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Base-excision repair and control of nucleotide pools safe-guard against permanent uracil accumulation in DNA relying on two key enzymes: uracil–DNA glycosylase and dUTPase. Lack of the major uracil–DNA glycosylase UNG gene from the fruit fly genome and dUTPase from fruit fly larvae prompted the hypotheses that i) uracil may accumulate in Drosophila genomic DNA where it may be well tolerated, and ii) this accumulation may affect development. Here we show that i) Drosophila melanogaster tolerates high levels of uracil in DNA; ii) such DNA is correctly interpreted in cell culture and embryo; and iii) under physiological spatio-temporal control, DNA from fruit fly larvae, pupae, and imago contain greatly elevated levels of uracil (200–2,000 uracil/million bases, quantified using a novel real-time PCR–based assay). Uracil is accumulated in genomic DNA of larval tissues during larval development, whereas DNA from imaginal tissues contains much less uracil. Upon pupation and metamorphosis, uracil content in DNA is significantly decreased. We propose that the observed developmental pattern of uracil–DNA is due to the lack of the key repair enzyme UNG from the Drosophila genome together with down-regulation of dUTPase in larval tissues. In agreement, we show that dUTPase silencing increases the uracil content in DNA of imaginal tissues and induces strong lethality at the early pupal stages, indicating that tolerance of highly uracil-substituted DNA is also stage-specific. Silencing of dUTPase perturbs the physiological pattern of uracil–DNA accumulation in Drosophila and leads to a strongly lethal phenotype in early pupal stages. These findings suggest a novel role of uracil-containing DNA in Drosophila development and metamorphosis and present a novel example for developmental effects of dUTPase silencing in multicellular eukaryotes. Importantly, we also show lack of the UNG gene in all available genomes of other Holometabola insects, indicating a potentially general tolerance and developmental role of uracil–DNA in this evolutionary clade.
Author Summary
The usual paradigm confines "normal" DNA of living cells to a well-defined restricted chemical space populated with only four bases (adenine, thymine, guanine, and cytosine) and some of their methylated derivatives (e.g. 5′-methyl-cytosine). Uracil is not considered to be a "normal" DNA base, except in several bacteriophages. On the contrary, uracil is generally considered to be an error in DNA. We show that Drosophila cells interpret uracil-substituted DNA as normal DNA, due to lack of two repair enzymes. Importantly, this unusual trait is under developmental control and applies only for animals before pupation. Metamorphosis is drastically perturbed by silencing of dUTPase, responsible for keeping uracil out of DNA. Our results argue that in Drosophila, and perhaps in other Holometabola insects as well, uracil–DNA plays a dedicated physiological role.
Citation: Muha V, Horváth A, Békési A, Pukáncsik M, Hodoscsek B, Merényi G, et al. (2012) Uracil-Containing DNA in Drosophila: Stability, Stage-Specific Accumulation, and Developmental Involvement. PLoS Genet 8(6): e1002738. doi:10.1371/journal.pgen.1002738
Editor: R. Scott Hawley, Stowers Institute for Medical Research, United States of America
Received: August 22, 2011; Accepted: April 13, 2012; Published: June 7, 2012
Copyright: © 2012 Muha et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Hungarian Scientific Research Fund (OTKA K68229, NK84008, CK78646, K75774, NKTH-OTKA H07-BEL74200), the National Innovation Office, the New Hungary Development Plan (TÁMOP-4.2.1/B-09/1/KMR-2010-0002), Howard Hughes Medical Institutes #55005628 and #55000342, and Alexander von Humboldt-Stiftung, Germany. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
In wild-type organisms, notably excepting some rare bacteriophages with thymineless DNA genomes [1], [2], uracil in DNA is thought to occur only transiently and at very low frequency (<20/million bases) as a damage product [3], [4]. Efficient base-excision DNA repair together with fine-tuned control of nucleotide pools safe-guard against uracil in DNA relying on two key enzymes: uracil–DNA glycosylase (UDG) [5] and dUTPase [6].
UDG deficiency, in combination with thymidylate synthase inhibition or depleted dUTPase activity, was reported to lead into notable uracil accumulation in DNA. However, deficiency in uracil–DNA glycosylase on its own contributes only slightly to the genomic uracil content [7]–[10]. Since dUTPase deficiency or silencing can be rescued by depleted activity of UDG [11]–[14], it can be argued that UDG is a major factor that renders uracil–DNA, formed in absence of dUTPase, intolerable for cells. Uracil–DNA glycosylases represent a superfamily that involves enzymes with specialized functions. Among the superfamily members, UNG is reported to be the most abundant one that also possesses the highest activity in removing uracils from any context of both single-stranded and double-stranded DNA [5]. SMUG has similar attributes to UNG but prefers uracil-containing ssDNA as substrate [15]. MBD4 and TDG recognize mismatched uracil or thymine bases that base-pair with guanine [16]. The latter enzyme is known to function within CpG islands, where thymine is formed after spontaneous methyl-cytosine deamination [17].
Through hydrolyzing dUTP to dUMP and pyrophosphate, dUTPase serves a dual role in cell physiology: it produces a precursor for thymidylate synthesis, and removes dUTP from the deoxynucleotide pool. Eukaryotic and bacterial DNA polymerases incorporate deoxyuridine into DNA with a rate that depends on the cellular dUTP concentration [6], [10]. Therefore dUTPase has a significant indirect impact in prevention of uracil incorporation into DNA. Lack of dUTPase is supposed to induce thymine to uracil substitution that does not alter the genetic code; however UDG still removes this kind of base modification supposedly resulting in genome instability. This process may serve as an explanation for overall lethality of dUTPase deficiency [6].
Here, we propose that a condition similar to simultaneous deficiency in both dUTPase and UDG is present in Drosophila larvae. The Drosophila genome does not encode the major uracil–DNA glycosylase UNG [18], therefore uracil–DNA tolerance may be expected. Drosophila larvae contain both proliferating primordial tissues of imago and differentiated tissues that degrade during metamorphosis. Our previous study reported Drosophila dUTPase [19], [20] expression only in the first one of the two kinds of tissues [21], [22]. We wished to investigate if the stage- and tissue-specific expression of dUTPase is translated into differences in the uracil content of genomic DNA and whether this putative pattern has developmental significance.
We now report high resistance of Drosophila cell lines to fluorodeoxyuridine (FdUR) that induced high uracil–DNA levels. We show that in addition to tolerance of uracil-substituted DNA, Drosophila cells correctly interpret this unusual DNA both in vitro and in vivo. Moreover, in wild type Drosophila we observe stage- and tissue-specific changes in uracil-content of DNA that are inversely correlated to dUTPase expression and cellular fate. We propose that the observed pattern is due to the lack of the ung gene from the Drosophila genome paralleled with developmental down-regulation of dUTPase in larval tissues. To check whether the absence of dUTPase may be causative for uracil accumulation in DNA, we show that silencing of dUTPase in larvae expands the uracil–DNA pattern to imaginal tissues, as well. Interestingly, dUTPase silencing results in abnormal DNA strand breaks, cell death and developmental arrest in early pupal stage. This may indicate that tolerance of highly uracil-containing DNA is also stage-specific, and other tolerance factors, in addition to the lack of UNG, may appear in this specific developmental phase. To our knowledge, our study is the first one that reports uracil–DNA appearance in absence of dUTPase in a wild type complex eukaryotic organism and demonstrates the developmental pattern of its tolerance and stability.
Since the Drosophila genome lacks UNG, we wished to test whether Drosophila cells show similar characteristics to UNG deficient organisms. The drug 5′-fluorodeoxyuridine (FdUR), frequently used as an inhibitor of thymidylate biosynthesis [23], [24], leads to perturbation of nucleotide levels and cell death. Loss of uracil–DNA glycosylase activity was found to lead to fluoropyrimidine resistance in E. coli [25], yeast [26] and C. elegans [27]. Deficient uracil–DNA glycosylase activity was also reported to be required for increased genomic uracil content after FdUR exposure in E. coli and mammalian cells [7]–[10]. Therefore, response to FdUR treatment may be an indicator for testing cellular uracil–DNA glycosylase activity. We observed that the Drosophila S2 cell line shows only small decrease in viability in the presence of 1 mM of FdUR, while HeLa cells, possessing the ung gene, show strong lethality at this drug concentration (Figure 1A). As a dose-dependent response to increasing concentrations of FdUR, uracil accumulation in genomic DNA of Drosophila S2 cells became highly elevated (up to approx. 450 uracil/million bases) (Figure 1B). Both the observed relatively high resistance for FdUR and the cellular response of genomic uracil incorporation may be explained by the fact that Drosophila cells lack significant uracil–DNA glycosylase activity.
(A) Dose-response curve upon FdUR treatment followed by Alamar Blue assay. (B) FdUR leads to uracil accumulation in DNA of Drosophila S2 cells. Data indicate that increased level of uracil is well-tolerated in Drosophila, but not in human cells. Data are presented as mean ± s.e.m. (C) Drosophila S2 (top panels) and human HeLa cells (bottom panels) were transfected with normal plasmid (left panels) or uracil-containing plasmid (right panels). Expression of YFP in Drosophila S2 cells or dsRedMonomer in HeLa cells indicates stability of the DNA. (D) Microinjection of uracil-plasmid into Drosophila embryo. Non-injected embryos served as control sample.
