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Isolation and characterization of IS1 circles
Y Sekine1, N Eisaki, K Kobayashi
1Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Japan.
Gene
|June 3, 1997
Summary
Insertion sequence IS1 frameshifting generates transposase. Modified IS1 efficiently produces transposase, leading to novel IS1 circles formed by excision from plasmids.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- Transposase from insertion sequence IS1 is produced via translational frameshifting.
- A specific adenine run in IS1 facilitates this frameshifting.
- IS1 transposition can lead to plasmid rearrangements.
Purpose of the Study:
- To investigate the products of an IS1 mutant with altered frameshifting efficiency.
- To characterize novel circular DNA molecules generated in cells containing the IS1 mutant.
- To elucidate the mechanism of formation for these circular DNA molecules.
Main Methods:
- Plasmid manipulation and transformation.
- DNA cloning and sequencing.
- Polymerase chain reaction (PCR) for DNA isolation.
Main Results:
- Besides miniplasmids, minicircles lacking replication regions were observed.
- Most minicircles were IS1 circles, comprising the entire IS1 sequence and a short intervening sequence.
- The intervening sequence in IS1 circles originated from flanking regions of the parental plasmid's IS1 terminal repeats.
- IS1 circles are generated by an excision event from the parental plasmid.
Conclusions:
- IS1 circles are novel DNA structures formed by excision from plasmids.
- The formation of IS1 circles may involve cointegration reactions within the plasmid.
- This study reveals a new mechanism of DNA circle formation mediated by insertion sequences.