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The genome of the stick insect Medauroidea extradentata is strongly methylated within genes and repetitive DNA.

Krauss V, Eisenhardt C, Unger T - PLoS ONE (2009)

Bottom Line: Bisulfite sequencing of one of these fragments and of parts of conserved protein-coding genes revealed a methylcytosine content of 12.6%, mostly found at CpG, but also at CpT and CpA dinucleotides.Both repetitive DNA and coding genes appear to contain high levels of methylcytosines.These results argue for similar functions of DNA methylation in stick insects as those already known for vertebrates.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Institute of Biology II, University of Leipzig, Leipzig, Germany. krauss@rz.uni-leipzig.de

ABSTRACT

Background: Cytosine DNA methylation has been detected in many eukaryotic organisms and has been shown to play an important role in development and disease of vertebrates including humans. Molecularly, DNA methylation appears to be involved in the suppression of initiation or of elongation of transcription. Resulting organismal functions are suggested to be the regulation of gene silencing, the suppression of transposon activity and the suppression of initiation of transcription within genes. However, some data concerning the distribution of methylcytosine in insect species appear to contradict such roles.

Principal findings: By comparison of MspI and HpaII restriction patterns in genomic DNA of several insects we show that stick insects (Phasmatodea) have highly methylated genomes. We isolated methylated DNA fragments from the Vietnamese Walking Stick Medauroidea extradentata (formerly known as Baculum extradentatum) and demonstrated that most of the corresponding sequences are repetitive. Bisulfite sequencing of one of these fragments and of parts of conserved protein-coding genes revealed a methylcytosine content of 12.6%, mostly found at CpG, but also at CpT and CpA dinucleotides. Corresponding depletions of CpG and enrichments of TpG and CpA dinucleotides in some highly conserved protein-coding genes of Medauroidea reach a similar degree as in vertebrates and show that CpG methylation has occurred in the germline of these insects.

Conclusions: Using four different methods, we demonstrate that the genome of Medauroidea extradentata is strongly methylated. Both repetitive DNA and coding genes appear to contain high levels of methylcytosines. These results argue for similar functions of DNA methylation in stick insects as those already known for vertebrates.

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Related in: MedlinePlus

MspI/HpaII restriction analysis of selected insect species.Equivalent amounts of insect genomic DNA were digested with MspI (black line) and HpaII (red line) and separated at adjacent lanes of an agarose gel. The ethidium bromide signals of both lanes were plotted in different colors into one plot. Results are shown for Drosophila melanogaster, Bombyx mori, Apis mellifera and the walking sticks Sipyloidea sipylus and Medauroidea extradentata.
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pone-0007223-g001: MspI/HpaII restriction analysis of selected insect species.Equivalent amounts of insect genomic DNA were digested with MspI (black line) and HpaII (red line) and separated at adjacent lanes of an agarose gel. The ethidium bromide signals of both lanes were plotted in different colors into one plot. Results are shown for Drosophila melanogaster, Bombyx mori, Apis mellifera and the walking sticks Sipyloidea sipylus and Medauroidea extradentata.

Mentions: We started our analysis with a comparative digestion of genomic insect DNA using the restriction enzymes MspI and HpaII. Both enzymes are isoschizomers that recognize the target sequence 5′-CCGG-3′, but only HpaII is inhibited by methylation of the inner cytosine of this sequence. If the digestion pattern of HpaII is shifted to higher molecular weights in relation to the MspI pattern, the corresponding genome contains mCpG (methylcytosine, followed by a guanine). The genomic DNA of two walking sticks (Sipyloidea sipylus and Medauroidea extradentata) clearly showed such a difference (Figure 1), while DNA of Drosophila melanogaster (Diptera), Bombyx mori (Lepidoptera) and Apis mellifera (Hymenoptera) was equally digested by MspI and HpaII. We note that for Apis and Bombyx CpG methylation was shown [17], [18], but the fraction of methylated CpG is probably rather low in both species.