We then asked if uracil-containing chemically unusual DNA may be tolerated and interpreted in Drosophila cells. Uracil–DNA specific cell response was provoked by transfecting cells with exogenous plasmid uracil–DNA. The expression signal of the fluorescent reporters (eYFP or dsRedMonomer) encoded by plasmids produced in wild type (normal plasmid) or dut-1, ung-1 (uracil-plasmid, approx. 5500 uracil/million bases [8], [10]) E. coli were followed. The gene span for the eYFP or DSRed-monomer reporters included the promoter (CMV immediate early or metallothionein), the reporter protein gene (DSRed-monomer or eYFP), and the SV40 polyadenylation signal. The total length of these gene spans comprises 1515 or 1299 nucleotides. If synthesized in the double mutant dut-1, ung-1 E.coli strain, the average number of uracil substitutions on a single strand within these gene spans corresponds to 8.3 and 7.1, respectively. Human (HeLa) cells, possessing UNG, showed appreciable fluorescent signal only when transformed with normal plasmid, indicating that uracil-containing plasmid was not interpreted probably due to its degradation. On the contrary, Drosophila S2 cells could express reporter genes encoded either on normal or uracil containing plasmid at comparable level (Figure 1C and Figure S1).
Having established that Drosophila cells in in vitro culture may tolerate and do correctly interpret uracil-containing DNA, we wished to assess the fruit fly physiological response to uracil–DNA at the organism level. Therefore similarly to the cell culture reporter-assay, uracil–DNA plasmid was introduced to Drosophila embryo. Upon microinjection of pP{Gal4VP16} expression vector (where the 1000 nt segment includes the P-element promoter, the Gal4-VP16 fusion gene and the hsp70 terminator that may have an average number of 5.5 uracil substitutions if produced in double mutant E. coli) into P{mw+UASeGFP} transgenic animals, the strong eGFP signal indicated the stability of exogenous DNA (Figure 1D). In pre-hatching embryos eGFP signal was detected from both normal and uracil-containing DNA with commeasurable intensities. In both cases the expression pattern of the reporter construct is similar and eGFP is most pronounced in the gut. These results can be explained only by assuming that the genetic information stored in uracil-containing DNA serves as a cognate code for transcription in Drosophila cells. Such ability of the fruitfly cells is most probably due to lack of UNG under which condition uracil–DNA does not get rapidly degraded.
The other key factor responsible for keeping uracil out of DNA is the enzyme dUTPase, the importance of which is even more substantiated in D. melanogaster that lacks UNG. During development of D. melanogaster, high dUTPase protein levels can be observed only in embryonic stages (Figure 2A). As shown by Western blotting and immunohistochemistry, starting from late embryonic phase, dUTPase expression level is decreased drastically and remains low during postembryonic stages. Under normal physiological circumstances of larval development, dUTPase is predominantly expressed in imaginal discs, the central nervous system and in the testis (Figure 2) but not in most larval tissues, like salivary gland and gut. In the ventriculus and in the salivary gland, some cells are associated with dUTPase expression – these seem to correspond to the imaginal cells (Figure 2B). In the imago, dUTPase is present in the ovary of females. Cellular localization of dUTPase is usually nuclear [21], [28]–[31], however cytoplasmic occurrence is also evident in the nurse cells of mature follicles within ovaries, as described previously [21]. The present results are in agreement with our more limited earlier observations at the protein level [21]. We also quantified the dUTPase mRNA level by real-time PCR and found that protein and mRNA levels show similar tendencies during development (Figure 3A and 3B).
Western blotting (A) and immunohistochemistry (B) was performed on selected developmental stages and tissues. Embryo 0–6 h (E1), embryo 0–24 h (E2), 1st larvae (1L), 2nd larvae (2L), early 3rd larvae (3L1), wandering 3rd 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.
(A) Changes of dUTPase mRNA level throughout fruitfly development: embryo (E), 1st larvae (L1), 2nd larvae (L2), late 3rd 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 D. melanogaster genome in different developmental stages: embryo (E), 1st larvae (L1), 2nd larvae (L2), late 3rd larvae (L3) and pupae (P). Embryonic sample was used as reference since it was shown to contain undetectable levels of uracil in DNA (cf. Figure S2). (D) Comparison of genomic uracil content in wild type imaginal disc and salivary gland of 3rd larvae. Data are presented as mean ± s.e.m.
We were interested to learn whether coincidence of uracil–DNA tolerance and dUTPase down-regulation results in uracil accumulation in the genome of affected developmental stages and larval tissues. Determination of genomic uracil level was carried out by applying a recent quantitative real-time PCR-based method [10]. Data presented in Figure S2 and Figure 3C argue that DNA purified from embryo is relatively uracil-free, whereas in larval stages, uracil becomes much accumulated. The presence of uracil–DNA in larvae was further confirmed by using an independent method, the UNG-ARP assay (Figure S3) [8]. Uracil content also varies according to tissue type within the larvae: the salivary gland, a representative tissue of pupal degradation, accumulates high levels of uracil; while imaginal discs, which do not undergo abundant metamorphosis-coupled cell death, contain much less uracil (Figure 3D). Uracil level in larval tissues is comparable to that previously reported in the double mutant dut1-1, ung-1 E. coli strain [8], [10], amounting to several thousands of uracils per million bases. The above discussed pattern of uracil accumulation in different stages and tissues shows a clear negative correlation to the expression pattern of dUTPase (compare the respective developmental stages in Figure 2 and Figure 3).
In order to investigate if perturbation of the wild-type pattern of uracil levels in DNA may interfere with normal development, we aimed to silence dUTPase in transgenic D. melanogaster strains (Table S1). Efficient silencing could be successfully achieved in a setup resulting in well distinguishable F1 phenotypes: non-silenced animals were characterized by GFP expression and curly wings, whereas silenced animals had no markers [32] (Figure S4). Overall silencing resulted in efficiently decreased dUTPase protein levels in larvae and pupae (Figure 4A). RNAi silencing may not operate appropriately in early embryo due to maternal effects, but this stage is out of the scope of our present experiment with transgenic strains. We observed that dUTPase silencing did not perturb normal life and development of larvae. The silenced versus non-silenced larvae of F1 generation were selected based on GFP expression, and the time interval between the egg laying and puparium formation did not show any significant alteration (Figure 4B). Importantly, effective silencing of dUTPase in imaginal discs and larval brain did not cause any observable morphological changes in tissue morphology (Figure 5A). At pupal stage, however, dUTPase silencing induced 100% lethality, i.e. no silenced animals could develop into imago (observation is based on counting 2350 curly winged control imagos resulting from the first generation of crossings) (Figure 4C).
Western blots in (A) show that the protein level of dUTPase is under detection limit in silenced animals. Actin served as loading control. (B) Curves show the relative number of silenced and non-silenced animals that have undergone puparium formation at the given time point after egg deposition. Inflection points of the curves represent the mean time of puparium formation characteristic for the given population. dUTPase silencing did not perturb the time interval required for puparium formation. (C) Graph shows the number of counted dead animals relative to number of hatched curly winged control flies. Among these dead animals, three groups with distinct morphological traits characteristic for wandering larvae (w3L), prepupae (preP), and pupal stage P5 (P5) were identified and counted. (D) Genomic uracil content of dUTPase silenced and control tissues from 3rd larvae.
One possible proof for RNAi specificity is a rescue by the corresponding complementary DNA [33], [34]. Therefore, to test the specificity of dUTPase RNAi, the RNAi construct was co-expressed with a dUTPase transgene that led to the expression of dUTPase protein, as detected in the rescued animals (cf. method described in Text S1 and shown on Figure S4C and S4D). We observed a full rescue of the lethality caused by the RNAi confirming that the RNAi phenotype was due to a reduction of dUTPase level (Table S2). Rescued animals underwent development and metamorphosis just like the wild type animals. In the larval, prepupal and imago stages, rescued animals were carefully investigated for morphology and no distinct traits were observed.
In the detailed phenotype analysis of dUTPase-silenced animals, morphologic observations indicated serious adverse effects and developmental arrest at or before the pupal stage P5 [35] (Figure 5B, Figures S5 and S6). Upon removing the puparium of the silenced animal, defects were identified in the inner texture of everted discs, and head sack, as well as in the development of adult eye. These defects are permanent, and are not simply due to slow down of normal development. Moreover, 3–4 days after puparium formation, tissues showing morphological traits associated with the larval stage were identified within dissected samples (Figure S7 versus Figure S8). Noteworthy, the typical organ structures of the wild type adult (legs, wings, Malpighian tubules, Yellow Body, eyes etc.) never appeared in the silenced animals. Darkened tissues that may result from cell death, necrosis or histolysis were also observed e.g. in the prothoracic region and organs (Figure 5B and Figure S8) [36]. This result argued that although dUTPase is dispensable in larval tissues [21], the presence of the enzyme at the developmental stage of early puparium formation is essential for normal development during metamorphosis.
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. Figure S5) 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).
