The results of analytical gel filtration and the determination of the oligomeric status of each truncated fragments.

<p>The results of analytical gel filtration and the determination of the oligomeric status of each truncated fragments.</p

Unidirectional truncation of UDE gene.

<p><b>(A)</b> The location of designed starting points along the UDE sequence indicating the predicted disordered segments and the conserved motifs. <b>(B)</b> The truncated UDE gene fragments generated by N-terminal truncation were fused in-frame with the biotin acceptor peptide and out-of-frame with hexahistidine tag, while fragments produced by C-terminal truncation were fused in-frame with hexahistidine tag and out-of-frame with BAP.</p

Screening expression level and solubility of UDE truncation libraries.

<p><b>(A)</b> Size fractionation of UDE fragments generated by unidirectional truncation on agarose gel. In the lanes next to the DNA ladders is the vector with total length UDE gene at higher position while the empty vector is at a lower position. N1–N3 and C1–C3 marked samples show by the exonuclease III truncation generated UDE constructs. <b>(B)</b> Assessment of UDE sublibraries size and diversity by PCR screen. <b>(C)</b> Separation of purified protein fractions on Ni²⁺-NTA resin from N-terminal (upper panels) and C-terminal (bottom panels) libraries on SDS-PAGE.</p

Alignment and scale-up of selected UDE truncated fragments.

<p><b>(A)</b> The restricted nine UDE truncated fragments from the identified protein clusters that were chosen for scale-up. Arrows show the expression compatible boundaries compared to the previously designated conserved motifs determined by the alignment of UDE homologues sequences. <b>(B)</b> The optimized expression of the nine UDE constructs in <i>E</i>. <i>coli</i> BL21 cells before (-) and after (+) IPTG induction. <b>(C)</b> Purification of recombinant UDE constructs by Ni²⁺-affinity chromathography. Gel slice images show the supernatant (termed as “Sup” on the figure) of cell lysis and the 300 mM imidazole elution (termed as “Elu” on the figure) fractions for each constructs. Note that the entire purification process can be followed on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156238#pone.0156238.s001" target="_blank">S1 Fig</a> that shows the whole SDS-PAGE gels, not only the supernatant and imidazole elution samples.</p

Secondary structure content determination of UDE and its fragments using VUVCD and SELCON3 program.

<p><b>(A)</b> Vacuum-ultraviolet circular dichroism (Δε) spectra of the UDE protein and its nine truncated fragments measured over the wavelength region of λ = 170–255 nm. The spectra are sorted into two panels for better visibility and spectra of UDE (red) is shown in both panels for reference <b>(B)</b> Decomposition of the CD spectra of UDE and its selected fragments using six secondary structure components; regular/distorted α-helix (rH/dH), regular/distorted β-strand (rS/dS), turn (T), and disordered structure (D). Upper panel: CD spectrum of UDE as measured and as fitted using the six components [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156238#pone.0156238.ref022" target="_blank">22</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156238#pone.0156238.ref024" target="_blank">24</a>]. Spectra of the components are also plotted with magnitudes proportional to their ratios in the full-length protein. Lower Panel: Difference spectra corresponding to UDE-NA2 and NG3-NA3 together with the fittings based on the spectra of the six basic components.</p

Spatial distribution of the secondary structure components along UDE protein.

<p>The location of α-helical segments <b>(A)</b> in the full-length (355 aa) UDE protein and <b>(B)</b> in its nine truncated fragments were determined from the CD spectra and the amino acid sequence using a neural network algorithm [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156238#pone.0156238.ref025" target="_blank">25</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156238#pone.0156238.ref026" target="_blank">26</a>]. The α-helical segments and β-strands are displayed in blue and red, respectively, while both turns and disordered parts appear in yellow. <b>(C)</b> Our final estimate for the secondary structure of UDE obtained as an average of the structure of UDE proposed in panel <b>(A)</b> and the structure of the fragments shown in panel <b>(B)</b> except for CA7. <b>(D)</b> The native structure of the N-terminal end of the full-length UDE was investigated using the evaluation of the CD spectrum of the N- terminal as the difference between the CD spectra of UDE and NA2 according to (Δε1UDE×N1UDE−Δε1NA2×N1NA2)/(N1UDE—N1NA2), where Δε is the molar ellipticity and N is the number of amino acids for UDE and NA2. The same subtraction method was performed with the highly overlapping fragments NG3 and NA3.</p

Occurrence of genes encoding dUTPase and UNG in different insects.

<p>The gene for dUTPase is ubiquitous, but the gene of the major uracil–DNA glycosylase, ung is not encoded in the genome of Holometabola species.</p>*<p>In the genome of <i>Aedes aegypti</i> strain Liverpool, an unexpected ung sequence was found, showing very high (87%–94%) similarity to the ung gene of Comamonadaceae, a family of Proteobacteria, arguing for its bacterial origin.</p

<i>D. melanogaster</i> genomic DNA uracil content inversely correlates with dUTPase expression.

<p>(A) Changes of dUTPase mRNA level throughout fruitfly development: embryo (E), 1^st larvae (L1), 2^nd larvae (L2), late 3^rd larvae (L3) and pupae (P). Note that dUTPase is down-regulated in larvae. (B) Comparison of dUTPase RNA level in the larval tissues salivary gland and imaginal tissue. Data are presented as mean of triplicates ± s.e.m. mRNA level was measured by RT-qPCR and dUTPase mRNA level was normalized to Rp49 mRNA level. (C) Uracil content of <i>D. melanogaster</i> genome in different developmental stages: embryo (E), 1^st larvae (L1), 2^nd larvae (L2), late 3^rd larvae (L3) and pupae (P). Embryonic sample was used as reference since it was shown to contain undetectable levels of uracil in DNA (cf. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002738#pgen.1002738.s002" target="_blank">Figure S2</a>). (D) Comparison of genomic uracil content in wild type imaginal disc and salivary gland of 3^rd larvae. Data are presented as mean ± s.e.m.</p

Stage- and tissue-specific distribution of dUTPase protein levels in <i>D. melanogaster</i>.

<p>Western blotting (A) and immunohistochemistry (B) was performed on selected developmental stages and tissues. Embryo 0–6 h (E1), embryo 0–24 h (E2), 1^st larvae (1L), 2^nd larvae (2L), early 3^rd larvae (3L1), wandering 3^rd larvae (3L2), pupae before head eversion (P1), pupae after head eversion (P2) and pupae 50–60 h after puparium formation (P3). For Western blotting, actin was used as loading control. Note that dUTPase protein levels are down-regulated during larval stages and expression is confined to specific tissues.</p

Morphological consequences of dUTPase silencing.

<p>In larvae (A) and pupae (B). (A) Immunohistochemistry of wing and eye discs, and brain of non-silenced (control) and silenced larvae for dUTPase (red) and DAPI staining for DNA (blue) demonstrate on one hand highly effective silencing; and on the other hand no observable morphological changes within these tissues. (B) Wild type pupae (control) in stage P6 (cf. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002738#pgen.1002738.s005" target="_blank">Figure S5</a>) and dUTPase silenced pupae at corresponding time after puparium formation in dorsal and ventral view are shown, after puparium removal. Wild type traits, Malpighian tubules (white arrows), Yellow Body (white asterix), developing adult eye (white arrowheads) are not observable on silenced animals. Instead, darkened (apoptotic/necrotic or melanized) tissues (red arrowheads) can be visualized on these pupae. Note the basically different inner texture of the everted discs (white boxes) and head sack (white circles).</p