The principal aim is to investigate the role of DNA methylation in cells of higher animals to see if it could be a factor in the aging syndrome. To do this we propose to modify DNA by methylation and then to put the DNA back in cells to see if the methylation pattern will be transmitted to DNA replicated from the modified template. We will also see if the modification affects the rate of transcription. We isolated and purified the methyl transferase from Haemophilus influenza to the point that it would modify supercoiled SV40 DNA without relaxing it. SV40 DNA was used initially since it has a single HpaII site (CCGG), very unusual for a DNA withmore than 5000 bp. The methylation of CCGG can be probed by digestion with two restriction endonucleases, HpaII which is inhibited by 5 methylcytosine (5mC) at the second position and MspI which is not. When the SV40 DNA was methylated and used to infect monkey kidney cells the newly replicated DNA failed to maintain the modification. We are now planning experiments with other systems. We have cloned random segments of frog DNA (Xenopus laevis) several of which increase the efficiency of replication of the plasmids containing these when injected into unfertilized Xenopus eggs. One segment has been sequenced and shown to contain 507 bp of moderately repetitive DNA belonging to the family of repeated sequences designated AluI in human DNA and Alui-equivalent sequence in other vertebrates. We plan to methylate the plasmid at both EcoRI and HpaII sites and follow the maintenance of both modifications during two rounds of replication. To test transcriptional effects we will methylate a plasmid containing the Herpes thymidine kinase gene and tests the efficiency of its transcription when injected into Xonopus oocyte nuclei. We are also sequencing DNA's from germline cells (bovine sperm) and a variety of somatic tissues to compare the methylation patterns. A 1400 bp EcoRI repeat from satellite I has been partially sequenced and shown to have the same sequence from sperm and somatic cells, but very different methylation patterns. We plan to transfer bovine satellite I with its natural methylation patterns into hamster cells (CHO) in culture to see how well the pattern will be maintained. The vector will be a plasmid containing the Herpes thymidine kinase gene.
| Hare, J T; Taylor, J H (1989) Methylation in eucaryotes influences the repair of G/T and A/C DNA basepair mismatches. Cell Biophys 15:29-40 |
| Taylor, J H (1987) Replication of DNA in eukaryotic chromosomes. Results Probl Cell Differ 14:173-97 |
| Hare, J T; Taylor, J H (1985) One role for DNA methylation in vertebrate cells is strand discrimination in mismatch repair. Proc Natl Acad Sci U S A 82:7350-4 |
| Hare, J; Taylor, J H (1985) Methylation directed strand discrimination in mismatch repair. Prog Clin Biol Res 198:37-44 |