Tissue specific silencing in the dorsal wing surface of imaginal discs was also developed as described in Figure S9 (cf. [37]). The great majority of silenced animals developed curly wings, and a significant portion of them even showed blisters on the wings. Curling and blistering of the wings was suggested to result from abnormal cell death in dorsal wing surface [38], [39]. These results suggest that dUTPase silencing within a well-defined tissue segment may be specifically and exclusively associated with phenotypic effects in the very same tissue segment.
To analyze whether dUTPase silencing changed the distribution of uracil–DNA within different larval tissues, we assayed genomic uracil content of imaginal discs from both non-silenced and silenced larvae of F1 generation. As Figure 4D shows, imaginal discs from dUTPase silenced larvae accumulated a high amount of uracil in their genome (1097+/−55 uracil/million bases, to be compared with 131+/−69 uracil/million bases in the non-silenced animals) that approximated the genomic uracil content of salivary gland. Further increase in uracil–DNA content of salivary gland DNA was not observed in dUTPase silenced larvae, indicating that silencing was effective in tissues that normally express dUTPase but had no effect on uracil–DNA in tissues that normally do not express dUTPase. This result confirms that uracil appearance in DNA depends on dUTPase expression, thus dUTPase activity is causative in maintaining DNA with low levels of uracil.
To analyze if the effects of dUTPase silencing may lead to DNA damage or cell death, we applied TUNEL and phospho-Histone H2Av assays [40], [41]. We observed that imaginal discs isolated from 3rd stage wandering larva of dUTPase-silenced animals show strong enrichment in TUNEL positive cells (Figure 6A and 6B). TUNEL staining in the tissues indicate primarily cell death in the phase of DNA fragmentation. To address the question whether dUTPase depletion violates genome integrity we stained nuclei for phospho-Histone H2Av. H2Av histone modification by the ATR/ATM kinases indicates DNA double strand breaks (DSBs) in the proximity of the foci [41]. We observed numerous phospho-H2Av foci in dUTPase silenced wing imaginal discs, while no such foci were visible in the wild type (Figure 6C). These results suggest a potential correlation between dUTPase activity and DNA integrity. The increased level of DNA damage observed in our experiments in the dUTPase-silenced animals may result from excessive processing of uracil-containing DNA that concludes to DNA fragmentation.
(A) Imaginal discs were isolated from wild type and dUTPase silenced wandering 3rd larvae and stained for TUNEL assay (shown as red dots). Discs from silenced animals showed highly increased TUNEL staining. (B) TUNEL positive cell counts in imaginal discs from wild type and dUTPase silenced wandering 3rd larvae. Error bars represent the standard error of mean. (C) Imaginal discs from wild type and dUTPase silenced 3rd wandering larvae stained against phospho-H2Av foci (white dots, some of these are appointed by white arrowheads). dUTPase depleted discs showed several nuclei with phospho-H2Av foci indicating DNA damage. Scale bar represents 50 µm.
Uracil–DNA measurements provided direct evidence that larval tissues of D. melanogaster that undergo developmental degradation accumulate uracil in genomic DNA. Up to date, presence of highly uracil-substituted genomic DNA within wild type organisms was reported only in some viruses; e.g. in bacteriophages [1], [2] and recently in HIV. To our knowledge, the present study may present a first example for naturally occurring permanent existence of uracil–DNA in a free-living complex eukaryotic organism (cf. Figure 3C and 3D). This condition requires developmental down-regulation of dUTPase and absence of UNG in the case of D. melanogaster. Involvement of dUTPase in determining genomic uracil content by regulating dUTP levels was confirmed by dUTPase silencing that resulted in appearance of uracil–DNA in imaginal disc tissues (cf. Figure 4D). Silencing of dUTPase resulted in developmental defects and DNA strand breaks (cf. Figure 5 and Figure 6). We also reported that uracil–DNA is tolerated and interpreted at least from embryonic to 3rd larval stages.
The extraordinary situation of tolerance and stability of uracil–DNA may not be exclusively present in D. melanogaster as absence of ung is ubiquitous among Holometabola (Table 1). As uracil–DNA naturally occurs in larval tissues that are sentenced to death, we consider that uracil–DNA might be linked to metamorphosis and tissue degradation. Further investigations should be taken to describe the mechanism, its impact and its putative role.
Pupal lethality was observed in flies affected by mutations or silencing of purine biosynthesis enzymes [42], [43]. These animals showed apoptosis in developing imaginal primordium during metamorphosis that were also observable as darkened tissues. In these studies, deficient deoxynucleotide biosynthesis may have resulted in imbalanced dNTP levels and increased ratio of mismatches in DNA [44]. Overrepresented DNA modifications like cytosine methylation also result in pupal lethality [45], [46]. In this case increased methylation pattern of DNA inhibits transcriptional activity and cell cycle progression.
We can suggest two hypotheses to explain pupal intolerance of uracil–DNA. First, similarly to the case of hypermethylated DNA, uracil–DNA may show a decreased response and interaction with transcriptional regulators, activators or other morphogenetic factors required specifically during pupal metamorphosis. Second, one or more factor(s), functional only in the pupal stage, may process uracil–DNA resulting in genome instability and defects in cell cycle progression or cell death [47], [48]. Beyond UNG that is missing from Drosophila, other uracil–DNA glycosylases would be suspect for this role. In agreement with this suggestion, expression patterns of other uracil–DNA glycosylases (SMUG and Thd1) and AP endonucleases (Rrp1, RpS3 and RpLP0) indicate relative upregulation during metamorphosis after their lowest expression in larval stages according to microarray data (Table S3) [49]. Developmental downregulation of these base-excision DNA repair pathway (BER) enzymes may contribute to uracil–DNA appearance and tolerance in larval stages, and their upregulation may initiate uracil–DNA processing, destabilization or degradation. The pupal uracil–DNA intolerance pathways hypothesized above are intended to be analyzed in further experiments.
Taken together, we suggest three different factors as being responsible for the stage-specific elevated level of uracil in larval DNA: 1) lack of ung gene, 2) absence of dUTPase and 3) decreased levels of enzymes involved in uracil removal. We also conclude that dUTPase is essential for the full completion of the Drosophila life cycle, although its absence may be tolerated in the larval stages. Although fruit flies, and perhaps other Holometabola insects, are unique in possessing a developmental period when uracil–DNA can be tolerated, preservation and transmission of genetic information for several generations still requires dUTPase.
Materials and Methods
Uracil–DNA stability assays
For the uracil–DNA stability assays, pRm-eYFP, pDsRedMonomer-N1, pP{Gal4VP16} (kind gift from László Sipos) were amplified in E.coli K12 XL1Blue strain and CJ236 dut-1, ung-1 strain (NEB). Plasmids were purified with Plasmid Midi Kit (QIAGEN). Uracil content of the plasmids was checked with UDG and AP endonuclease treatment followed by standard agarose gel electrophoresis [48].
Uracil-containing plasmid stability in cell culture.
Transfection of pRm-eYFP-N-C* into Drosophila S2 cells was carried out in the presence of Cellfectine (Invitrogen) following the manufacturer's instructions and expression was induced from the metallothionein promoter at 25°C by addition of 700 µM CuSO4 and overnight incubation. pDsRedMonomer-N1 was transfected into HeLa cells by using Lipofectamine (Invitrogen). Samples were visualized with a Leica DMLS fluorescence microscope 48 hours after transfection.
Uracil-containing plasmid stability in Drosophila embryos.
pP{Gal4VP16} plasmids were injected into 0–30 min pP{mw+UASeGFP} transgenic Drosophila embryos (kind gift of László Sipos). For each experiment, app. 40 dechorionated embryos were microinjected. They were aligned on a glass coverslip, dried for 30 min, than covered with 10S Voltalef oil before injection. Plasmid concentration was adjusted to 1 mg/ml, by dilution in standard injection buffer. GFP signal was detected in pre-hatching embryos, 22 h after injection. Embryos without injection served as a negative control.
Cell viability assay
Drosophila Schneider 2 (S2) cells and human HeLa cells were cultured in 96 well plates at 5×104cells/well or 2×103 cells/well, respectively. The culture media used were Serum Free Insect Medium (Sigma, S3777) supplemented with 1% penicillin–streptomycin solution for S2 cells; and DMEM-F12 (Sigma, D8437) supplemented with 10% FBS and 1% penicillin–streptomycin for HeLa cells. FdUR (Sigma) was added at a final concentration range of 0.1–1000 µM. After 96 hours in culture, cell viability was quantified by Alamar Blue assay (BioSource). The experiment was repeated in triplicates.
Quantitative measurement of the uracil content in DNA samples
In order to quantify uracil content of DNA, a real-time quantitative PCR-based assay was used [10]. Genomic DNA was isolated and digested with NheI. DNA fragments of 4–5 kb were purified from gel. Real-time PCR was performed on Mx3000P qPCR System (Agilent Technologies) using EvaGreen dye (Biotium) and PfuTurbo Hotstart DNA polymerase and PfuTurbo Cx Hotstart DNA polymerase (Stratagene). A segment with 963 base length defined by the primers (puBSd-Fw 5′-TCGGGATGACTTTTGGGTTCTG-3′ and puBSd985R 5′-CGCGGTTTAACACAGCGTCGG-3′) is amplified during the PCR reaction. Two-fold dilution series were prepared from DNA samples. Three or more parallel measurements were performed.
dUTPase silencing by RNA interference and rescue of RNAi
UAS-IR stocks were obtained from Vienna Drosophila RNAi Centre (VDRC) [32]. Strain #21883 and #21884 was used for dUTPase silencing. Rescue was performed by co-expression of the RNAi construct with a dUTPase transgene. For details see Text S1, Tables S1 and S2, Figures S4 and S9.
