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In vitro transposition of ISY100, a bacterial insertion sequence belonging to the Tc1/mariner family.

Feng X, Colloms SD - Mol. Microbiol. (2007)

Bottom Line: Transposase made double-strand breaks on a supercoiled DNA molecule containing a mini-ISY100 transposon, cleaving exactly at the transposon 3' ends and two nucleotides inside the 5' ends.Cleavage of short linear substrates containing a single transposon end was less precise.Transposase also catalysed strand transfer, covalently joining the transposon 3' end to the target DNA.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedical and Life Sciences, Division of Molecular Genetics, University of Glasgow, Anderson College, 56 Dumbarton Rd, Glasgow G11 6NU, Scotland, UK.

ABSTRACT
The Synechocystis sp. PCC6803 insertion sequence ISY100 (ISTcSa) belongs to the Tc1/mariner/IS630 family of transposable elements. ISY100 transposase was purified and shown to promote transposition in vitro. Transposase binds specifically to ISY100 terminal inverted repeat sequences via an N-terminal DNA-binding domain containing two helix-turn-helix motifs. Transposase is the only protein required for excision and integration of ISY100. Transposase made double-strand breaks on a supercoiled DNA molecule containing a mini-ISY100 transposon, cleaving exactly at the transposon 3' ends and two nucleotides inside the 5' ends. Cleavage of short linear substrates containing a single transposon end was less precise. Transposase also catalysed strand transfer, covalently joining the transposon 3' end to the target DNA. When a donor plasmid carrying a mini-ISY100 was incubated with a target plasmid and transposase, the most common products were insertions of one transposon end into the target DNA, but insertions of both ends at a single target site could be recovered after transformation into Escherichia coli. Insertions were almost exclusively into TA dinucleotides, and the target TA was duplicated on insertion. Our results demonstrate that there are no fundamental differences between the transposition mechanisms of IS630 family elements in bacteria and Tc1/mariner elements in higher eukaryotes.

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Mapping cleavage positions on supercoiled substrates. A. Transposase was incubated with supercoiled plasmids containing ISY100 or ISY100-kan to give ETF and vector fragments, which were purified from an agarose gel. 3′ OH ends were tailed using terminal transferase and dCTP or dGTP. Tailed products were amplified by PCR using the indicated primers and cloned by topo-TA cloning. The end-tail junction sequences obtained and their frequencies are shown. B. The vector fragment was circularized with T4 DNA ligase, giving the junction sequence shown.
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fig07: Mapping cleavage positions on supercoiled substrates. A. Transposase was incubated with supercoiled plasmids containing ISY100 or ISY100-kan to give ETF and vector fragments, which were purified from an agarose gel. 3′ OH ends were tailed using terminal transferase and dCTP or dGTP. Tailed products were amplified by PCR using the indicated primers and cloned by topo-TA cloning. The end-tail junction sequences obtained and their frequencies are shown. B. The vector fragment was circularized with T4 DNA ligase, giving the junction sequence shown.

Mentions: To see if cleavage one nucleotide inside the transposon end occurs only on small linear substrates, transposase cleavage sites were mapped on plasmid substrates containing mini-transposons. After in vitro cleavage with transposase, the ETF and the vector backbone fragments were purified by gel electrophoresis and G- or C-tailed using terminal transferase. End-tail junctions were amplified by PCR with poly C or poly G primers together with vector- or transposon-specific primers (Fig. 7A). PCR products were ligated into a cloning vector and isolated by transformation into E. coli. Individual end-tail junctions were then determined by DNA sequencing. This analysis indicated that cleavage on plasmid substrates occurred exactly at the transposon 3′ ends (24 out of 24 IRR and IRL G-tail junctions; Fig. 7A) and almost always two nucleotides inside the transposon 5′ ends (13 out of 16 vector C-tail junctions; Fig. 7A). Consistent with this result, gel-purified vector backbone produced by transposase-mediated cleavage of pISY100-kan was efficiently circularized with T4 DNA ligase and could be recovered after transformation into E. coli, yielding plasmids with the predicted TATATA junction sequence (Fig. 7B).


