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Identification of Methylated Loci in the Trypanosoma brucei Genome
Author(s) -
Jayakar Sangeeta K,
Jensen Tina K,
Hennick Stacy M,
Militello Kevin T
Publication year - 2009
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.23.1_supplement.lb190
Subject(s) - methylated dna immunoprecipitation , biology , gene , genome , dna methylation , genetics , retrotransposon , bisulfite sequencing , microbiology and biotechnology , trypanosoma brucei , dna , transposable element , gene expression
Previous mass spectrometry and blotting experiments in our laboratory have shown that Trypanosoma brucei , the causative agent of African Sleeping Sickness, contains low levels of 5‐methylcytosine in its genomic DNA. In order to determine the location of 5‐methylcytosine in the genome, an immunoprecipitation strategy was utilized. T. brucei DNA was cut with Dpn II, modified by linker addition, and immunoprecipitated with an antibody against 5‐methylcytosine. Immunoprecipitated DNAs were amplified by PCR, inserted into the pGEM‐T Easy plasmid, and analyzed by DNA sequencing. In total, greater than 100 clones have been analyzed representing DNA from the insect stage and bloodstream stage of the parasite. To date, all methylated loci are in the nuclear genome. Several loci were found in the immunoprecipitate multiple times, including variant surface glycoprotein genes and retrotransposon genes. Thus, DNA methylation may regulate expression of these genes. However, housekeeping genes were also found in the immunoprecipitate indicating that DNA methylation is not restricted to a single locus. To pinpoint the position of 5‐methylcytosine within specific genes, sodium bisulfite sequencing of retrotransposon genes was initiated. In total, our data demonstrate that 5‐methylcytosine is present in the nuclear genome and may be overrepresented in multi‐gene families and repeated sequences. This work is supported by National Institutes of Health award AI074035‐01 to K.T.M.