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The Detection of 5‐hydroxymethylcytosine in Trypanosoma brucei DNA
Author(s) -
Valentine Erika,
Smindak Richard,
Militello Kevin T
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.981.7
Subject(s) - 5 hydroxymethylcytosine , trypanosoma brucei , dna , biology , base pair , genomic dna , genome , microbiology and biotechnology , computational biology , gene , chemistry , biochemistry , dna methylation , gene expression
The goal of this project is to determine whether or not the modified base 5‐hydroxymethylcytosine is present in T. brucei nuclear DNA. T. brucei is the causative agent of African Sleeping Sickness, and has been previously shown to contain 5‐methylcytosine, 5‐hydroxymethyluracil, and base J. The T. brucei J‐base binding proteins (JBPs) synthesize 5‐hydroxymethyluracil, and are homologous to the mammalian TET (ten‐eleven translocation) proteins which synthesize 5‐hydroxymethylcytosine. Thus, we believe is it possible that the T. brucei JBPs synthesize 5‐ hydroxmethylcytosine in addition to 5‐hydroxymethyluracil. To test this model, an immunoblot assay was developed to detect 5‐hydroxymethylcytosine in DNAs spotted onto a nylon membrane. Our data indicate that both T. brucei bloodstream form and insect form DNA contain low but detectable levels of 5‐hydroxymethylcytosine. The same results were also generated using ELISA assays specific for 5‐hydroxymethylcytosine. We are currently using LC MS/MS to confirm the experiments above, and hydroxymethylated DNA immunoprecipitation experiments to determine the location of 5‐hydroxymethylcytosine in the T. brucei genome.