In vitro transposition of ISY100, a bacterial insertion sequence belonging to the Tc1/mariner family.

Feng X, Colloms SD - Mol. Microbiol. (2007)

Mapping cleavage positions on supercoiled substrates. A. Transposase was incubated with supercoiled plasmids containing ISY100 or ISY100-kan to give ETF and vector fragments, which were purified from an agarose gel. 3′ OH ends were tailed using terminal transferase and dCTP or dGTP. Tailed products were amplified by PCR using the indicated primers and cloned by topo-TA cloning. The end-tail junction sequences obtained and their frequencies are shown. B. The vector fragment was circularized with T4 DNA ligase, giving the junction sequence shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2170065&req=5

fig07: Mapping cleavage positions on supercoiled substrates. A. Transposase was incubated with supercoiled plasmids containing ISY100 or ISY100-kan to give ETF and vector fragments, which were purified from an agarose gel. 3′ OH ends were tailed using terminal transferase and dCTP or dGTP. Tailed products were amplified by PCR using the indicated primers and cloned by topo-TA cloning. The end-tail junction sequences obtained and their frequencies are shown. B. The vector fragment was circularized with T4 DNA ligase, giving the junction sequence shown.
Mentions: To see if cleavage one nucleotide inside the transposon end occurs only on small linear substrates, transposase cleavage sites were mapped on plasmid substrates containing mini-transposons. After in vitro cleavage with transposase, the ETF and the vector backbone fragments were purified by gel electrophoresis and G- or C-tailed using terminal transferase. End-tail junctions were amplified by PCR with poly C or poly G primers together with vector- or transposon-specific primers (Fig. 7A). PCR products were ligated into a cloning vector and isolated by transformation into E. coli. Individual end-tail junctions were then determined by DNA sequencing. This analysis indicated that cleavage on plasmid substrates occurred exactly at the transposon 3′ ends (24 out of 24 IRR and IRL G-tail junctions; Fig. 7A) and almost always two nucleotides inside the transposon 5′ ends (13 out of 16 vector C-tail junctions; Fig. 7A). Consistent with this result, gel-purified vector backbone produced by transposase-mediated cleavage of pISY100-kan was efficiently circularized with T4 DNA ligase and could be recovered after transformation into E. coli, yielding plasmids with the predicted TATATA junction sequence (Fig. 7B).

Bottom Line: Transposase made double-strand breaks on a supercoiled DNA molecule containing a mini-ISY100 transposon, cleaving exactly at the transposon 3' ends and two nucleotides inside the 5' ends.Cleavage of short linear substrates containing a single transposon end was less precise.Transposase also catalysed strand transfer, covalently joining the transposon 3' end to the target DNA.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedical and Life Sciences, Division of Molecular Genetics, University of Glasgow, Anderson College, 56 Dumbarton Rd, Glasgow G11 6NU, Scotland, UK.

ABSTRACT
The Synechocystis sp. PCC6803 insertion sequence ISY100 (ISTcSa) belongs to the Tc1/mariner/IS630 family of transposable elements. ISY100 transposase was purified and shown to promote transposition in vitro. Transposase binds specifically to ISY100 terminal inverted repeat sequences via an N-terminal DNA-binding domain containing two helix-turn-helix motifs. Transposase is the only protein required for excision and integration of ISY100. Transposase made double-strand breaks on a supercoiled DNA molecule containing a mini-ISY100 transposon, cleaving exactly at the transposon 3' ends and two nucleotides inside the 5' ends. Cleavage of short linear substrates containing a single transposon end was less precise. Transposase also catalysed strand transfer, covalently joining the transposon 3' end to the target DNA. When a donor plasmid carrying a mini-ISY100 was incubated with a target plasmid and transposase, the most common products were insertions of one transposon end into the target DNA, but insertions of both ends at a single target site could be recovered after transformation into Escherichia coli. Insertions were almost exclusively into TA dinucleotides, and the target TA was duplicated on insertion. Our results demonstrate that there are no fundamental differences between the transposition mechanisms of IS630 family elements in bacteria and Tc1/mariner elements in higher eukaryotes.

Show MeSH
Related in: MedlinePlus