1985. Locus: Hprt; +/-: +; Probe name/PCR: pHPT5; Reference: Konecki et al. 1982. Locus: Cdr; +/-: +; Probe name/PCR: pCDR-9; Reference: Hirst et al. 1991. Locus: DXS296; +/-: +; Probe name/PCR: VK21C; Reference: Suthers et al. 1989. Locus: DXS52h; +/-: +; Probe name/PCR: St14; Reference: Oberle et al. 1987. Locus: Gabra3; +/-: +; Probe name/PCR: pGabra; Reference: Levitan et al. 1988. Locus: Gdx; +/-: +; Probe name/PCR: (a); Reference: Herman et al. 1990. Locus: DXNds3; +/-: +; Probe name/PCR: PCR; Reference: Todd et al. 1991. Locus: DXS393h; +/-: +; Probe name/PCR: pE1-174; Reference: Reed et al. in preparation Locus: Zfx; +/-: +; Probe name/PCR: CMPXY1; Reference: Affara at al. 1989. Locus: Xist; +/-: +; Probe name/PCR: 8A11; Reference: Brown et al. 1991. Locus: Pgk- 1; +/-: +; Probe name/PCR: PCR; Reference: Brown et al. 1992. Locus: DXPas2; +/-: +; Probe name/PCR: (a); Reference: Amar et al. 1985. Locus: Plp; +/-: +; Probe name/PCR: rPlp; Reference: Milner et al. 1985. Locus: DXPas1; +/-: +; Probe name/PCR: (a); Reference: Amar et al. 1985. Locus: Pdha- 1; +/-: -; Probe name/PCR: pPDH1; Reference: Brown et al. 1990. Locus: Amg; +/-: -; Probe name/PCR: pAmg; Reference: Snead et al. 1985. Notes: The approximate locations of probes are depicted in Figure 1. (a) DXPas1, DXPas2, PXPas3 and Gdx were shown to be present in clone 8.0 by Amar et al. 1985 and Herman et al. (1991). *The probe pH3-7 detects a family of genetic elements in man and mouse at least of one of which co-segregates with DXWas70 in the mouse; four of these elements were absent in DNA prepared from clone 8.0. Methods Probes were labelled by nick-translation or random priming and hybridized to EcoRI or TaqI digested DNA displayed on Hybond N + filters (Amersham International) as previously described (Lava1 et al. 1991). Further details concerning the probes used in this study can be obtained from the references given in Table 1. All probes detected restriction fragments in the mouse genome which could be clearly distinguished from those in human DNA and thus the presence/absence of the murine locus in clone 8.0 could be established. Pgk-1 and DXNds3 were detected using PCR in standard conditions with annealing temperatures of 55 degrees C (Brown et al. 1992; Todd et al. 1991). Results and Discussion Analysis of DNA prepared with clone 8.0 and parental human and mouse controls revealed that murine X-linked loci which lay proximal to Timp were absent, those which lay between, and including, Timp and Plp were present, and those distal to Plp were absent (Table 1; Figure 1). Combining our results with those of Herman et al. 1991, we can conclude that the mouse X chromosome present in clone 8.0 contains two breakpoints, one in ~1.0cM interval between Maoa and Timp and the other between DXPas1 and Pdha-1. Furthermore this information suggests that Pdha1 lies distal to DXPas1, which is consistent with its localisation by in situ hybridization (Brown et al. 1990) and our information on genetic linkage (Blair et al., in preparation). Figure 1 (Legend). Region of the mouse X chromosome present in clone 8.0. Right: Approximate localizations of the loci relevant to this study are indicated on the probe map of the mouse X chromosome (Brown et al. 1992). Centre: The ideogram of the mouse X chromosome with lines indicating those loci whose position has been determined on the cytogenetic map by in situ hybridisation (Evans, 1989; Lyon & Kirby, 1992). Left: The extent of the X chromosome material present in clone 8.0. Although our data and that of others suggest that the portion of the mouse X chromosome contained within clone 8.0 is intact, we cannot exclude the possibility that very small interstitial deletions are present within the material contained in the hybrid. However the definition of the regions absent in the hybrid make it of use in mapping loci known to lie in the proximal and distal regions of the mouse X chromosome. For example, we are able to place Pdha-1 distal to DXPasl from our mapping studies and to suggest that at least four of the repeat elements which compose the DXF34h locus lie proximal to Timp (see also Laval & Boyd, 1992). The information presented here also permits further refinement of loci detected by clones which have been derived from clone 8.0. For example, Brockdorff et al. (1990) mapped 73 linking clones obtained from clone 8 to one of four different intervals using a panel of mouse x hamster somatic cell hybrids which retained X; autosome translocation chromosomes. Thirty-six of these clones were mapped to the interval between the T16H breakpoint and the centromere; our data indicate that these loci must lie in the smaller interval between the Maoa locus and the T16H breakpoint. Similarly, the 10 linking clones which map to the distal region of the X chromosome most probably lie between the T6R1 breakpoint and the Pdha-1 locus which has been mapped to XF3-Xter by in situ hybridization (Brown et al. 1989) and between Plp and Amg in an interspecific backcross (Blair et al. in preparation). Conclusion The mouse X chromosome content of the somatic cell hybrid, clone 8.0, has been characterized in detail. The hybrid appears to contain an intact portion of the X chromosome from a point somewhere between the Maoa and Timp loci to a point somewhere between DXPas1 and Pdha-1 as depicted in Figure 1. Acknowledgements We are grateful to Dr. Ian Craig for supplying the hybrid clone 8.0; all queries concerning the availability should be made to Dr. Craig at the Genetics Laboratory. Department of Biochemistry, South Parks Road, Oxford OX1 3QU, UK. We also thank N. Affare, A. Ballabio, Z. Chan, H-H. Dahl, K. Davies, A. Doherty, M. Lyon, L. Mitchell for supplying probes. This work was supported in part by a grant from the MRC HGMP. References Affara et al. Cytogen. Cell Gen. 51:948, 1989. Amar et al. Embo J. 4:3695-3700, 1985. Brockdorff et al. Genomics 7: 573-578, 1990. Brown et al. Somat. Cell Mol. Genet. 16: 487-492, 1990. Brown et al. Mammalian Genome, in press, 1992. Brown et al. Nature 349: 38-44, 1991. Evans, in Lyon & Searle, GVSLM, Oxford University Press. Herman et al. Genomics 10: 961-970, 1991. Hirst et al. Am. J. Med. Genet. 38: 354-356, 1991. Jackson et al. NAR 15: 4357, 1987. Konecki et al. NAR 10: 6763-6775, 1982. Laval et al. Genomics 10: 1030-1034, 1991. Laval & Boyd Genomics: in press, 1992. Levitan et al. Nature 335: 76-79, 1988. Lyon & Kirby The Mouse Chromosome Atlas, 1992. Milner et al. Cell 42: 931-939, 1985. Oberle et al. Hum. Genet. 77: 60-65, 1987. Snead et al. Biochem. Biophys. Res. Commun. 129: 812-819, 1985. Suthers et al. Science 246: 1298-1300. Todd et al. Nature 351: 542-547, 1991. |
