In vivo DNA methylation of Escherichia coli DH5α and top10F’ strains by Bacterial cytosine 5-Methyltransferase M. Msp1

At the chromatin level, methylated CpG dinucleotides are resistant compared with nonmethylated counterparts. The DNA of two strains was analyzed following transformationwith bacterial cytosine-5-methyltransferase gene in mammalian transfection vector pcDNA3. | Tr. J. of Biology 22 (1998) 143-151 © TÜBİTAK In vivo DNA Methylation of Escherichia coli DH5α and Top10F’ Strains by Bacterial Cytosine-5 Methyltransferase M. Msp1* Öztürk ÖZDEMİR Cumhuriyet Üniversitesi Tıp Fakultesi Tıbbi Biyoloji ve Genetik . Sivas-TURKEY David HORNBY Department of Molecular Biology and Biotechnology University of Sheffield Received: Abstract: At the chromatin level, methylated CpG dinucleotides are resistant compared with nonmethylated counterparts. The DNA of two strains was analyzed following transformationwith bacterial cytosine-5-methyltransferase gene in mammalian transfection vector pcDNA3. Expression of the was tested by digestion. The results suggest that the DNA of both strains was fully methylated at the CCGG sequences, by the active enzyme under the control of T7 and cytomegalovirus (CMV) promoters. Methylated DNA can not be digested and it exhibits higher fragment sizes in 1% agarose gel in contrast to the untransformed cell DNAs in vivo. Key Words: Bacterial MTase , in vivo DNA methylation Escherichia coli DH5a ve Top10F’ Hücre DNA’larının Bakteri Sitozin-5Metiltransferaz Enzimi, M. Msp1, Tarafından In vivo Metilasyonu Özet: Kromatin düzeyinde metillenmiş CpG dinükleotidleri, metil grubu taşımayan nükleotidlerin aksine bir modifiye restriksiyon enzimi olan R. Msp1 enzimine dirençlidirler. Yapısında metillenmiş dinükleotid (mCpG) taşıyan bu serbest DNA aynı restriksiyon enzimince kesilmez. Bu araştırmada, metillenmiş nükleotidlerin bu dirençli olma özelliğinden yararlanarak başka bir bakteri türüne ait (Moroxella spp.), rekombine metiltransferaz enziminin, farklı hücrelerde benzer metilaz aktivite gösterip göstermeyeceği araştırılmak istenmiştir. Bunun için izogenik olmayan iki suşu (DH5α ve Top 10F’), PCR ile 5’ ucuna omurgalı nükleer transport sinyali SV40 VP1 ve markır enzim glutatyonS-transferaz (GST) gen dizileri takılmış bakteriyel sitozin-5-spesifik .