Immunohistochemistry and Western blot analysis
Western blot analysis of larval organs and stage specific dUTPase expression was performed as described previously [21]. For immunohistochemistry, applied primary antibodies were anti-dUTPase (1∶10000) or mouse anti-phospho-Histone H2A.X (Ser139) (Millipore) (1∶250). The latter was shown to recognize Drosophila phospho-Histone H2Av [50]. Tissues were fixed in 50% n-Heptane and 50% PEM-formaldehyde (100 mM PIPES, 1 mM MgCl2, 1 mM EGTA, 2.5% Tween-20, 4% PFA, pH = 6.9) for 30 minutes, with vigorous shacking at room temperature (RT). Samples were washed with inactivating buffer (50 mM TRIS, 150 mM NaCl, 0.5% Tween-20, pH = 7.4). Blocking was performed in the following: 5% goat serum, 1.5% BSA, 0.1% Tween-20, 1% Triton-X 100, 0,001% NaN3, in PBS, pH = 7.4 for 4 hours at RT. Tissues were incubated in primary antibody diluted in blocking buffer (1∶10000), at 4°C for 16 h. Samples were further washed with blocking buffer for 8 h at RT. Secondary antibody was applied to visualize dUTPase staining (Alexa 543 conjugated anti rabbit IgG, Molecular Probes) in blocking buffer (1∶1000) for 2 h, RT. DAPI was used for DNA staining. After further washing steps, samples were mounted in FluorSave (Calbiochem). Images were either acquired with a Zeiss LSCM 710 or a Leica DM IL LED Fluo microscope equipped with a Leica DFC345 FX monochrome camera.
TUNEL assay
TUNEL assay was carried out as described in [40]. After careful dissection in PBS, imaginal discs were fixed (0.1M PIPES, pH = 6.9, 1 mM EDTA, 1% Triton X-100, 2 mM MgSO4, 1% formaldehyde) for 30 min at RT, washed for times with PBS buffer also containing 0.1% Triton X-100 (PBT) and two times with PBT 5X (PBS, 0.5% Triton X-100) (10 min each), and transferred into permeabilization solution (0.1M sodium citrate in PBT) for 30 min at 65°C. Discs were washed twice with PBT 5X, three times in PBT and incubated in reaction buffer (30 mM Tris-HCl, pH = 7.2, 140 mM sodium cacodylate, 1 mM cobalt chloride) for 30 min RT. Reaction was carried out in reaction buffer containing 0.2 unit/microL terminal deoxynucleotidyl transferase (NEB) and 5 microM Cy5-dUTP (GE Healthcare) for 1 hour RT. The reaction was stopped with PBT 5X and washed three times with PBT and finally with PBS. Nuclei were stained by Hoechst. Discs were imaged by laser scanning confocal microscopy (Zeiss LSCM 710). The number of TUNEL positive cells was determined by ImageJ (Rasband, W.S., ImageJ, National Institutes of Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/, 1997–2004.)
Supporting Information
Figure S1.
Percentage of fluorescent cells upon transfection with normal (T pl.) or uracil-substituted plasmids (U pl.). (A) Drosophila S2 cells, (B) HeLa cells. The number of observed fluorescent cells is also presented within the bars together with the total number of scored cells (shown in brackets).
Figure S2.
Genomic uracil content of embryo is under detection limit. Uracil content of Drosophila embryonic genome compared to that of DNA plasmid purified from wild-type E. coli. Both of the samples showed a value under the detection limit.
Figure S3.
Ung-ARP assay. UNG-ARP assay shows presence of uracil–DNA in Drosophila larvae. For negative and positive controls, genomic DNA samples from XL1 Blue and CJ236 ung-1, dut-1 E.coli strains were used respectively. CJ236 ung-1, dut-1 E.coli strain produces DNA with high uracil content (approx. 5500 uracil/million bases [8], [10]).
Figure S4.
Scheme of crossing for silencing of dUTPase in Drosophila larvae and pupae and for rescue of dUTPase RNAi. Crossing schemes are shown on panel A and C: Act-Gal4 means Gal4 gene coupled with actin 5C promoter that result in ubiquitous and constitutive expression of yeast transcription factor, Gal4 in transgenic D. melanogaster driving transcription of silencing element (IR) following the UAS promoter. F1 generation has two genotypes: Act-Gal4/UAS-IR animals express dsRNA for dUTPase silencing, and have no markers; in UAS-IR/CyO, GFP animals, the silencing element is not activate, curly wing (CyO) and GFP markers expressed. Silenced and non-silenced animals are distinguishable at larvae/pupae and imago stages on the basis of GFP (panel B) and CyO markers, respectively. Crossing scheme for silencing is shown on panel C: UAS-dUTPase-FLAG stands for the rescue construct. Two relevant categories of the F1 generation can be unambiguously distinguished based on the phenotype of the marker mutations of the CyO, SM6b, and TM3 balancer chromosomes. The TM3 phenotype marks the gene silenced progenies, while the rescued animals show noTM3 phenotype. Panel D shows Western blot for dUTPase in silenced versus rescued animals. Note the absence of dUTPase protein in silenced animals (silencing alleles 21883 and 21884), whereas the presence of dUTPase proteins in the rescued animals (rescuing alleles DMDUT20 and DMDUT29). Equivalent total protein loading was verified by developing the blot also against tubulin using anti-Tubulin (E7, provided by M. Klymkowsky; Developmental Studies Hybridoma Bank, University of Iowa, Iowa city, IA).
Figure S5.
Summary of pupal developmental processes. Red arrow shows the stage P5 (around 12–14 h after puparium formation) until lethality due to dUTPase silencing appear.
Figure S6.
Developmental arrest caused by dUTPase silencing in Drosophila pupae. Wild type (upper panels) and dUTPase silenced (bottom panels) pupae were compared in stages P4, P5–6, P6–7, and P9. Every panel shows four views of the same pupa: dorsal (upper two) and ventral (bottom two) with and without its puparium. Specific differences appear at or before P5: Malpighian tubules (arrows) and Yellow Body (asterices) never appears in dUTPase silenced pupae.
Figure S7.
Wild type structures of pharate adults 3 days after puparium formation. Wild type pupa was dissected at stage P11 where adult organs have already developed (A). Dissected Malpighian tubules (arrows on B) and Yellow Body (asterices on B) of wild type pupa these organs have never identified within dUTPase silenced pupae.
Figure S8.
Larval traits in dissected silenced pupae 3 days after puparium formation. Three days after puparium formation, dissected tissues of silenced pupae still preserve larval traits: testis is oval (A), foregut and gastric caeca show larval characteristics (B, D, asterices), Malpighian tubules (B, arrows) are thin characteristic for larval ones, and brain (C, white arrowhead) also preserves the basic structure of larval one. Darkened tissues may have resulted from necrosis, apoptosis or melanisation [36].
Figure S9.
Scheme of crossing for silencing of dUTPase in the dorsal compartment of Drosophila wing imaginal discs. Crossing scheme is shown on panel (A): virgin females of the MS1096 Gal4 enhancer trap line expressing Gal4 preferentially in the dorsal compartment of the wing and carrying UAS-Dicer2 in homozygous form on the second chromosome (Bloomington stock No. 25706) were crossed to males carrying the Gal4 inducible silencing element (UAS-IR) in homozygous form on the second chromosome. The silencing element was activated by the MS1096 driver [37] in female progenies only while F1 males served as an internal negative control where no silencing occurred. Silenced females exhibited dorsally curled wing phenotype (panel B) often with blisters. The penetrance of the phenotype was around 85%. About 35% of the silenced female progeny also showed blistering wings (panel C). Male progenies had no wing phenotype. Panel D shows the expression pattern of the MS1096 driver in the dorsal compartment of the wing disc visualized by crossing MS1096 females to UAS-MoesinCherry [51] males (panel D) (red fluorescent staining in the wing disc). MoesinCherry overexpressing female progeny had no wing phenotype.
Table S1.
Genomic position of UAS-IR constructs in dUTPase RNAi stocks.
Table S2.
dUTPase transgene rescues the dUTPase RNAi phenotype. Table shows the results of the rescue crosses. UAS-IR/SM6b; UAS-dUTPase-FLAG/TM3 males were crossed to Act-Gal4/CyO females (Figure S4). Two UAS-IR (21883 and 21884) and two transgenic rescue lines (DMDUT20 and DMDUT29) were combined. Number of progenies of the relevant F1 categories is shown. Gene silencing was complete since no UAS-IR/Act-GAl4; TM3/+ adult progeny was observed. However, when the dUTPase transgene was present, rescued animals survived to adulthood.
Table S3.
