Base J originally found in kinetoplastida is also a minor constituent of nuclear DNA of Euglena gracilis - PubMed (original) (raw)

Comparative Study

Base J originally found in kinetoplastida is also a minor constituent of nuclear DNA of Euglena gracilis

D Dooijes et al. Nucleic Acids Res. 2000.

Abstract

We have analyzed DNA of EUGLENA: gracilis for the presence of the unusual minor base beta-D-glucosyl-hydroxymethyluracil or J, thus far only found in kinetoplastid flagellates and in DIPLONEMA: Using antibodies specific for J and post-labeling of DNA digests followed by two-dimensional thin-layer chromatography of labeled nucleotides, we show that approximately 0.2 mole percent of EUGLENA: DNA consists of J, an amount similar to that found in DNA of Trypanosoma brucei. By staining permeabilized EUGLENA: cells with anti-J antibodies, we show that J is rather uniformly distributed in the EUGLENA: nucleus, and does not co-localize to a substantial extent with (GGGTTA)(n) repeats, the putative telomeric repeats of EUGLENA: Hence, most of J in EUGLENA: appears to be non-telomeric. Our results add to the existing evidence for a close phylogenetic relation between kinetoplastids and euglenids.

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Figures

Figure 1

Detection of J in E.gracilis. (A) Detection of J-containing DNA on filters with polyclonal anti-J antibodies. The dot-blot with serial dilutions of total genomic DNA was incubated with rabbit antiserum 538αJ and bound antibodies were detected as described in Materials and Methods. DNA loading was checked by ethidium bromide staining of the DNA samples. From left to right: procyclic (insect form) T.brucei (PC), bloodstream form T.brucei (BF) and E.gracilis DNA. PC trypanosomes do not contain J. (B) Analysis of E.gracilis DNA by 32P-nucleotide post-labeling combined with 2D-TLC (D1 and D2 are indicated), as described in Materials and Methods. The positions of the labeled 5′-deoxynucleosidemonophosphates are indicated by filled circles, 5′-ribonucleoside monophosphates are indicated by open circles, and background spots (open circles with dotted lines) are explained in panel I. Panel II: labeling of total DNA of E.gracilis. The position of J is indicated by an arrow.

Figure 2

Immunofluorescence assay of cells with anti-J antiserum combined with fluorescence in situ hybridisation with a telomeric repeat probe. The preparations were analysed with a Leica Confocal Laser Scanning Microscope. The scale bars represents 10 µm. From left to right: T.brucei procyclic form (PC), T.brucei bloodstream form (BF), E.gracilis, and unstained E.gracilis (as negative control for the cytoplasmic red autofluorescence). From top to bottom: transmission image, detection of J with anti-J antibodies, detection of (GGGTTA)n repeats with a (GGGTTA)n probe, and merge of the J and (GGGTTA)n images (green and red, respectively). Cells incubated with the pre-immune serum (or another rabbit antiserum) showed no staining in the case of T.brucei and only red autofluorescence in the case of E.gracilis (not shown).

Figure 3

Fluorescent images of a E.gracilis nucleus hybridized with the GGGTTA repeat probe (green) and counterstained with TO-PRO-3 (red). (A) CLSM section of a nucleus showing the understained region (the putative nucleolus). (B) Same section as (A) but showing hybridization with the GGGTTA probe around the putative nucleolus. (C) The superposition of all sections of a different Euglena nucleus, in which we count a total of 93 spots. Note that the edge of the nucleus counterstains weakly and that all spots are in the nucleus. The scale bars represent 2 µm. See Materials and Methods for details.

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