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Heteroduplex electron microscopy of phage Mu mutants containing IS1 insertions and chloramphenicol resistance
Abstract:
We have examined by electron microscopy the DNA heteroduplexes of six bacteriophage Mu mutants, Mu X cam, generated by the insertion of the Tn9 transposon for chloramphenicol resistance. Tn9 was found to be 2.8 +/- 0.2 kilobases (kb) in length and to consist of a cam determinant flanked by two IS1 sequences arranged in a direct order. In two of the six Mu X cam mutants, the Tn9 insertion was at a fixed location, 3.9 kb from the left, or c, end. In the other four mutants, the position of the insertion varied, even though the lysogenic cultures induced were grown from single colonies. The insertion was located at either 3.3 kb, 3.9 kb, or, less frequently, at 4.4 kb from the left end of the DNA. Furthermore, at low frequencies, the insertions were found to be in an orientation opposite to what predominated in the preparation. Thus, Tn9 in the Mu X cam mutants examined could appear to undergo rapid rearrangements during Mu growth or over a few generations of cell growth. One of the Tn9 insertion sites was apparently the same as that for a 0.8 kb insertion found in a Mu X mutant. This latter insertion was identified as an IS1 sequence. The DNA molecules from all the Mu X cam mutant phage particles were found to be missing the bacterial DNA at the S (right) end, along with a variable amount of the adjoining Mu DNA in the beta region. This observation supports the headful packaging model for Mu DNA.
Insights
Bacteriophage Mu mutants with Tn9 insertions showed rapid DNA rearrangements, with Tn9 elements frequently changing position and orientation. This suggests extensive genetic instability within the Mu genome during replication.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- Bacteriophage Mu is a versatile genetic element.
- Transposons like Tn9 facilitate genetic analysis and manipulation.
- Understanding DNA rearrangements is crucial for phage biology.
Purpose of the Study:
- To characterize the structure and behavior of Tn9 insertions in bacteriophage Mu.
- To investigate the stability and potential rearrangements of transposon elements within the Mu genome.
- To explore the implications for Mu DNA packaging and replication.
Main Methods:
- Electron microscopy of DNA heteroduplexes.
- Analysis of bacteriophage Mu X cam mutants containing Tn9.
- Mapping of Tn9 insertion sites and orientations.
Main Results:
- Tn9 is 2.8 kb, containing a chloramphenicol resistance gene flanked by IS1 sequences.
- Tn9 insertions in Mu X cam mutants exhibited variable positions and orientations.
- Rearrangements of Tn9 were observed during Mu growth or cell division.
- Mu X cam phage DNA showed deletions at the right end, supporting headful packaging.
Conclusions:
- Tn9 elements within bacteriophage Mu display significant instability and undergo rapid rearrangements.
- The observed deletions support the headful packaging model for Mu DNA.
- These findings provide insights into the dynamic nature of bacteriophage Mu genomes.