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Transpositionally active episomal hAT elements.

O'Brochta DA, Stosic CD, Pilitt K, Subramanian RA, Hice RH, Atkinson PW - BMC Mol. Biol. (2009)

Bottom Line: V(D)J signal joints, which resemble episomal transposable elements, have been considered non-recombinogenic products of V(D)J recombination and a safe way to dispose of excised chromosomal sequences.Up to 50% of hobo/Hermes episomes contained two intact, inverted-terminal repeats and 86% of these contained from 1-1000 bp of intercalary DNA.Episomal hobo/Hermes elements were recovered from Musca domestica (a natural host of Hermes), Drosophila melanogaster (a natural host of hobo) and transgenic Drosophila melanogaster and Aedes aegypti (with autonomous Hermes elements).

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Biosystems Research, University of Maryland Biotechnology Institute, Rockville, MD 20850, USA. obrochta@umbi.umd.edu

ABSTRACT

Background: hAT elements and V(D)J recombination may have evolved from a common ancestral transposable element system. Extrachromosomal, circular forms of transposable elements (referred to here as episomal forms) have been reported yet their biological significance remains unknown. V(D)J signal joints, which resemble episomal transposable elements, have been considered non-recombinogenic products of V(D)J recombination and a safe way to dispose of excised chromosomal sequences. V(D)J signal joints can, however, participate in recombination reactions and the purpose of this study was to determine if hobo and Hermes episomal elements are also recombinogenic.

Results: Up to 50% of hobo/Hermes episomes contained two intact, inverted-terminal repeats and 86% of these contained from 1-1000 bp of intercalary DNA. Episomal hobo/Hermes elements were recovered from Musca domestica (a natural host of Hermes), Drosophila melanogaster (a natural host of hobo) and transgenic Drosophila melanogaster and Aedes aegypti (with autonomous Hermes elements). Episomal Hermes elements were recovered from unfertilized eggs of M. domestica and D. melanogaster demonstrating their potential for extrachromosomal, maternal transmission. Reintegration of episomal Hermes elements was observed in vitro and in vivo and the presence of Hermes episomes resulted in lower rates of canonical Hermes transposition in vivo.

Conclusion: Episomal hobo/Hermes elements are common products of element excision and can be maternally transmitted. Episomal forms of Hermes are capable of integration and also of influencing the transposition of canonical elements suggesting biological roles for these extrachromosomal elements in element transmission and regulation.

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Structure of the autonomous Hermes elements used in this study. Hermes terminal sequences are shown as thick black arrows with associated nucleotide numbers in parenthesis. Primers used are thin arrows with their corresponding name. The position of relevant restriction endonuclease sites are shown using conventional abbreviations (BamHI, EcoRI, MspI). Hermes ORF - the complete Hermes transposase open reading frame. hsp70 5'-promoter from the hsp70 gene of D. melanogaster. hsp70 3' - 3' untranslated region of the hsp70 gene of D. melanogaster. Actin5C - promoter from the Actin5C gene of D. melanogaster. EGFP- the complete coding region for Enhanced Green Fluorescent Protein. SV40 3'- 3' untranslated region of Simian Virus 40. Ori - ColE 1 origin of replication. Kanamycin - kanamycin resistance gene.
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Figure 1: Structure of the autonomous Hermes elements used in this study. Hermes terminal sequences are shown as thick black arrows with associated nucleotide numbers in parenthesis. Primers used are thin arrows with their corresponding name. The position of relevant restriction endonuclease sites are shown using conventional abbreviations (BamHI, EcoRI, MspI). Hermes ORF - the complete Hermes transposase open reading frame. hsp70 5'-promoter from the hsp70 gene of D. melanogaster. hsp70 3' - 3' untranslated region of the hsp70 gene of D. melanogaster. Actin5C - promoter from the Actin5C gene of D. melanogaster. EGFP- the complete coding region for Enhanced Green Fluorescent Protein. SV40 3'- 3' untranslated region of Simian Virus 40. Ori - ColE 1 origin of replication. Kanamycin - kanamycin resistance gene.

Mentions: Hermes episomes were recovered from two transgenic D. melanogaster containing integrated, self-mobilizable Hermes elements (Hermes7011 and Hermes198H70-1) (Figure 1). Episomes in insects containing Hermes7011 were detected using a nested PCR strategy using two pairs of Hermes terminus-specific primers oriented so that PCR products will only arise when the termini are joined end-to-end (see Methods, Figure 1). The DNA sequence was determined for a sample of the cloned products arising from this PCR reaction (Table 2). Thirty-five percent (9/26) of the episomes recovered using this method had intact inverted repeats and all of the sequence information necessary for transposition. Only two of the recovered episomes were perfect end-to-end joins. Thirty-one percent (8/26) had only intact left inverted terminal repeats with variable amounts of the right end being deleted while 19% (5/26) had a reciprocal structure with intact right inverted terminal repeats and variable amounts of the left end being deleted. Fifteen percent of the recovered episomes were missing both inverted terminal repeats and adjacent sub-terminal sequences.


Transpositionally active episomal hAT elements.

