| First Author | Fowler KJ | Year | 1992 |
| Journal | Mouse Genome | Volume | 90 |
| Issue | 2 | Pages | 220-221 |
| Mgi Jnum | J:1274 | Mgi Id | MGI:49804 |
| Citation | Fowler KJ (1992) PstI allelic polymorphism in the mouse haemopoietic cell kinase gene. Mouse Genome 90(2):220-221 |
| abstractText | Full text of Mouse Genome contribution: Pst I ALLELIC POLYMORPHISM IN THE MOUSE HAEMOPOIETIC CELL KINASE GENE (Hck). Kerry J. Fowler, Melbourne Tumour Biology Branch, Ludwig Institute for Cancer Research, Australia, 3050. Introduction The haemopoietic cell kinase gene (Hck) is a member of the Src family of protein tyrosine kinases and is expressed in haemopoietic cells. Although two different immuno-reactive gene products have been described for both the single human and murine Hck genes, their biological function remains unknown (1, 2, 3). Hck has been localized to human chromosome 20q11-12 (1) and to the distal portion of chromosome 2 in the mouse. In the latter study, the pHK24 human cDNA probe identified two additional loci (provisionally named Hck-2, Hck-3) located on chromosome 4 (4). Further examination has shown that Hck-2 and Hck-3 actually correspond to two other Src family members, Lyn and Lck respectively (5; Siracusa, personal communication). In the present study, genomic blot analysis has been used to search for restriction fragment length polyrnorphisms (RFLP's) in the single murine Hck gene. Materials and Methods Genomic DNA was prepared from the tails (6) of random-bred Swiss and inbred mouse strains AKR, BALB/c, CBA/CaH and SJL/J purchased from Animal Resource Centre, Willetton, Western Australia, Australia, 6155. Genomic DNA from the inbred mouse strains 129/SvJcch/cchc/c, A/J, AKR/J, BALB/cBy, C3H/HeJ, C57L/J, DBA/2J and SWR/J was purchased from Mouse DNA Resource, The Jackson Laboratory, Bar Harbor, Maine 04609, USA. 129/SvHe mice (7) were kindly provided by the Animal House at the Ludwig Institute. 20-30ug of genomic DNA was digested to completion according to the manufacturer's instructions (Boehringer, Mannheim), electrophoresed on 0.8% agarose gels, and later transferred in 0.4M NaOH onto Biotrace HP nylon membranes (Gelman Sciences, Ann Arbor, MI 48106). The blots were incubated for 2 hr at 65 degrees C in a solution of 3xSSC, 1% SDS, 0.04% non-fat milk powder and 0.5ug/ml freshly denatured salmon sperm DNA (1xSSC contains 0.15M NaCl and 0.015M sodium citrate). The blots were then hybridized overnight with a 600 bp Bam HI-Sac I fragment corresponding to sequences of the intron between exons 4 and 5 of the murine Hck gene (8; Lock et al., unpublished observations) labeled with [alpha32p] dATP (4000 Ci/mmol, Bresatec, Adelaide) using the Bresatec Oligo-labelling system (9). Post-hybridization washes contained 1% SDS and were taken to a stringency of 0. lxSSC at 65 degrees C. Size markers are Lambda DNA digested with Hind III (New England BioLabs). Results Psi I identified two alleles with bands at 3.6kb in the A/J and DBA/2J inbred mouse strains and bands at 4.3kb in 8 different inbred strains. Genomic DNA from 9 individual random-bred Swiss mice digested with Pst I also produced the 4,3kb band (Figure 1). Figure 1 (Legend). Pst I RFLP recognized by the murine Hck probe in the A/J and DBA/2J inbred mouse strains using Southern blot analysis. The blot was auto-radiographed using Kodak XAR5 film at -70°C with 2 intensifying screens for 3 days. No RFLP's were detected using Southern blot analysis of genomic DNA digested with the following restriction enzymes: Bam HI, Bgl II, Eco RI and Hind III (Table 1). Table 1. Inbred mouse strains not polymorphic for Bam HI, Bgl II, Eco RI and Hind III. Restriction Enzyme: Bam HI; Mouse Strains Examined: AKR/J, C3H/HeJ, DBA/2J, SWR/J; Restriction Enzyme: Bgl II; Mouse Strains Examined: 129/SvHe, AKR/J, BALB/c, C3H/HeJ, C57L/J, DBA/2J, SWR/J; Restriction Enzyme: Eco RI; Mouse Strains Examined: AKR/J, C3H/HeJ, C57L/J, DBA/2J, SWR/J; Restriction Enzyme: Hind III; Mouse Strains Examined: 129/SvHe, AKR, BALB/c, C3H/HeJ, C57L/J, DBA/2J. Acknowledgements I thank M. L. Hibbs and P. Lock for providing the Hck fragment for use as a probe in these analyses. References 1. Quintrell, N., Lebo, R., Varmus, H., Bishop, J.M., Pattenati, M.J., LeBeau, M.M., Diaz, M.O. and Rowley, J.D. (1987) Mol. Cell. Biol. 7: 2267-2275. 2. Ziegler, S.F., Marth. J.D., Lewis, D.B. and Perlmutter, R.M. (1987) Mol. Cell. Biol. 7:2276-2285. 3. Holtzman, D.A., Cook. W.D. and Dunn, A.R. (1987) PNAS USA 84: 8325-8329. 4. Siracusa, L.D., Buchberg, A.M., Copeland, N.G. and Jenkins, N.A. (1989) Genetics 122:669-679. 5. Ceci, J.D., Siracusa, L.D,, Jenkins, N.A., and Copeland, N.G. (1989) Genomics 5:699-709. 6. Hogan, B., Constantini, F, and Lacy, E. (1986) In: Manipulating the Mouse Embryo, A Laboratory Manual, Cold Spring Harbor Laboratory, New York. 7. Pease, S., Braghetta, P., Gearing, D., Grail, D. and Williams, R.L. (1990) Dev. Biol, 141: 344-352. 8. Ziegler, S.F., Pleiman, C.M., and Perlmutter, R.M. (1991) Oncogene 6:283-288. 9. Felnberg, A.P. and Vogelstein, B. (1983) Anal. Biochem. 132:6-13. |
