| First Author | Irving NG | Year | 1991 |
| Journal | Mouse Genome | Volume | 89 |
| Issue | 4 | Pages | 854-856 |
| Mgi Jnum | J:2353 | Mgi Id | MGI:50877 |
| Citation | Irving NG, et al. (1991) A mouse chromosome 16 inter-repeat PCR product from C57BL/10 and Mus spretus forms a heteroduplex in interspecific backcross progeny. Mouse Genome 89(4):854-856 |
| abstractText | Full text of Mouse Genome contribution: A MOUSE CHROMOSOME 16 INTER-REPEAT PCR PRODUCT FROM C57BL/10 AND Mus spretus FORMS A HETERODUPLEX IN INTERSPECIFIC BACKCROSS PROGENY. N.G. Irving and S.D.M. Brown; Department of Biochemistry and Molecular Genetics, St Mary's Hospital Medical School, London W2 1 PG, UK. Introduction The formation of human heteroduplex PCR products that can be distinguished by their altered migration in agarose gels has frequently been reported (Nagamine et al., 1989). In some cases this has enabled the detection of single base changes (Keen et al., 1991). We have previously reported the generation of mouse chromosome specific markers from Chinese hamster/mouse somatic cell hybrids using inter-repeat PCR between mouse Ll elements (Irving and Brown, 1991). Using Chinese hamster/ mouse somatic cell hybrids and genetic linkage analysis two inter-repeat PCR markers of 580 bp and 250 bp have been localized and mapped to specific regions of mouse chromosome 16. Hybridisation of the isolated 250bp marker to inter-Ll repeat PCR products from C57BL/6 and C57BL/10 detected a major hybridising band at 250bp and a weakly hybridizing band at 180bp. Hybridisation to Mus spretus inter-Ll repeat PCR products revealed a major hybridising band at 240bp and a weakly hydridising band at 180bp. The 240bp Mus spretus band was difficult to resolve from the 250bp C57BL band after blotting and hybridisation, even on 3% agarose gels. However analysis of interspecific backcross progeny from the cross (C57BL/10 x M. spretus ) x C57BL/10 revealed an additional hybridising band of 280bp, in 50% of the progeny, which appeared to segregate as a Mus spretus specific allele on MMU16. Only the 250bp and 180bp C57BL bands were detected in homozygous mice. In contrast heterozygous mice displayed the 250bp C57BL band, the 240bp Mus spretus band, the weakly hybridsing 180bp band and an additional band at 280 bp. Similar results were observed upon analysis of progeny from an interspecific backcross (C57BL/6/M spretus) x M. spretus backcrossed to Mus spretus. In this case the extra 280bp hybridising band appeared to segregate as a Mus domesticus specific allele, mapping to MMU16. The formation of the 280bp heterozygote specific band enabled the marker to be rapidly mapped through a panel of 41 mice from the cross (C57BL/10/M. spretus) x C57BL/10 and 27 mice from the interspecific cross (C57BL/6/M. spretus) x M. spretus, backcrossed to Mus spretus. Genetic linkage analysis indicated that this inter repeat PCR marker mapped between the loci Dl6Smh6 and the D21S16h, App, Pgk1-ps1 cluster on mouse chromosome 16 (Irving and Brown, 1991). The heterozygote specific 280bp hybridising PCR product could also be generated from an equimolar mixture of C57BL/10 and Mus spretus DNA (Irving and Brown, 1991). This would suggest that this novel band was produced by the formation of a heteroduplex between the C57BL/10 and Mus spretus products. An alternative explanation is that small differences in sequence organisation, for example a frameshift of sequences, allow incomplete parental PCR products to cross hybridise with one or more overhanging end, which could then be filled-in by the polymerase to generate the longer heterozygote specific product. The sequence of the C57BL/10 PCR product has previously been determined (Irving and Brown, 1991). In order to determine the reasons for the formation of the heterozygote specific band we have isolated and seauenced the Mus spretus PCR product. In addition we have demonstrated the generation of this novel band by reannealling a mixture of the isolated parental PCR products in the absence of amplification. These data lead us to the conclusion that the novel 280bp heterozygote specific PCR product is in fact a heteroduplex of the divergent C57BL/10 and Mus spretus PCR products. Results The novel 280bp PCR product is a heteroduplex. The 250bp C57BL/10 and the 240bp Mus spretus PCR products were isolated from agarose gels. The two products were mixed in approximately equimolar amounts in PCR reaction buffer. No primers, dNTPs or polymerase were added. After four cycles of the original thermocycler program the products were run on a 3% agarose gel along with the parental PCR products that had been treated in the same way. The novel band migrating at 280bp was only generated from the mixed PCR products, see Figure 1. Sequence determination. The Mus spretus 240bp PCR product was subcloned into T-tailed bluescript as previously described and sequenced from the vector T3 and T7 primers (Irving and Brown, 1991). Two clones in opposite orientations were sequenced on both strands, the full 236bp sequence is presented in Figure 2. As in the case of the homologous C57BL/10 sequence, the inter-repeat region was found to be A+T rich (72.4%) and contained numerous short mononucleotide repeats. An alignment of the two sequences was computed and is presented in Figure 2. The ends of the Ll element consensus sequence are provided in the figure for comparison. Mononucleotide repeats, of variable length were present at positions 80 to 85 and 171 to 178. In addition, a mononucleotide repeat found only in the C57BL/10 sequence was observed at position 99 to 104. Variation due to the deletion and duplication of short sequence motifs was also revealed at positions 155 and to 199 in the L1 repeat sequence. Discussion Inter-repeat PCR markers have been widely utilised in humans and more recently in the mouse (Nelson et al., 1989; Ledbetter et al., 1990; Irving and Brown, 1991). The use of T-tailed bluescript vectors has aided the rapid cloning and sequence determination of such