O'Brochta DA, Stosic CD, Pilitt K, Subramanian RA, Hice RH, Atkinson PW - BMC Mol. Biol. (2009)

Structure of the autonomous Hermes elements used in this study. Hermes terminal sequences are shown as thick black arrows with associated nucleotide numbers in parenthesis. Primers used are thin arrows with their corresponding name. The position of relevant restriction endonuclease sites are shown using conventional abbreviations (BamHI, EcoRI, MspI). Hermes ORF - the complete Hermes transposase open reading frame. hsp70 5'-promoter from the hsp70 gene of D. melanogaster. hsp70 3' - 3' untranslated region of the hsp70 gene of D. melanogaster. Actin5C - promoter from the Actin5C gene of D. melanogaster. EGFP- the complete coding region for Enhanced Green Fluorescent Protein. SV40 3'- 3' untranslated region of Simian Virus 40. Ori - ColE 1 origin of replication. Kanamycin - kanamycin resistance gene.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2803484&req=5

Figure 1: Structure of the autonomous Hermes elements used in this study. Hermes terminal sequences are shown as thick black arrows with associated nucleotide numbers in parenthesis. Primers used are thin arrows with their corresponding name. The position of relevant restriction endonuclease sites are shown using conventional abbreviations (BamHI, EcoRI, MspI). Hermes ORF - the complete Hermes transposase open reading frame. hsp70 5'-promoter from the hsp70 gene of D. melanogaster. hsp70 3' - 3' untranslated region of the hsp70 gene of D. melanogaster. Actin5C - promoter from the Actin5C gene of D. melanogaster. EGFP- the complete coding region for Enhanced Green Fluorescent Protein. SV40 3'- 3' untranslated region of Simian Virus 40. Ori - ColE 1 origin of replication. Kanamycin - kanamycin resistance gene.
Mentions: Hermes episomes were recovered from two transgenic D. melanogaster containing integrated, self-mobilizable Hermes elements (Hermes7011 and Hermes198H70-1) (Figure 1). Episomes in insects containing Hermes7011 were detected using a nested PCR strategy using two pairs of Hermes terminus-specific primers oriented so that PCR products will only arise when the termini are joined end-to-end (see Methods, Figure 1). The DNA sequence was determined for a sample of the cloned products arising from this PCR reaction (Table 2). Thirty-five percent (9/26) of the episomes recovered using this method had intact inverted repeats and all of the sequence information necessary for transposition. Only two of the recovered episomes were perfect end-to-end joins. Thirty-one percent (8/26) had only intact left inverted terminal repeats with variable amounts of the right end being deleted while 19% (5/26) had a reciprocal structure with intact right inverted terminal repeats and variable amounts of the left end being deleted. Fifteen percent of the recovered episomes were missing both inverted terminal repeats and adjacent sub-terminal sequences.

Bottom Line: V(D)J signal joints, which resemble episomal transposable elements, have been considered non-recombinogenic products of V(D)J recombination and a safe way to dispose of excised chromosomal sequences.Up to 50% of hobo/Hermes episomes contained two intact, inverted-terminal repeats and 86% of these contained from 1-1000 bp of intercalary DNA.Episomal hobo/Hermes elements were recovered from Musca domestica (a natural host of Hermes), Drosophila melanogaster (a natural host of hobo) and transgenic Drosophila melanogaster and Aedes aegypti (with autonomous Hermes elements).

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Biosystems Research, University of Maryland Biotechnology Institute, Rockville, MD 20850, USA. obrochta@umbi.umd.edu

ABSTRACT

Background: hAT elements and V(D)J recombination may have evolved from a common ancestral transposable element system. Extrachromosomal, circular forms of transposable elements (referred to here as episomal forms) have been reported yet their biological significance remains unknown. V(D)J signal joints, which resemble episomal transposable elements, have been considered non-recombinogenic products of V(D)J recombination and a safe way to dispose of excised chromosomal sequences. V(D)J signal joints can, however, participate in recombination reactions and the purpose of this study was to determine if hobo and Hermes episomal elements are also recombinogenic.

Results: Up to 50% of hobo/Hermes episomes contained two intact, inverted-terminal repeats and 86% of these contained from 1-1000 bp of intercalary DNA. Episomal hobo/Hermes elements were recovered from Musca domestica (a natural host of Hermes), Drosophila melanogaster (a natural host of hobo) and transgenic Drosophila melanogaster and Aedes aegypti (with autonomous Hermes elements). Episomal Hermes elements were recovered from unfertilized eggs of M. domestica and D. melanogaster demonstrating their potential for extrachromosomal, maternal transmission. Reintegration of episomal Hermes elements was observed in vitro and in vivo and the presence of Hermes episomes resulted in lower rates of canonical Hermes transposition in vivo.

Conclusion: Episomal hobo/Hermes elements are common products of element excision and can be maternally transmitted. Episomal forms of Hermes are capable of integration and also of influencing the transposition of canonical elements suggesting biological roles for these extrachromosomal elements in element transmission and regulation.

Show MeSH
Related in: MedlinePlus