A novel DNA nucleotide inTrypanosoma bruceionly present in the mammalian phase of the life-cycle

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Abstract
The existence of an unusual form of DNA modification in the bloodstream form of the African trypanosome Trypanosoma brucei has been inferred from partial resistance to cleavage of nuclear DNA with Pstl and Pvull (Bemards et al, 1984; Pays et al, 1984). This putative modification is correlated with the shut-off of telomeric Variant-specific Surface Glycoprotein (VSG) gene expression sites (ESs). The modification only affects inactive VSG genes with a telomeric location, and it is absent in procyclic (Insect form) trypanosomes in which no VSG is made at all. Previous attempts to detect unusual nucleosides in T.brucei DNA were unsuccessful, but we now report the detection of two unusual nucleotides, called pdV and pdV, in T.brucei DNA, using the 32 P-postlabeling technique. Nucleotide pdV was present in both bloodstream form and procyclic T.brucei DNA and co-migrated in two different two-dimensional thin layer chromatography (2D-TLC) systems with hydroxymethyideoxyurldine 5′-mono-phosphate (pHOMedU). in contrast, nucleotide pdV was exclusively present in bloodstream form trypanosomal DNA. Levels of pdj were higher in DNA enriched for teiomerlc sequences than in total genomic DNA and pdV was also detected in other Kinetoplastida species exhibiting antigenic variation. Postlabeling and 2D-TLC analyses showed base J to be dtfferent from the known eukaryotic unusual DNA bases 5-methylcytosine, N 6 and hydroxymethyluracil, and also from (glucosylated) hydroxymethylcytosine, uracil, a putrescinyithymine, 5-dihydroxypentyluracil and N 6 carbamoylmethyladenine. We conclude that pdV is a novel eukaryotic DNA nucleotide and that It is probably responsible for the partial resistance to cleavage by Pvull and Pstl of inactive telomeric VSG genes. It may therefore be involved in the regulation of ES activity in bloodstream form trypanosomes.