markers. We have now examined such an inter repeat region in both Mus domesticus (C57BL/10) and Mus spretus in order to determine the mechanism of formation of a novel heterozygote specific band migrating at 280bp. This has also enabled us to investigate the principle sources of interspecies variation in such regions of the mouse genome. We have demonstrated that the heterozygote specific 280bp band could be genenerated from reannealed C57BL/10 and Mus spretus PCR products in the absence of amplification by Taq polymerase. This suggests that it is produced due to the formation of a heteroduplex PCR product, with altered electrophoretic mobility, rather than artefactual PCR primings. Much of the interspecies variation is due to elongation and contraction of the mononucleotide repeats. For example the (A)6 repeat at position 80 to 85 in the C57BL/10 sequence is increased to (A)9 in the homologous Mus spretus sequence. In addition the (T)10 repeat at position 171 to 180 has been compressed to (T)8 in Mus spretus. A short (A)6 repeat, not present in Mus spretus, was found at position 99 to 104 of the C57BL/10 sequence. Longer mononucleotide repeats have been reported to be highly variable both within and between mouse species (Aitman et al., 1991). Our observations suggest that shorter mononucleotide repeats may also be highly variable, at least between inbred laboratory strains and Mus spretus. A GAA motif at position 154 was duplicated in Mus spretus along with a TATT motif at position 196. The evolutionary changes observed between the two species could readily be explained by slippage replication and/or unequal exchange during replication. The alignment of the two sequences in figure 2 also gives an indication of the probable causes of the altered mobility of the novel 280bp heteroduplex. The variable length mononucleotide repeats and the small structural sequence changes may result in looped out regions which retard the electrophoretic migration of the heteroduplex. The formation of heteroduplex PCR products, with altered migration, has been utilised to enable the detection of single base changes in otherwise identical DNA fragments (Keen, et al. 1991). In this case the formation of the 280bp heteroduplex has facilitated the segregation analysis of this marker, in the interspecific backcross progeny, using conventional agarose gels. The overall size difference between the C57BL/10 and Mus spretus PCR products is only 8bp, making them difficult to resolve under normal conditions. However the 280bp heteroduplex could clearly be distinguished from the parental PCR products, after blotting and hybridisation. Using this technique interspecific backcross progeny could readily be scored for this inter repeat marker. Heteroduplex PCR products may be generated in a similar way from other inter-repeat sequences. The exploitation of such heteroduplex products may be of general value in the mapping of inter-repeat PCR markers in mammalian genomes. References 1. Aitman, T.J., Hearne, C.H., McAleer, M.A. and Todd, J.A. (1991) Mononucleotide Repeats are an abundant source of length variants in mouse genomic DNA. Genomics in press. 2. Irving, N.G. and Brown, S.D.M. (1991) Mouse chromosome-specific markers generated by PCR and their mapping through interspecific backcrosses. Genomics in press. 3. Keen, J., Lester, D., Inglehearn, C., Curtis, A. and Bhattacharya, S. (1991) Rapid detection of single base mismatches as heteroduplexes on hydrolink gels. Trends Genet. 7: 5. 4. Ledbetter, S.A., Nelson, D.L, Warren, S.T. and Ledbetter, D.H. (1990) Rapid Isolation of DNA Probes within Specific Chromosome Regions by Interspersed Repetitive Sequence Polymerase Chain Reaction. Genomics. 6: 475-481. 5. Nagamine, C.M., Chan, K. and Lau, Y.C. (1989) A PCR Artifact: Generation of Heteroduplexes. Am. J. Hum. Genet. 45: 337-339. 6. Nelson, D.L., Ledbetter, S.A., Corbo, L., Victoria, M.F., Ramirez-Solis, R., Webster, T.D., Ledbetter, D.H. and Caskey, C.T. (1989) Alu polymerase chain reaction: A method for rapid isolation of human-specific sequences from complex DNA sources. P.N.A.S. USA: 86: 6686-6690. 7. Simmler, M.C., Cox, R.D. and Avner, P.A. (1991) Adaption of the Interspersed Repetitive Sequence Polymerase Chain Reaction to the Isolation of Mouse DNA Probes from Somatic Cell Hybrids on a Hamster Background. Genomics 10: 770-778. Figure I. (Legend). Isolated PCR products from C57BL/10 and Mus spretus separated on a 3% agarose gel. Fragment sizes are given in base pairs (bp). The C57BL/10 track contains only the isolated 250bp Mus domesticus PCR product. The Mus spretus track contains only the isolated 240bp Mus spretus PCR product. The mixed products track contains an approximately equimolar mixture of the C57BL/10 and Mus spretus PCR products after four cycles of the original thermocycler program. No primers, dNTP's or polymerase were added. Three bands were observed in the mixed products track. The 250bp Mus domesticus PCR product, 240bp Mus spretus PCR product and the novel band migrating at 280bp. The 280bp heterozygote specific band was only observed in the mixed PCR products track. Figure 2. (Legend). PCR fragments were subcloned and sequenced as previously described (See Ref. 2). Two clones in opposite orientations were sequenced on both strands. The full 236bp Mus spretus sequence is presented (bottom line) along with the sequence of the homologous C57BL/10 PCR product (middle line). Nucleotides are numbered in relation to the longer 244bp C57BL/10 product. The ends of the Ll element consensus sequence are provided in the figure for comparison. The position and sequence of the LI element PCR primers are indicated by boxes. Mononucleotide repeats, of variable length were present at positions 80 to 85 (a) and 171 to 178 (c). In addition a mononucleotide repeat found only in the C57BL/10 sequence was observed at position 99 to 104 (b). A GAA motif at position 154 was duplicated in Mus spretus along with a TATT motif at position 196, indicated by underlining. |
