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Cointegration and resolution mediated by IS101 present in plasmid pSC101.
Molecular & General Genetics : MGG
|January 1, 1985
Summary
Cointegrate plasmids formed via insertion sequence IS101 in E. coli, mediated by the gamma-delta transposon. Gamma-delta and Tn3 transposons resolved these cointegrates, highlighting sequence homology in recombination sites.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- Cointegrate plasmids can arise from the interaction of different plasmid elements within a host cell.
- Insertion sequences (IS) and transposons play crucial roles in mediating DNA rearrangements and plasmid evolution.
- Understanding the mechanisms of cointegrate formation and resolution is vital for genetic engineering and plasmid stability.
Purpose of the Study:
- To investigate the mechanism of cointegrate plasmid formation between a pSC101 derivative and pBV320 in E. coli.
- To identify the specific genetic elements involved in both the formation and resolution of these cointegrates.
- To analyze the sequence homology between the involved genetic elements, particularly insertion sequence IS101 and transposons gamma-delta and Tn3.
Main Methods:
- Cleavage analysis of cointegrate plasmids.
- DNA sequencing to identify recombination junctions and involved sequences.
- Comparative sequence analysis of IS101, gamma-delta, and Tn3.
- Functional analysis of transposon-mediated cointegration and resolution.
Main Results:
- Cointegrate plasmids were formed between pSC101 derivative and pBV320 in E. coli.
- Direct repeats of insertion sequence IS101 were found at the recombination junctions, indicating IS101 mediation.
- Cointegration occurred only in cells containing the gamma-delta transposon, suggesting its role in providing transposase.
- Cointegrates were resolved by gamma-delta and Tn3 transposons (providing resolvase) in cells containing these elements.
- Sequence analysis revealed homology between IS101 and gamma-delta/Tn3 at resolvase binding sites and terminal inverted repeats, but not at the internal resolution site.
Conclusions:
- The formation of cointegrate plasmids in this system is mediated by the insertion sequence IS101.
- The gamma-delta transposon is essential for the cointegration process, likely by providing transposase activity.
- Both gamma-delta and Tn3 transposons are capable of resolving the cointegrates, indicating their resolvase functions are involved.
- Sequence homology in specific regions of IS101 and the gamma-delta/Tn3 transposons underlies the observed recombination events.