Related Experiment Videos
Association of Mu-containing plasmids with the Escherichia coli chromosome upon prophage induction
Abstract:
To determine the structure of a prophage-containing plasmid during Mu transposition, we have monitored the physical state of pSC101[unk]Mucts after thermoinduction. We have also examined the fate of a mini Mu plasmid constructed in vitro by deleting 27 kilobases from the center of the Mu prophage in pSC101[unk]Mucts. At various times after prophage induction, DNA was extracted from Mu or mini Mu plasmid-containing strains and subjected to electrophoresis in low concentration agarose gels followed by transfer of the DNA to nitrocellulose paper. Separate hybridization with (32)P-labeled pSC101 and Mu DNA revealed the position of the plasmids and the replication of Mu DNA. At times after induction when Mu replication was clearly visible, Mu and mini Mu plasmids were found to migrate with Escherchia coli DNA. This Mu-specific association requires the phage coded A and B proteins. Electron microscopy has shown that some of the associated DNA is comprised of circular plasmid molecules which appear to be in contact with the chromosomal DNA. These structures may represent intermediates or end products of the replication-integration process. The finding that Mu and mini Mu plasmids do not give rise to any detectable excision products and apparently remain intact during Mu transposition supports our proposal that the predominant event after Mu induction is the replication of Mu DNA in situ to generate integrative intermediates.
Insights
Bacteriophage Mu transposition involves plasmid DNA replication in situ, forming integrative intermediates. This process requires phage-coded proteins and results in Mu and mini Mu plasmids associating with chromosomal DNA.
Area of Science:
- Molecular Biology
- Genetics
Background:
- Bacteriophage Mu is a transposable element that integrates into the host genome.
- Understanding the mechanism of Mu transposition is crucial for genetic engineering and gene therapy.
Purpose of the Study:
- To investigate the physical state of plasmids during Mu transposition.
- To elucidate the role of phage-coded proteins in Mu transposition.
- To determine the fate of Mu and mini Mu plasmids during transposition.
Main Methods:
- Thermoinduction of prophage in pSC101[unk]Mucts.
- Construction and analysis of a mini Mu plasmid.
- DNA extraction, agarose gel electrophoresis, and Southern blotting.
- Hybridization with labeled pSC101 and Mu DNA.
- Electron microscopy of associated DNA structures.
Main Results:
- Mu and mini Mu plasmids associate with E. coli chromosomal DNA after induction.
- This association requires the phage-coded A and B proteins.
- Electron microscopy reveals circular plasmid molecules in contact with chromosomal DNA.
- No detectable excision products of Mu and mini Mu plasmids were observed.
Conclusions:
- Mu transposition predominantly involves in situ replication of Mu DNA to form integrative intermediates.
- Plasmid-associated Mu DNA replication is a key step in the transposition process.
- The A and B proteins are essential for the Mu-specific association with chromosomal DNA.