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Uracil-containing DNA in Drosophila: stability, stage-specific accumulation, and developmental involvement.

Muha V, Horváth A, Békési A, Pukáncsik M, Hodoscsek B, Merényi G, Róna G, Batki J, Kiss I, Jankovics F, Vilmos P, Erdélyi M, Vértessy BG - PLoS Genet. (2012)

Bottom Line: Upon pupation and metamorphosis, uracil content in DNA is significantly decreased.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Enzymology, Research Center for Natural Sciences, Hungarian Academy of Science, Budapest, Hungary.

ABSTRACT
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.

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Silencing of dUTPase in Drosophila larvae and pupae.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.
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pgen-1002738-g004: Silencing of dUTPase in Drosophila larvae and pupae.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.

Mentions: 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).


Uracil-containing DNA in Drosophila: stability, stage-specific accumulation, and developmental involvement.

Muha V, Horváth A, Békési A, Pukáncsik M, Hodoscsek B, Merényi G, Róna G, Batki J, Kiss I, Jankovics F, Vilmos P, Erdélyi M, Vértessy BG - PLoS Genet. (2012)

Silencing of dUTPase in Drosophila larvae and pupae.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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3369950&req=5

pgen-1002738-g004: Silencing of dUTPase in Drosophila larvae and pupae.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.
Mentions: 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).

Bottom Line: Upon pupation and metamorphosis, uracil content in DNA is significantly decreased.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Enzymology, Research Center for Natural Sciences, Hungarian Academy of Science, Budapest, Hungary.

ABSTRACT
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.

Show MeSH