Uracil–DNA repair is perturbed in Drosophila. Microarray data available on FlyBase were used. Table shows mRNA level for genes involved in different DNA repair pathways, elements of uracil–DNA repair are highlighted on grey background. ↓ indicates mRNA level decrease, ↑ mRNA level increase, ≈ no stage-specific change. Note that the overall base excision repair is down-regulated during larval development, but other DNA repair processes are not.
Text S1.
Supplementary information. Includes Supplementary Materials and Methods and Supplementary References.
We thank Prof. Tibor Vellai and Ágnes Regős for kind help in using the Zeiss stereomicroscope and László Sipos for kind help in embryo microinjections.
Author Contributions
Conceived and designed the experiments: VM AH AB MP GR JB PV ME BGV. Performed the experiments: VM AH AB MP BH JB IK GM GR FJ PV ME. Analyzed the data: VM AH AB JB MP GM GR JB IK FJ PV ME BGV. Wrote the paper: VM AH AB MP GR PV ME BGV.
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Field Notes Brand and The Deck Network. Visit them please.
Fresh Signals
"Our agency is getting big. That's something to be happy about. But it's something to worry about, too, and I don't mind telling you I'm damned worried." Bill Bernbach wrote to his bosses in 1947. -jc.05.15.14 #
A post from the daily flow of linkage called Fresh Signals, at Coudal.com.

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Lynne Spears is aghast at the injustice of the way she and her teenage daughter, Jamie Lynn Spears, were treated compared to Sarah Palin when her teenage daughter, Bristol Palin's pregnancy was revealed.
She has a point. Two mothers. Two pregnant teenage daughters who have — and will — keep their babies. Two very different public outcries.
Lynn was widely vilified as a bad mother who hadn't raised Jamie Lynn with proper morals, who probably encouraged her daughter to keep her baby so she could use them as a money-making device (selling the pregnancy story and baby photos to OK!). Jamie Lynn was accused of having grown up too fast, gone Hollywood and for having sex at such a young age.
By contrast, Alaska Gov. Palin, John McCain's VP running mate, staunch advocate against abortion and for abstinence, was heralded as being a good mother for supporting her pregnant daughter and advising her to keep her baby. Few thought she'd been irresponsible for not teaching her daughter about birth control.
Bristol has received the velvet glove treatment and goes unscathed by criticism for her morals or her behavior, even when she did her command performance at the Republican National Convention with her boyfriend/baby daddy.
Lynne talks about all this in an interview in Newsweek to promote her new book about her daughters Jamie Lynn and Britney Spears, "Through the Storm: A Real Story of Fame and Family in a Tabloid World."
Here are some excerpts from the Newsweek interview with Lynne Spears:
"It's as if [Sarah Palin] became celebrated. I mean, the mother, Palin, was celebrated for this. Every woman in the world has applauded her strength and her convictions and poor little old Jamie Lynn, you saw how she was crucified — everybody did, firsthand. I just feel like it's been a very hypocritical situation."
Keep reading for Lynne's answer to the criticism that Jamie Lynn's situation might glamorize teen pregnancy:
To the criticism that Jamie Lynn's situation –- having a healthy happy baby –- might glamorize teen pregnancy:
"We don't want this said, 'cause this would be glamorizing, but I will tell you a secret: Her baby does sleep all night. What do you think about those apples? Maddie is the best baby I have ever seen. She is like a little angel. She's so content. She laughs and she coos and [is] ahead of herself with her stages. But of course we can't tell that because then we would just glamorize it more."
Advice to the Palin family:
"Everybody's situation is different. I would never tell anybody else what to do with their child. I would never attempt to make them think I knew more about their child. Even if they were to ask, I would tell them dig deep, what do you think?"
And about that OK! cover:
"Sometimes, when you're a celebrity, you have to choose the lesser of two evils. So she decided to do the OK! Magazine, give a spread, give some pictures, and it really did ease off the paparazzi. It has lightened up her load a lot …. Did it make it glamorous? It showed her with some cute little shots, having some fun moments with her little family. If they call it glamorous, well I'm sorry. But everybody knows that once you get a new baby, it's the most cherished time of your life — but it is not glamorous."
What do you think? Did the public and the media treat Jamie Lynne and her mom differently than they treated Bristol Palin and Sarah? Should they have? Why? Or why not?
Photos: Top, Lynne Spears with Jamie Lynn Spears, and Jamie Lynn alone: both WireImage. Bristol Palin in February, Brian Wallace: Juneau Empire


text:
- 1GB 64-Bit GDDR3
- Core Clock 625 MHz
- 1 x DVI 1 x HDMI
- 160 Stream Processors
- PCI Express 2.1 x16
Excellent HTPC GPU 12/01/2013
This review is from: DIAMOND Radeon HD 6450 DirectX 11 6450PE31GSS 1GB 64-Bit GDDR3 PCI Express 2.1 x16 HDCP Ready Video Card
Bought this for my HTPC... works beautifully! No muss no fuss install... install, install the drivers, update and you are good.
None so far.
Other Thoughts:
This is not a gaming GPU... I think it struggles with Solitaire, but as a streamer it works very, very well.
I did get my rebate from Diamond.
1 people found this review helpful. Did you? Yes / No Thanks for the valuable feedback you provided!


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Hi,I just fresh installed a FC5-t3 (2.6.15-1.1955_FC5) on a ppc64 system and noticed the following behavior with auditctl:
Inserting an audit rule in following manner works (ie. there is record for rule addition, and it generates a record when the syscall is executed)
auditctl -a action,list -S syscallHowever, the following does not work (ie. there is a record that a rule was added in log, but no record is generated when syscall is executed)
auditctl -a action,list -F arch=b32 -S syscall or auditctl -a action,list -F arch=b64 -S syscall The version of auditctl on the system is audit-1.1.4-5.1Michael tried this on an i386 FC5-t3 and he sees the same problem. But on an i386 with latest lspp.10 kernel everything works fine.
Has anyone experienced this problem? - Loulwa


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US: +1 630 333 9501 UK: +44 208 123 1770 email@example.com
Global Mobile GIS Market 2016-2020
Outlook of the global mobile GIS market
Technavio's market research analyst predicts the global mobile geographical information system (GIS) market to grow steadily at a CAGR of around 10% during the forecast period. The rising need to access data by field workers across industries is the primary growth driver for this market. The implementation of GIS in mobile devices will help organizations to leverage maps and streamline field workflows. Mobile GIS can also be used to aid utility workers in locating gas or water networks. The ability of mobile GIS to improve the overall efficiency of the field workforce will result in this market's steady growth during the forecast period.
The integration facility provided by mobile devices is the key driver for this market. Most mobile devices offer a multitude of integrated features that can be used to carry out various GIS tasks. For instance, the camera embedded in a mobile device can be used to take pictures of the working field. This eliminates the need to carry a desktop device and a camera separately. The widespread application of mobile GIS solutions across major end-user industries like government, military, natural resources, and utilities will lead to this market's growth during the predicted period.
End-user segmentation of the mobile GIS market
- Government
- Military
- Natural resources
- Utilities
In this market study, analysts have estimated the government sector to be the largest end-user segment of this market. At present, this segment accounts for more than 21% of the total market share and is envisaged to retain its hold over the market until 2020 owing to the augmented need for enhanced national security. Increasing investments in GIS technology by various governments from across the globe will spur the prospects for market growth in this segment during the forecast period.
Geographical segmentation of the mobile GIS market
- Americas
In terms of geography, the Americas accounted for approximately 49% of the total market share in 2015 and is anticipated to grow at a CAGR of around 9% until 2020. The presence of major GIS vendors in the US is a critical factor that spurs the prospects for market growth in this region. The government in this region is also spending heavily on GIS technology as it will help the utilities and the natural resources industries to bolster their efficiency.
Competitive landscape and key vendors
The global mobile GIS market is fragmented owing to the presence of a large number of vendors. Many vendors have started to enhance their market potential through strategies, which include developing mobile applications for mobile users. Since smaller companies have also started providing open-source mobile GIS applications for free, the possibility of new vendors entering this market is high.
Leading vendors in the market are -
- Hexagon
Other prominent vendors in the market include GIS Cloud, Rockwell Spatial, Trimble, Takor Group, Garafa, and Supergeo Technologies.
Key questions answered in the report include
- What will the market size and the growth rate be in 2020?
- What are the key factors driving the global mobile GIS market?
- What are the key market trends impacting the growth of the mobile GIS market?
- What are the challenges to market growth?
- Who are the key vendors in this market space?
- What are the market opportunities and threats faced by the vendors in the global mobile GIS market?
- Trending factors influencing the market shares of the Americas, APAC, and EMEA.
- What are the key outcomes of the five forces analysis of the mobile GIS market?
Technavio also offers customization on reports based on specific client requirement.


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Hardee Electric strives to be the most reliable and professional electrical repair company for your home or office located in the greater Fort Worth, TX area. Our skilled and courteous techs provide the expertise, passion and dependability to complete every project quickly and efficiently, on time and on budget. Our electric repair company has a proven reputation of only providing the best home and office wiring and electrical service in the greater Fort Worth area.
Hardee Electric is your trusted Fort Worth and surrounding areas electrical professionals. We will competitively bid any project while only providing the highest quality workmanship. Stay within your budget – Contact us now for your free estimate. We look forward to solving all your electrical problems for both home and office.


