5hmC-like modification is the predominant cytosine modification in the Plasmodium falciparum genome. Immunostaining of Ring (R) or Schizont (S) stage P. falciparum 3D7-G7 parasites with anti-5hmC (top) or anti-5mC (bottom) antibodies. Anti-histone H3 antibodies were used as a positive control for nuclear staining. BF = Brightfield. To examine the presence of unexplored cytosine modifications in P. falciparum, we denatured genomic DNA prepared from parasites of the 3D7-G7 strain grown in WBC-free blood, spotted them onto nylon membranes and probed the membrane with either anti-5mC or anti-5hmC antibodies from different sources. Genomic DNA prepared from all three stages (synchronized at the Ring (R; 8–14 h post-invasion (hpi)), Trophozoite (T; 24–28 hpi) and Schizont (S; 38–42 hpi)), reacted with the anti-5hmC antibodies, indicating the presence of a new DNA modification, 5hmC or 5hmC-like, in the parasite.e Additionally, we performed immunofluorescence assays on fixed 3D7-G7 parasites using primary antibodies and observed that the fluorescence signal was restricted to the nucleus, further supporting the existence of this novel DNA modification in vivo.

Hammam E, Ananda G, Sinha A, Scheidig-Benatar C, Bohec M, Preiser PR, Dedon PC, Scherf A, Vembar SS. Discovery of a new predominant cytosine DNA modification that is linked to gene expression in malaria parasites. Nucleic Acids Res. 2019 Nov 28.