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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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Episomal detection using PCR. A. PCR reaction products from 8 individual transgenic adult D. melanogaster (Canton S) with the autonomous element Hermes 7011 (lanes 1-8). Lanes 9-12 contain the products of identical PCR reactions using the equal amounts of genomic DNA from non-transgenic Canton S individuals. The diagram below schematically illustrates the structure and size of PCR products arising from Hermes elements with their terminal inverted repeats (thick arrows) joined as shown using the primers indicated (66R, 2681F) with variable (n) numbers of nucleotides of intercalary DNA. The positions and size in basepairs of molecular weight standards are indicated. B. PCR reaction products from 9 individual M. domestica adults from 3 geographically distinct natural populations. The diagram below schematically illustrates the structure and size of PCR products arising from Hermes elements with their terminal inverted repeats (thick arrows) joined as shown using the primers indicated (66R, 2431F) with variable (n) numbers of nucleotides of intercalary DNA. Molecular weight markers (m) and their sizes in basepairs are shown. Roman numerals refer to bands that were excised, reamplified, cloned and sequenced. The results are shown in Table 5. C. PCR reaction products from 6 individual M. domestica adults from the laboratory colony, Cs. DNA from unfertilized eggs (e) from this colony was also used as template in these PCR reactions. The positions and size in basepairs of molecular weight standards are indicated.
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Figure 2: Episomal detection using PCR. A. PCR reaction products from 8 individual transgenic adult D. melanogaster (Canton S) with the autonomous element Hermes 7011 (lanes 1-8). Lanes 9-12 contain the products of identical PCR reactions using the equal amounts of genomic DNA from non-transgenic Canton S individuals. The diagram below schematically illustrates the structure and size of PCR products arising from Hermes elements with their terminal inverted repeats (thick arrows) joined as shown using the primers indicated (66R, 2681F) with variable (n) numbers of nucleotides of intercalary DNA. The positions and size in basepairs of molecular weight standards are indicated. B. PCR reaction products from 9 individual M. domestica adults from 3 geographically distinct natural populations. The diagram below schematically illustrates the structure and size of PCR products arising from Hermes elements with their terminal inverted repeats (thick arrows) joined as shown using the primers indicated (66R, 2431F) with variable (n) numbers of nucleotides of intercalary DNA. Molecular weight markers (m) and their sizes in basepairs are shown. Roman numerals refer to bands that were excised, reamplified, cloned and sequenced. The results are shown in Table 5. C. PCR reaction products from 6 individual M. domestica adults from the laboratory colony, Cs. DNA from unfertilized eggs (e) from this colony was also used as template in these PCR reactions. The positions and size in basepairs of molecular weight standards are indicated.

Mentions: Hermes is a natural inhabitant of the genome of M. domestica and has been detected in all individuals (n = 65) sampled from 13 populations from four continents [36]. Because the Hermes element in these populations did not naturally contain a functional origin of replication, episomes could not be recovered by plasmid rescue and could only be detected using a PCR-based method that relied on the use of PCR primers specific to the right and left terminal sequences of the element that would result in PCR products only when the termini were joined end to end. Total genomic DNA isolated from individual M. domestica from three populations had evidence of episomal Hermes elements as indicated by the recovery of PCR products using end-specific primers (Figure 2B). The most abundant forms were perfect (434 bp) or near perfect (> 434 bp) end-to-end joints containing all of the information necessary for transposition (Figure 2B; Table 5). As in transgenic D. melanogaster, 'defective' forms were also recovered with variable amounts of terminal sequences deleted from PCR products less than 434 bp (Figure 2B). All 'defective' forms recovered were missing the right inverted terminal repeat and, in one case, both termini were absent (Table 5). Finally, episomal Hermes elements were detected in unfertilized eggs of M. domestica (Figure 2C).


Transpositionally active episomal hAT elements.

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

Episomal detection using PCR. A. PCR reaction products from 8 individual transgenic adult D. melanogaster (Canton S) with the autonomous element Hermes 7011 (lanes 1-8). Lanes 9-12 contain the products of identical PCR reactions using the equal amounts of genomic DNA from non-transgenic Canton S individuals. The diagram below schematically illustrates the structure and size of PCR products arising from Hermes elements with their terminal inverted repeats (thick arrows) joined as shown using the primers indicated (66R, 2681F) with variable (n) numbers of nucleotides of intercalary DNA. The positions and size in basepairs of molecular weight standards are indicated. B. PCR reaction products from 9 individual M. domestica adults from 3 geographically distinct natural populations. The diagram below schematically illustrates the structure and size of PCR products arising from Hermes elements with their terminal inverted repeats (thick arrows) joined as shown using the primers indicated (66R, 2431F) with variable (n) numbers of nucleotides of intercalary DNA. Molecular weight markers (m) and their sizes in basepairs are shown. Roman numerals refer to bands that were excised, reamplified, cloned and sequenced. The results are shown in Table 5. C. PCR reaction products from 6 individual M. domestica adults from the laboratory colony, Cs. DNA from unfertilized eggs (e) from this colony was also used as template in these PCR reactions. The positions and size in basepairs of molecular weight standards are indicated.
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Figure 2: Episomal detection using PCR. A. PCR reaction products from 8 individual transgenic adult D. melanogaster (Canton S) with the autonomous element Hermes 7011 (lanes 1-8). Lanes 9-12 contain the products of identical PCR reactions using the equal amounts of genomic DNA from non-transgenic Canton S individuals. The diagram below schematically illustrates the structure and size of PCR products arising from Hermes elements with their terminal inverted repeats (thick arrows) joined as shown using the primers indicated (66R, 2681F) with variable (n) numbers of nucleotides of intercalary DNA. The positions and size in basepairs of molecular weight standards are indicated. B. PCR reaction products from 9 individual M. domestica adults from 3 geographically distinct natural populations. The diagram below schematically illustrates the structure and size of PCR products arising from Hermes elements with their terminal inverted repeats (thick arrows) joined as shown using the primers indicated (66R, 2431F) with variable (n) numbers of nucleotides of intercalary DNA. Molecular weight markers (m) and their sizes in basepairs are shown. Roman numerals refer to bands that were excised, reamplified, cloned and sequenced. The results are shown in Table 5. C. PCR reaction products from 6 individual M. domestica adults from the laboratory colony, Cs. DNA from unfertilized eggs (e) from this colony was also used as template in these PCR reactions. The positions and size in basepairs of molecular weight standards are indicated.
Mentions: Hermes is a natural inhabitant of the genome of M. domestica and has been detected in all individuals (n = 65) sampled from 13 populations from four continents [36]. Because the Hermes element in these populations did not naturally contain a functional origin of replication, episomes could not be recovered by plasmid rescue and could only be detected using a PCR-based method that relied on the use of PCR primers specific to the right and left terminal sequences of the element that would result in PCR products only when the termini were joined end to end. Total genomic DNA isolated from individual M. domestica from three populations had evidence of episomal Hermes elements as indicated by the recovery of PCR products using end-specific primers (Figure 2B). The most abundant forms were perfect (434 bp) or near perfect (> 434 bp) end-to-end joints containing all of the information necessary for transposition (Figure 2B; Table 5). As in transgenic D. melanogaster, 'defective' forms were also recovered with variable amounts of terminal sequences deleted from PCR products less than 434 bp (Figure 2B). All 'defective' forms recovered were missing the right inverted terminal repeat and, in one case, both termini were absent (Table 5). Finally, episomal Hermes elements were detected in unfertilized eggs of M. domestica (Figure 2C).

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