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Molecular Biology Reports

, Volume 42, Issue 12, pp 1615–1621 | Cite as

Presence of DNA methyltransferase activity and CpC methylation in Drosophila melanogaster

  • Chitra S. Panikar
  • Shriram N. Rajpathak
  • Varada Abhyankar
  • Saniya Deshmukh
  • Deepti D. DeobagkarEmail author
Manuscript

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.

Keywords

DNA methyltransferase activity CpC methylation 14K oligo microarray Restriction enzyme mediated PCR 

Notes

Acknowledgments

We acknowledge the valuable suggestions by Dr. D. N Deobagkar. The research was funded by grants from SPPU (Savitribai Phule Pune University) and UGC-GOI (University Grant Commission Govt. of India). VA is CSIR (Council of Scientific and Industrial Research)-SRF.

Supplementary material

11033_2015_3931_MOESM1_ESM.pdf (282 kb)
Supplementary material 1 (PDF 282 kb) Supplementary Figure S1: Graph representing colony count (CFU/mL) log values versus time in hours for control and antibiotic treated adult D. melanogaster Supplementary Figure S2: Global DNA methylation and DNMT activity (values are represented in log2 scale) in protein extract from control and antibiotic treated adult D. melanogaster Supplementary Figure S3:MspI/HpaII mediated PCR of FBgn0034232:Lanes A and C contain amplicons from MspI and HpaII digested DNA respectively obtained from control adult Drosophila melanogaster and Lanes B and D contain amplicons from MspI and HpaII digested DNA respectively obtained from antibiotic treated adult Drosophila melanogaster Supplementary Figure S4: Sensitivity of MspI/HpaII: Gel represents PCR results after MspI/HpaII digestion of FBgn0038325 gene amplicon. Lanes A and B do not show amplification after digesting the unmethylated product with MspI/HpaII respectively. Lanes C and D show amplification as the product was methylated with MspI, digested with MspI and methylated with HpaII, digested with HpaII. Lane E shows no amplification as the HpaII methylated product was digested with MspI while Lane F shows amplification as the MspI methylated product is digested with HpaII. Lane L corresponds to 100 bp NEB Ladder. The product was methylated using MspI/HpaII methyltransferase (NEB) as per the manufactures instructions and digested with MspI/HpaII (NEB) as mentioned in materials and method section of this paper. This indeed confirms the efficiency of restrictions enzymes used for experiments and ruled out the possibility of any false positive detection Supplementary Figure S5: NcoI mediated PCR of FBgn0034232: Lanes A and B contain amplicons from NcoI digested and undigested DNA respectively obtained from control adult D. melanogaster. NcoI is not sensitive to any form of DNA methylation. Digestion was performed with enzyme units and incubation period identical to MspI/HpaII digestion. The absence of an amplicon in lane A demonstrated that the digestion protocol used for methylation sensitive MspI and HpaII digestion ensures total cleavage of genomic DNA and leaving no intact template for PCR
11033_2015_3931_MOESM2_ESM.docx (13 kb)
Supplementary Table S1 List of true positive oligos identified by in vitro methyltransferase assay. Supplementary material 2 (DOCX 13 kb)
11033_2015_3931_MOESM3_ESM.docx (18 kb)
Supplementary Table S2 List of primers used for methylation sensitive restriction endonuclease mediated PCR. Supplementary material 3 (DOCX 18 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Chitra S. Panikar
    • 1
  • Shriram N. Rajpathak
    • 1
  • Varada Abhyankar
    • 1
  • Saniya Deshmukh
    • 1
  • Deepti D. Deobagkar
    • 1
    • 2
    Email author
  1. 1.Molecular Biology Research Laboratory, Department of Zoology, Centre for Advanced StudiesUniversity of PunePuneIndia
  2. 2.Bioinformatics CenterUniversity of PunePuneIndia

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