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Britain is one of very few developed countries which will actually cut emissions. Others, including the United States, Canada and Australia, look set to break their promise and increase their output of carbon dioxide over the decade. Britain's success is mainly due to the drastic slump in the use of coal to generate electricity and its replacement by gas which produces much less carbon dioxide.
Mr Gummer is urging industrialised countries to commit themselves to cut emissions by between 5 and 10 per cent between 2000 and 2010. Last year, United Kingdom carbon dioxide emissions actually rose slightly, according to the latest statistics, because gas consumption rocketed by 17 per cent and oil by 2 per cent. So, while Britain does seem set to keep its international promise come 2000, the trend then is likely to be upwards.
Climate Change, UK Programme, Cm3558, HMSO. Nicholas Schoon
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Item added successfully to the Shopping Cartcontinue shopping proceed to checkout
There is a concealed zip fastening at the back and the dresses come complete with a slim black suede effect belt
The dresses are true 1950s length (approximately 43 inches from shoulder to hem in a Size 8)
We have photographed the dress using a 1950s style 27" petticoat, which helps to show the fullness of the skirt
Although not essential to wear with one, they really do create an extremely Vintage look
We only use the best quality materials in our dresses, and this Cotton Sateen has excellent stretch
This dress is not lined
Manufactured by Lady V London and Made in London
97% Cotton
Machine washable at 30 degrees (delicate wash)
Do not hand wash
Do not soak
Do not tumble dry
Shipping Policy:
Ships from India within 7 to 10 days.
Returns Policy:
No questions asked return or exchange*: items may be returned or exchanged for a full refund in the original form of payment or an Elitify Credit within 14 days after shipping. However, for items in final sale no exchange or return will be initiated. Your satisfaction is our biggest prize, so we do everything to ensure that you are happy with your purchase. All products are 100% authentic, procured from brands directly and are backed by our purchase protection with 10 times money back guarantee


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This mall has collectibles, antiques, primitives, and locally handmade goods. On West Frankfort Main Street. Only 10 miles south of Rend Lake.
CITY:West Frankfort
More than 10,000 square feet of antiques and collectibles located seven miles south of Marion on Route 37 and west on Whitecotton Rd., or take exit 45 on interstate 57 and follow the signs north.
Whimsy, A Children's Clothing Store
This children's clothing store features boutique clothing at reasonable pricing. They carry a wide variety of styles from newborn to children's size 8. Boutique brands they carry include (but not limited to): Banana Split, Three Sisters, Persnickety, Sweet Petunia, Chibella, Peaches & Cream, Mustard Pie, Flit and Flitter, Moxie & Mabel, and Matilda Jane
Weller florist has all your floral needs for weddings, funerals, or just any occasion. Professional designers are on staff to help you create the perfect piece for your home or occasion in silk or fresh flowers.
With over 15 years of experience and a lifelong passion for flowers, They carry a large selection of quality flowers to suit your taste and style. A rainbow of roses, wildflowers and seasonal blooms yield never ending combinations to create unique designs. Gourmet Baskets, Fruit Baskets, Chocolate and Theme Baskets are custom made to order. European Dish Gardens, Orchid Plants and seasonal flowering. We also carry inspirational, home decor and baby items to make the perfect gift.
More than 11,000 square feet of booths in a century-plus old warehouse offers a variety of antiques and collectibles.
Enclosed mall with ten stores. Walkers welcome. Restaurants nearby.
CITY:West Frankfort
From Our Hands to Yours, another unique DuQuoin Main Street shoppers delight. Find antiques, crafts and gifts of all sorts.
CITY:Du Quoin
Shopping mall with Sears, JC Penney, Peebles, and a variety of specialty shops.
CITY:Mount Vernon
Huge selection of yarn and knitting items including needles, workbooks, patterns, and much more. Store offers beginners classes, knit alongs, and open work station time. Hundreds of colors to choose from.
This primitive shop includes candles, country lighting, painted and upholstered furniture, Willow Tree, country gifts and accessories.
A gift shop that brings out the kid in you. Specializing in hand crafted gifts, doll clothes and doll accessories, inspirational signs and country decor. Doll clothes to fit the 18 inch and 14-15 inch dolls, like American Girl dolls. You can also stuff your own plush animal. Birthday parties are available.
Stevens Floral Gifts and Framing
Stevens offers a large selection of floral supplies, gifts and has a framing center onsite to fit all of your crafting needs. It is a great place to find gift ideas and is also a Vera Bradley retailer.
Southern Illinois Artisans Shop and Visitors Center
More than 25 categories of arts and crafts from fine art to the traditional crafts from more than 900 Illinois artisans. Also, home to the Southern Illinois Art Gallery. Special workshops and events throughout the year.
Located off Route 37 in Johnston City, this antique store has glassware, kitchenware, collectibles, and much more. Lots of variety in this newly established business.
CITY:Johnston City
Schwartz Orchard in Mt. Vernon
This orchard features fresh-picked apples, peaches, a pumpkin patch, cider and special events for children during apple season.
With over 50 vendor spaces, Rosebud Antique Mall offers high quality antiques and collectibles for all budgets. Primitives, fine antique furniture, quilts, china and glassware, pottery, jewelry, toys, framed art, comics, stained glass and many other unique items fill our clean and well-lit space.
Rend Lake College Market Place
Large golf outlet store plus clothing, brass, crafts and sporting goods store. Formerly Jent factory outlet mall.
CITY:Mount Vernon
Fine antiques, furniture, and collectibles in old historic building on Main Street of Sesser.
The Rabbit Patch Cottage is a unique gift store located in beautiful downtown Herrin. They are know for having Boyd's bears, candles, country lighting, painted and upholstered furniture, Willow Tree statues, country gifts, and accessories.
Pit Road Racing is Southern Illinois' largest Nascar shop. We offer a variety of Nascar Collectibles, memorabilia, nascar apparel, and hard to find items. Specialization in Diecast 1/24 scale and Apparel. If you can't find it in our store, we'll do our best to find it for you. We also offer custom apparel and window decals for your school, church, business, and everything in between.
Pickers Paradise Antiques, Crafts and Collectibles
Pickers Paradise has a variety of antiques, crafts and collectibles. Look for one of a kind items like an antique loom, furniture and much more.
Pickers Outlet Mall is a new 5,000 square foot building located on the busy Herrin Road (1.5 miles west of I-57). The mall can accommodate about 43 vendors. It is set-up like an antique mall, but has more to offer than antiques. It is a fun place to shop where the merchandise is changing daily.
CITY:Johnston City
Perfectly Posh Boutique and Gifts
Boutique & gift shop carrying clothing, shoes, handbags, accessories, jewelry, gourmet foods, candles, and various other gift items.


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Name: Belle Malkin
Born: July 28th, 1980
Age: 12
Physical description: she has deep set chestnut colored eyes; her hair is the color of burnished wood and is thick-curly and chest-lengthed; her skin is a soft-cream with rose colored cheeks
Species: human-shifter
Elemental: earth
Form: wolf
Parents: Kapricia (Nee: Hughes/mother) and Kay Malkin (father)
Siblings: Irvin Malkin (brother)
Grandparents: Isabelle and Lionel Hughes (mother side) Juno and James Malkin (father side)
Husband: Charles Hale
© Copyright 2017 Meggie Lynn. All rights reserved.
Status: Finished
Genre: Fantasy
Poem / Fantasy
Poem / Fantasy
Poem / Horror
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The Advanced Fighting Centre
10 High Bridge, Newcastle Upon Tyne, NE1 1EN
My name is John Atkin I Live In Newcastle (England) with my wife Kerry and our 2 daughters Molly & Erin, we are the proud owners of the AFC Newcastle.
I first started training at the age of 13, my first passion was Ninjutsu.
In the past I have trained not only all over England, but in Europe, Japan & America. I continue training and traveling as much as possible.
I got my 1st Dan in 1990 and have never looked back, I am now a 10th Dan in Ninjutsu, 4th Dan BCA (British Combat Association) and 4th Dan BASK (British all styles kickboxing) I am still learning new things every day.
I started teaching at the age of 18 in schools and church halls, but always dreamt of opening my own gym. This became a reality when in 1993 I signed a lease and was on my way…Read More About John Atkin
Latest News
Donec imperdiet lacinia justo
Instructors history, bio and disciplines
John AtkinAge: 47Rank: Owner/Instructor
Lee MairAge: 40Rank: Instructor
Rob RebairAge: 50Rank: Instructor
Our Classes
Contact us for more information
Ninjutsu taught today as part of Bunjinkan Budo Taijutsu, is a martial art with a traceable history stretching back 900…Read More
The grappling club offers all aspects of GI and None GI grappling Inc: – Ju-jitsu submissions Wrestling Mixed Martial Arts…Read More
A.F.C. Kickboxing is governed by the British All Styles Kickboxing Association… Click the link below for more information on our kickboxing classesRead More
The Weapons club covers Traditional Sword to Filipino Stick Work, so we go from the oldest traditional weapon up to…Read More
This club is designed to compliment all of the A.F.C. classes into oneRead More
Read more about kids classes in the link belowRead More
Private tuition caters to the individual needs. These are 1 hour classes, they have to be pre-booked as the fill…Read More


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FireEye Blogs
Information and insight on today's advanced threats from the leader in advanced threat prevention.