The genome of the stick insect Medauroidea extradentata is strongly methylated within genes and repetitive DNA.

Krauss V, Eisenhardt C, Unger T - PLoS ONE (2009)

MspI/HpaII restriction analysis of selected insect species.Equivalent amounts of insect genomic DNA were digested with MspI (black line) and HpaII (red line) and separated at adjacent lanes of an agarose gel. The ethidium bromide signals of both lanes were plotted in different colors into one plot. Results are shown for Drosophila melanogaster, Bombyx mori, Apis mellifera and the walking sticks Sipyloidea sipylus and Medauroidea extradentata.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2747282&req=5

pone-0007223-g001: MspI/HpaII restriction analysis of selected insect species.Equivalent amounts of insect genomic DNA were digested with MspI (black line) and HpaII (red line) and separated at adjacent lanes of an agarose gel. The ethidium bromide signals of both lanes were plotted in different colors into one plot. Results are shown for Drosophila melanogaster, Bombyx mori, Apis mellifera and the walking sticks Sipyloidea sipylus and Medauroidea extradentata.
Mentions: We started our analysis with a comparative digestion of genomic insect DNA using the restriction enzymes MspI and HpaII. Both enzymes are isoschizomers that recognize the target sequence 5′-CCGG-3′, but only HpaII is inhibited by methylation of the inner cytosine of this sequence. If the digestion pattern of HpaII is shifted to higher molecular weights in relation to the MspI pattern, the corresponding genome contains mCpG (methylcytosine, followed by a guanine). The genomic DNA of two walking sticks (Sipyloidea sipylus and Medauroidea extradentata) clearly showed such a difference (Figure 1), while DNA of Drosophila melanogaster (Diptera), Bombyx mori (Lepidoptera) and Apis mellifera (Hymenoptera) was equally digested by MspI and HpaII. We note that for Apis and Bombyx CpG methylation was shown [17], [18], but the fraction of methylated CpG is probably rather low in both species.

Bottom Line: Bisulfite sequencing of one of these fragments and of parts of conserved protein-coding genes revealed a methylcytosine content of 12.6%, mostly found at CpG, but also at CpT and CpA dinucleotides.Both repetitive DNA and coding genes appear to contain high levels of methylcytosines.These results argue for similar functions of DNA methylation in stick insects as those already known for vertebrates.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Institute of Biology II, University of Leipzig, Leipzig, Germany. krauss@rz.uni-leipzig.de

ABSTRACT

Background: Cytosine DNA methylation has been detected in many eukaryotic organisms and has been shown to play an important role in development and disease of vertebrates including humans. Molecularly, DNA methylation appears to be involved in the suppression of initiation or of elongation of transcription. Resulting organismal functions are suggested to be the regulation of gene silencing, the suppression of transposon activity and the suppression of initiation of transcription within genes. However, some data concerning the distribution of methylcytosine in insect species appear to contradict such roles.

Principal findings: By comparison of MspI and HpaII restriction patterns in genomic DNA of several insects we show that stick insects (Phasmatodea) have highly methylated genomes. We isolated methylated DNA fragments from the Vietnamese Walking Stick Medauroidea extradentata (formerly known as Baculum extradentatum) and demonstrated that most of the corresponding sequences are repetitive. Bisulfite sequencing of one of these fragments and of parts of conserved protein-coding genes revealed a methylcytosine content of 12.6%, mostly found at CpG, but also at CpT and CpA dinucleotides. Corresponding depletions of CpG and enrichments of TpG and CpA dinucleotides in some highly conserved protein-coding genes of Medauroidea reach a similar degree as in vertebrates and show that CpG methylation has occurred in the germline of these insects.

Conclusions: Using four different methods, we demonstrate that the genome of Medauroidea extradentata is strongly methylated. Both repetitive DNA and coding genes appear to contain high levels of methylcytosines. These results argue for similar functions of DNA methylation in stick insects as those already known for vertebrates.

Show MeSH
Related in: MedlinePlus