| First Author | Jenuth JP | Year | 1993 |
| Journal | Mouse Genome | Volume | 91 |
| Issue | 4 | Pages | 873-75 |
| Mgi Jnum | J:16280 | Mgi Id | MGI:64364 |
| Citation | Jenuth JP, et al. (1993) Molecular analysis of the strain distribution pattern for purine nucleoside phosphorylase (Np) alleles in the BXD and BXH recombinant inbred strains. Mouse Genome 91(4):873-75 |
| abstractText | Full text of Mouse Genome contribution: SHORT PAPERS. MOLECULAR ANALYSIS OF THE STRAIN DISTRIBUTION PATTERN FOR PURINE NUCLEOSIDE PHOSPHORYLASE (Np) ALLELES IN THE BXD AND BXH RECOMBINANT INBRED STRAINS. Jack P. Jenuth and Floyd F. Snyder; Departments of Medical Biochemistry and Paediatrics, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada. INTRODUCTION Purine nucleoside phosphorylase has been mapped to chromosome 14 in the mouse (1). Mice of the C57 and C58 background can be distinguished from many other inbred strains for purine nucleoside phosphorylase by the presence of a second erythrocytic band of activity on starch gel electrophoresis (2), and by quantitatively greater enzyme activity (3). The segregation of the second band of activity on starch gel electrophoresis led to the designation of a second purine nucleoside phosphorylase locus, Np-2 (2), although the data was not inconsistent with there being a single locus having multiple alleles. The determinant of the NP band visible only on starch electrophoresis was mapped to chromosome 14 (4) and no recombinants have been observed between Np-1 and Np-2 (5). We have recently cloned and sequenced the cDNA for mouse purine nucleoside phosphorylase (6). Subsequent cDNA sequence analysis, genomic amplification and restriction patterns are all consistent with there being a single Np locus in the mouse having multiple alleles (7). We have proposed that the purine nucleoside phosphorylase locus classification be modified to reflect these findings with most inbred strains such as DBA/2J and C3H/HeJ being Npa and strains of the C57 and C58 background, C57BL/6J, C57L/J and C58/J, being Npb. Two other alleles were sequenced, those for M.m. molossinus, Npc, and M. spretus, Npd (7), for which protein electrophoretic polymorphs were previously described (1, 8). Recombinant inbred lines have been typed by starch gel electrophoresis for what has been referred to as the NP-2 electromorph (4, 9). We report here the molecular typing for Np of the BXD and BXH recombinant inbred strains using genomic DNA. MATERIALS AND METHODS DNA. Mouse DNA was purchased directly from the Jackson Laboratory as prepared from: DBA/2J, C57BL/6J, C3H/HeJ, the 26 BXD and 12 BXH recombinant inbred strain sets. Molecular Analysis PCR analysis was performed with two primers previously described (7). The 5' and 3' primer sequences and their location (relative to initiation of translation) are given below, each having one mismatch (underlined) creating internal Sac I sites. The PCR reaction consisted of 1.0 \ug genomic DNA, the two primers each at 3.5 pmol, 0.2 mM each dNTP, 2.5 U Taq polymerase (Boehringer Mannheim), in a buffer of 10 mM Tris (pH 8.3 at 20 degrees C) 50 mM KC1, 2.5 mM MgCl2, 0.1 mg/ml gelatine (Boehringer Mannheim). The amplification was performed by 6 min initial denaturation at 94 degrees C, followed by 35 cycles of 40 sec. at 58 degrees C, 1 min at 72 degrees C, and 40 sec at 94 degrees C. All samples were chloroform extracted, ethanol precipitated and resuspended in 20 ul of water. 5'NP: dGGTTTGGAGCTCGTTTTCCTGC; location, 461-482. 