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- Spear Phishing Techniques Used in Attacks Targeting the Mongolian Government
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- APT28: At the Center of the Storm
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The latest updates on FireEye products and services from the teams building the FireEye Security platform.
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Covers the latest news and trends in cyber threats and cyber security focusing on the impact to business.
- Every day we see #endpointfail (and we built something better)
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- FireEye and Marsh & McLennan at the World Economic Form – A Closer Look at the Perfect Storm Threatening Europe
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- Defining a Security-Conscious Boardroom


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Iberostar Averroes, Hammamet
- Free breakfast, free WiFi, and free parking
Main amenities
- 266 guestrooms
- On the beach
- 3 restaurants
- Full-service spa
- Indoor pool and outdoor pool
- Health club
- Children's club
- 24-hour front desk
- Air conditioning
- Daily housekeeping
- Garden
- Car rentals on site
For families
- Free cribs/infant beds
- Children's pool
- Children's club
- Playground on site
- Children's club (surcharge)
- Private bathroom
What's around
- In Yasmine
- Yasmine Beach (0.2 mi / 0.4 km)
- Casino La Medina (0.4 mi / 0.6 km)
- Carthage Land (0.6 mi / 1 km)
- Yasmine Hammamet (0.7 mi / 1.1 km)
- Paintball Tunisie (1 mi / 1.6 km)
Iberostar Averroes, Hammametfrom $43
- Double Room, Garden View (Single Use)
- Twin Room, Sea View
- Double Room, Garden View
- Twin Room, Partial Sea View
- Double Room, Garden View
More great choices
At a glance
Key facts
Hotel size
- This hotel has 266 rooms
- This hotel is arranged over 4 floors
- Check-in time 2 PM-midnight
- Check-out time is noon
Required at check in
- Credit card or cash deposit required
- Government-issued photo ID required
Travelling with others
- Children's club *
- Pets not allowed
- Free WiFi in public areas
- Free WiFi in rooms
- Free self parking (limited)
Other information
- All-inclusive rates available
Nearby Four Star Hammamet hotels
In the hotel
- Children's club (surcharge)
- Free breakfast
- 3 restaurants
- Bar/lounge
- Coffee shop/café
- Snack bar/deli
- Poolside bar
- Indoor pool
- Outdoor pool
- Health club
- Tennis on site
- Full-service spa
- Sauna
- Children's pool
- Meeting rooms 2
- 24-hour front desk
- Concierge services
- Laundry facilities
- Hair salon
- Multilingual staff
- Number of buildings/towers - 1
- Year Built 2001
- Elevator/lift
- Garden
- In-room accessibility
In the room
- In-room climate control (air conditioning)
- Air conditioning
- Minibar
- Balcony
- Private bathroom
- Bathtub only
- Bidet
- Free toiletries
- Hair dryer
- Plasma TV
- Satellite TV channels
- Desk
- Free WiFi
- Phone
- Daily housekeeping
- In-room safe
Top Hammamet hotels
Recently reviewed hotels in Hammamet
- Sentido Le SultanFrom
"I was on a buisiness trip, left early, came mainly back in the evening. Staff allways friendly and helpfull, very comfortable stay for me. Impressive seavieuw! The breakfast…"Nov 4, 2016
- Hotel Bel Azur Thalasso & BungalowsFrom
"Staff if always pleasant, food is good with plenty of choices, pools are great, and the location is good for sunrises and sunsets."Oct 23, 2016
- Le Royal Hotels & Resorts - HammametFrom
"very nice with great beach acsess and nice pools and good food. "Sep 17, 2016
- Vincci Nozha Beach and SpaFrom
"Nice hotel very good place excellent Resturant , no wi Fi in rooms , nice staff , near beach , the price it's high "Jul 15, 2016
Nearby hotels
Also popular with travelers from the USA
Special features
All inclusive
All-inclusive room rates at Iberostar Averroes are available. These rates are higher because they include onsite food and beverages in the room rate (some restrictions may apply).
Guests can indulge in a pampering treatment at the hotel's full-service spa. Services include body treatments.
DiningLaurier Restaurant - This buffet restaurant serves breakfast, lunch, and dinner.
Olivier Italian Restauran - This theme restaurant serves dinner only.
Iberostar Averroes, Hammamet's small print
Also known as
- Iberostar Averroes
- Iberostar Averroes Hammamet
- Iberostar Averroes Hotel
- Iberostar Averroes Hotel Hammamet
- Iberostar Hotel Averroes
- Iberostar Hotel Hammamet
Optional extras
Children's club access is available for an extra charge
We have included all charges provided to us by this hotel. However, charges can vary, for example, based on length of stay or the room you book.
Recent reviews
Excellent 4.3 /5 from 70 reviews
the best hotel in hammamet ,thanks for all stuff, very friendly and courteous , especially Saoussen ikn the front desk , she is an amazing person
I /we found thr staff and hotel excellent. As Ive stayed in alot of 4 star hotels, I felt this was under rated. We moved to another 4 star that was over rated!! All the staff made us feel like valued guests and I was very sad to be leaving
All inclusive resort in Yasmine Hammamet. I stayed in a couple of hotels in the same area during my trip and this one was the best- it was modern, clean, safe, nice beach. Only thing worth improving is the wifi
amazing hotel with highly professional service team , the reception team are very friendly and helpful with nice communication skills, the animation team are wonderful, the food is delicious , lovely sand beach
very nice hotel friendly staff clean rooms I really enjoyed my stay and will come back for sure. Mr.Abbes was the best among all the staff I would like to thank him for his help and for being nice to us.
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Atlanta Litigation Lawyer, Georgia
Thomas Reynolds✓ VERIFIED
Credentials and Bar Status verified by Lawyer.com on January 10, 2017 Click here for more details
Mr. Reynolds was educated at Morehouse College in Atlanta, GA, where he graduated with honors before... (more)
Dorey Cole✓ VERIFIED
Credentials and Bar Status verified by Lawyer.com on March 28, 2014 Click here for more details
Cole Law Group, LLC is a multi-jurisdictional law firm that has represented and recovered damages for... (more)
I am an Atlanta attorney with a background in litigation, business and corporate law, securities... (more)
Alexander Gray Hait✓ VERIFIED
Credentials and Bar Status verified by Lawyer.com on August 03, 2014 Click here for more details
Mr. Alexander Gray Hait (âAlexâ) graduated from Georgia Tech in 1989 and went straight into Law School... (more)
Donald D. J. Stack
Don Stack brings to the law a knowledge of and respect for the... (more)
Richard J. Joseph
Richard Joseph heads SLC's Bankruptcy Law and Appellate Practice sections. Mr. Joseph has extensive... (more)
Thomas V. Keough
Tom Keough practices primarily in the areas of commercial law, bankruptcy, and general civil litigation.... (more)
Marion B. Stokes
Marion Stokes heads SLC's Creditor Rights & Commercial Law practice group. Mr. Stokes primarily... (more)
Michael J. Ernst
Michael Ernst heads both SLC's Business Litigation practice group and its Labor and Employment Law practice... (more)


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KUALA LUMPUR, Sept 22 (Reuters) - Malaysia's top carmaker, state-run Perusahaan Otomobil Nasional Bhd (Proton), said it has no plan to cut prices when the country lifts tariff barriers in 2005, the Edge weekly reported on Sunday. Proton's Chief Executive Mahaleel Tengku Ariff told the Edge in an interview there was no reasons for Malaysians to wait until 2005 to buy the Waja model, which was first introduced in 2000 and currently priced at 58,000 ringgit ($15,263) per unit. Malaysia, one ...
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Rutland Guest House
No prices found through our partners. Please contact the business directly or check some of our recommended alternatives.
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TripAdvisor Reviews Rutland Guest House St. Leonards-on-Sea
Travel Blogs from St. Leonards-on-Sea
Still at sea
... breakfast, whilst at sea, of Eggs Benedict. On our breakfast table was a couple from Williamsburg, Virginia who had flown to England to come on their 6th Mystery Cruise with Saga. They told us that Saga is the only company doing such cruises. They also visit London every year and take the opportunity to visit theatres, including the Royal Opera House. This year they had seen Krol Roger. There was, of course, an extended conversation over breakfast. Then it was ...
Afternoon Tea
... and Lily was packed up all ready to venture out. Unfortunately the weather in the UK lived up to its reputation, and the day was wet and cold. This wasnt going to dampen out spirits. Mum tucked herself up in the back with a duvet and Merv and I wrapped up in the front. The drive went without a hiccup and we found ourselves a lovely little spot in the carkpark. As it wasnt the greatest day weatherwise, we managed ...
What To Make Of It All?
... there seems to be a concerted effort from the Government to wean these down through education and installing a desire amongst the youth to make better for themselves.This will eventually happen and when it does expect Brazil to be one of the most influential countries in the world.Its nature,sights and sounds make a lasting impression that will make you want to come back again and again.Soon we will find this a country we will want to emigrate to.
Bad Beanie Day at Horsted Keynes.
