title: Presence of DNA methyltransferase activity and CpC methylation in Drosophila melanogaster.
abstract: Drosophila melanogaster lacks DNMT1/DNMT3 based methylation machinery. Despite recent reports confirming the presence of low DNA methylation in Drosophila; little is known about the methyltransferase. Therefore, in this study, we have aimed to investigate the possible functioning of DNA methyltransferase in Drosophila. The 14 K oligo microarray slide was incubated with native cell extract from adult Drosophila to check the presence of the methyltransferase activity. After incubation under appropriate conditions, the methylated oligo sequences were identified by the binding of anti 5-methylcytosine monoclonal antibody. The antibody bound to the methylated oligos was detected using Cy3 labeled secondary antibody. Methylation sensitive restriction enzyme mediated PCR was used to assess the methylation at a few selected loci identified on the array. It could be seen that a few of the total oligos got methylated under the assay conditions. Analysis of methylated oligo sequences provides evidence for the presence of de novo methyltransferase activity and allows identification of its sequence specificity in adult Drosophila. With the help of methylation sensitive enzymes we could detect presence of CpC methylation in the selected genomic regions. This study reports presence of an active DNA methyltransferase in adult Drosophila, which exhibits sequence specificity confirmed by presence of asymmetric methylation at corresponding sites in the genomic DNA. It also provides an innovative approach to investigate methylation specificity of a native methyltransferase.
authors.affiliation: Molecular Biology Research Laboratory, Department of Zoology, Centre for Advanced Studies, University of Pune, Pune, 411007, India.
authors.forename: Chitra S
authors.initials: CS
authors.lastname: Panikar
authors.affiliation: Molecular Biology Research Laboratory, Department of Zoology, Centre for Advanced Studies, University of Pune, Pune, 411007, India.
authors.forename: Shriram N
authors.initials: SN
authors.lastname: Rajpathak
authors.affiliation: Molecular Biology Research Laboratory, Department of Zoology, Centre for Advanced Studies, University of Pune, Pune, 411007, India.
authors.forename: Varada
authors.initials: V
authors.lastname: Abhyankar
authors.affiliation: Molecular Biology Research Laboratory, Department of Zoology, Centre for Advanced Studies, University of Pune, Pune, 411007, India.
authors.forename: Saniya
authors.initials: S
authors.lastname: Deshmukh
authors.affiliation: Molecular Biology Research Laboratory, Department of Zoology, Centre for Advanced Studies, University of Pune, Pune, 411007, India. deepti.deobagkar@gmail.com.|Bioinformatics Center, University of Pune, Pune, 411007, India. deepti.deobagkar@gmail.com.
authors.forename: Deepti D
authors.initials: DD
authors.lastname: Deobagkar
mesh_terms: D044503:5-Methylcytosine / Q000032:analysis / Q000276:immunology; D000818:Animals; D004247:DNA / Q000378:metabolism; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism*; D019175:DNA Methylation; D004262:DNA Restriction Enzymes; D029721:Drosophila Proteins / Q000378:metabolism; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics; D007120:Immunochemistry; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction
chemical_list: D029721:Drosophila Proteins; D044503:5-Methylcytosine; D004247:DNA; D004248:DNA (Cytosine-5-)-Methyltransferases; D004262:DNA Restriction Enzymes
keywords: 14K oligo microarray; CpC methylation; DNA methyltransferase activity; Restriction enzyme mediated PCR