3'NP: dAGCAGCGGAGCTCTCATTGC; location, 894-874. PCR products, 5 ul, were digested in buffer B (Boehringer Mannheim) with 10 U Taq I (Boehringer Mannheim) for 1 hr at 65 degrees C. Samples were electrophoresed on a 1.5% agarose gel containing ethidium bromide. RESULTS AND DISCUSSION Within the purine nucleoside phosphorylase coding region there are five nucleotide differences between the Npa and Npb alleles (7). Only one of these substitutions results in a codon and deduced amino acid change, threonine 176 to serine. Among these differences are two silent changes at nucleotides 699 and 702 relative to initiation of translation which are for Npa, represented by DBA/2J and C3H/HeJ, CTTCGA, and for Npb, C57BL/6J CTCCGT. These substitutions eliminate a Taq I site in C57BL/6J DNA. An 800 bp product is obtained by PCR amplification of genomic DNA using the 5Õ and 3'NP primers, which flank an intron splice site at nucleotide 652 and encompass approximately 376 bp of intron. Digestion of the 800 bp amplified product with Taq I gives 195 and 605 bp digestion products as predicted for the DBA/2J and C3H/HeJ sequence, whereas C57BL/6J DNA is refractory to cleavage (Fig. 1). The strain distribution pattern for the recombinant inbred strains, BXD and BXH, as observed by molecular analysis is shown in Figure 1 and is given below. The strain distribution pattern is identical to that obtained using the starch gel electromorph (4, 9), and previously classified as Np-2. Table 1. Strain Distribution Pattern for BXD and BXH recombinant inbred strains at the Np locus. BXD STRAINS 1: B; 2: D; 5: B; 6: D; 8: D; 9: D; 11: D; 12: D; 13: B; 14: D; 15: D; 16: D; 18: D; 19: D; 20: B; 21: B; 22: B; 23: B; 24: B; 25: B; 27: B; 28: B; 29: D; 30: B; 31: D; 32: D. BXH STRAINS 2: H; 3: B; 4: H; 6: B; 7: B; 8: H; 9: B; 10: B; 11: B; 12: B; 14: H; 19: H. The recombinant inbred strains have previously been typed only for Np-2, using the starch gel electromorph (4, 9). We report here the results of a PCR-RFLP based DNA typing of the BXD and BXH recombinant inbred strains for Npa and Npb, thereby illustrating a nucleotide polymorphism which may be used conveniently in other mapping applications. Figure 1. (Legend). PCR-RFLP analysis of the BXD and BXH recombinant inbred strain sets and the progenitor strains for purine nucleoside phosphorylase. An 800 bp fragment was amplified from genomic DNA and the Taq I site present in the Npa allele, DBA/2J and C3H/HeJ, is distinguished from its absence in the Npb allele, C57BL/6J. DNA molecular-weight marker V (Boehringer Mannheim) is present in the outside lanes. ACKNOWLEDGEMENTS This work was supported by grant MT-6376 from the Medical Research Council of Canada. REFERENCES 1. Womack, J.E., Davisson, M.T., Eicher, E.M., Kendall, D.A. (1977) Biochem. Genet. 15, 347-355. 2. Bremner, T.A., Premkumar, R., Nayar, K., Kouri, E. (1978) Biochem. Genet. 16, 1143-1151. 3. Snyder, F.F., Biddle, F.G., Lukey, T., Sparling, M.J. (1983) Biochem. Genet. 21, 323-332. 4. Taylor, B.A. (1981) Mouse News Letter 65, 28. 5. Lukey, T., Neote, K., Loman, J.F., Unger, A.E., Biddle, F.G., Snyder, F.F. (1985) Biochem. Genet. 23, 347-356. 6. Jenuth. J.P. and Snyder, F.F. (1991) Nucleic Acids Res. 19, 1708. 7. Jenuth, J.P., Mangat, R.K., Snyder, F.F. (1993) Mammalian Genome (in press). 8. Mably, E.R., Carter-Edwards, T., Biddle, F.G., Snyder, F.F. (1988) Comp. Biochem. Physiol. 89B, 427-431. 9. Dembic, Z., Bannwarth, W., Taylor, B.A., Steinmetz, M. (1985) Nature 314, 271-273. |