... of them to ensure they both survive. My experience of pubs in remote Oz is that people will always favour one or the other to the point of heated rivalry, so it was nice to hear this.
Heading back towards Mary's home, 'Woodwinds', I spotted myself in the reflection of her front door. I realised then why the spotty teenager thought I was an oddball; my beanie. The whole village probably knows about the grave disturbing Garden Gnome from Down Under now... B.
This place is fabulous...
... is what I have)? No. Do they have to pay to see the doctor? No. Do they have to pay extra for pre-existing conditions? NO. Do they have to pay for contraception? No. Do Britons go broke because they get sick? No.
Are Brits healthier than Americans? Yep. Strange how that works...
And before you go bonkers and say... "But, but, but... they pay more in taxes." Um, no. Most Brits pay a whole lot less than I do! Seriously. No joke. No lie.


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LEADVILLE, Colo. — Dana M. Hurkes, 54, died Saturday, Aug. 10, 2013, in Leadville.
Services were Aug. 16 at the Bailey-Kent Funeral Home.
A reception was held in the Eagles Lodge.
He was born Sept. 1, 1958, in Watertown, S.D. When he was 2 years old, his family moved to Colorado.
Survivors include his parents, Alfred and Helen Hurkes; five siblings, David, Dustin, Deb, Diane and Dorene; and one son, Craig.


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PAK-IT Heavy-Duty All-Purpose CleanerMultipurpose Cleaners
Liquid Packets - Pleasant Scent - 20/ Pack - View Complete Details
Product Number: BIG574420002240
In Stock - Hurry, only 227 left.
Estimated Delivery - 1 business day(s) - Tue 2/28 2 business days - Tue 2/28
Shipping - This item is eligible for Free Shipping on orders over $45
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Product Details
Click for PDF
Click the link below to download a Material Safety Data Sheet for this product.
PAK-IT Heavy-Duty All-Purpose Cleaner - Simply drop one pre-measured purple Heavy Duty All Purpose Cleaner PAK-IT® into its matching purple quart trigger spray bottle (sold separately) and add cold, tepid or warm water. Wait two minutes and shake. Adjust nozzle to mist setting and simply spray and wipe off with a clean microfiber cloth or ideally a paper towel. Non-aerosol, phosphate-free, ammonia-free formula still packs a serious punch making it both safe and effective on all surfaces.
- Pre-measured portion PAK-It®s each create 32 ounces of commercial strength Heavy Duty All Purpose degreaser.
- No need to cut, tear or open—simply drop 1 PAK-IT® into it's matching color-coded quart trigger spray bottle and add water!
- Powerful heavy-duty formula cuts through grease and grime on all surfaces; ideal stainless steel cleaner for use on stoves, grills, sinks, ovens, fridges, freezers and commercial equipment.
- Create a RTU industrial strength degreaser in under two minutes at a fraction of the cost of traditional degreasers and all purpose cleaners.
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LORNA JANE PRESS PLAY TANK - MAGENTAFeatures:
› Womens Active Singlet
› Made from Stretch Cotton Blend Rib
› Skinny T-back straps to give you freedom to move
› Contrast twin needle stitch on neckline and armholes
› Inspirational print to keep you motivated
› LJ metal badge
› Colour: Magenta
Size & Fit Guide:
› Fitted singlet top
› Model's height 173cm (5'8")
› Model's bust 86cm (32")
› Model's waist is 63.63cm (25")
› Model is wearing a size 8
About Lorna Jane:
Betty's not so much about fashion, she's set to become a wardrobe essential! Layer this piece with that, Betty has covered every type of undergarment you can imagine. Smooth, seamless, nylon elastane, she's anything but basic! Shop Lorna Jane Online.


text:
Tropical Fish Keeping - Aquarium fish care and resources (http://www.tropicalfishkeeping.com/forum.php)
- Beginner Saltwater Aquariums (http://www.tropicalfishkeeping.com/beginner-saltwater-aquariums/)
- - found some critters (http://www.tropicalfishkeeping.com/beginner-saltwater-aquariums/found-some-critters-5518/)
found some critters
So iv been finding more creatures every day. I found these clear sacks of jelly on my glass, about 5 mm long and in the shape of a tooth. You can see there internal organs. I also found these small shrimp looking creatures in my lr. They are clear and about 4mm long. kinda look like small mantis shrimps. I also found a bristle worm but its orange so i dont know if its a bristle worm.
How long intill my lr cures? its been in there for about 2 weeks and im getting impatient. No amonia yet.
Sack of jelly
I have no real idea what is on your glass, Your pic is fuzzy. My guesses: obligate tunicates, hydroids, aptasia, nudibranch.
I have seen that also from time to time on mine , Mine looks like pink in color with 2 entannas coming from the front, My guess was it was a baby Snail, But i'm not sure it moves quick for something so little :dunno:
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This review is from: ENERMAX COENUS GUNMETAL GREY ECA3290A-G ATX MID TOWER COMPUTER CASE
Pros: I am very satisfied and very pleased to have bought this Enermax Mid Box. It has a lot of the needed space to install extra parts and work area for what ever your putting together! I will be getting more when needed.
Cons: None so far!READ FULL REVIEW
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Pros: Needless to say I am very Pleased with your product. It has many small indivigual storing slots needed for storing and carring with you all of my needed electrical connectors for computer work and as well for Ipads and Iphones. Thanks again I will be back for more when needed!
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Clarke draws little comfort
Published 04/08/2009
So, it meandered peacefully to a draw. Without alarms, twists or any of the other drama that habitually attends the Ashes, England were comfortably denied by Australia yesterday.
An innings of effortless grandeur by Michael Clarke, at least until its final rites, and another by Marcus North that was less opulent but equally effective ensured that the third npower Test petered out by mid-afternoon. Australia will be mightily relieved that they were able to repel their opponents with less fuss than it takes to make a cup of tea but the fact remains that they are still 1-0 down with only two matches left.
To retain the urn the tourists have to win at least once, while continuing to deprive England. They have hardly looked like doing so and for the second successive match had to try to hang on after a dreadful first innings.
Clarke, who reached his 12th Test hundred with the last shot of the match, and North, who failed by four runs to acquire his third, ensured that the match ended in a draw with 13.3 overs left, by which time Australia were 262 runs ahead. Had there been a sixth day it might have been rivetting.
If there are firm indications that some individual Australians are in form — Clarke above all — the capturing of 20 wickets looks as though it will remain a conundrum, though that goes for both sides. Although England will have been disappointed not to make more inroads yesterday, the ball resolutely refusing to swing as it had in the first innings, they will regroup at Headingley tomorrow content that they have once more forced their old foes on to the back foot.
Many of the next few hours and days, as between the first and second and second and third Tests, will be taken up with concerns of the state of Andrew Flintoff's right knee. His movement in the field yesterday could hardly have been more restricted had he been bound from head to foot in chains.
Perhaps he will be picked in the squad, perhaps he can be patched up sufficiently to take his place in the team but it will be a near thing either way.
Few Ashes matches of recent years have ended as tamely as this. Of course, we have all been spoiled by 2005 and by the Australians' pre-eminence on either side of it, and throughout the Sixties when draws were almost obligatory it would have been seen as the height of excitement.
It was in any case entirely forgivable because the first four days of the match brought little more than two days' play. In effect, therefore, Australia were saving the match on the third day — and only eight per cent of all Test matches scheduled to last five days have ended that soon.
England definitely fancied their chances. Having taken two wickets overnight they were well aware that two more early on would open the match up. None came in the first hour, however, and Andrew Strauss made the mistake of expecting too much of Flintoff, rather than entrusting the ball to Jimmy Anderson, so potent in the first innings here.
Anderson was summoned only after Flintoff had toiled for seven overs and struck immediately when Shane Watson drove at a straight one. Before lunch Mike Hussey had gone in similar fashion, lured into reaching for a ball from Stuart Broad. Hussey had done enough to rediscover form, not quite sufficient to make Australia safe.
But England needed quick wickets after lunch and they never looked like coming. Clarke was splendid, his strokeplay crisp and precise. North is a more functional sort of player as five English counties will attest but he has made everything of his late call to Australia's colours.
Long before the evening session the match was realistically out of England's reach and it seemed that the day would be called not long after both men reached their second centuries of the series. North was in a rush to achieve his and plundered an over from Ravi Bopara for three successive fours, with the best three strokes of his innings — a back foot straight drive, a cover drive and a deftly placed cut.
A slashed drive at the other end off Broad seemed certain to give him his 16th four and his third Test hundred, except that Anderson, in the decidedly unconventional position of wide fly slip took off to his right and plucked a stunning catch.
Clarke was suddenly much more circumspect. Strokes which had been refined suddenly became ugly. He too might have gone on 96, the victim of another superb catch from Anderson behind the wicket, but Ravi Bopara had overstepped the popping crease and a no ball was called.
The teams shook hands on the draw as soon as Clarke pulled his 192nd ball for four, his 14th, in Bopara's next over. England had needed eight wickets in the day but they had taken only three on a benign pitch. They lacked zing if there ain't no swing.
England will be desperate to keep what they have but Headingley may not be the place to protect but to be bold. Above all, they must continue to believe they can beat Australia and some of that belief may have been drained.
Belfast Telegraph