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Cloning in Streptococcus pneumoniae of the gene for DpnII DNA methylase
Abstract:
The gene coding for the pneumococcal DNA adenine methylase that recognizes the sequence 5'-GATC-3' was cloned in a strain of Streptococcus pneumoniae that lacked both restriction endonucleases DpnI and DpnII. The gene was cloned as a 3.7-kilobase fragment of chromosomal DNA from a DpnII-containing strain inserted in both possible orientations in the multicopy plasmid vector pMP5 to give recombinant plasmids pMP8 and pMP10. Recombinant plasmids were selected by their resistance to DpnII cleavage. Cells carrying the recombinant plasmids modified phage in vivo so that it was restricted by DpnI- but not DpnII-containing hosts. They also showed levels of DNA methylase activity five times higher than that in cells of the original DpnII strain. No DpnII activity was observed in the clones; therefore, it was concluded that the insert did not contain an intact DpnII endonuclease gene and that methylation of host DNA did not turn on a latent form of the gene.
Insights
Researchers cloned the pneumococcal DNA adenine methylase gene into Streptococcus pneumoniae. This enhanced DNA methylation activity and modified phage DNA, demonstrating successful gene cloning and function.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- Restriction-modification systems are crucial for bacterial defense and DNA regulation.
- Pneumococcal DNA adenine methylase (dam) methylates the 5'-GATC-3' sequence.
- Understanding dam gene function is key to bacterial genetics.
Purpose of the Study:
- To clone and express the pneumococcal DNA adenine methylase gene in a heterologous host.
- To characterize the functional expression of the cloned dam gene.
- To investigate the effects of dam gene expression on phage DNA modification.
Main Methods:
- Cloning of a 3.7-kilobase chromosomal DNA fragment containing the dam gene into a plasmid vector (pMP5).
- Transformation of a restriction-endonuclease-deficient Streptococcus pneumoniae strain with recombinant plasmids (pMP8, pMP10).
- Selection of transformants based on resistance to DpnII cleavage and assessment of DNA methylase activity.
Main Results:
- Successful cloning and stable maintenance of the dam gene on multicopy plasmids.
- Recombinant plasmids conferred a five-fold increase in DNA adenine methylase activity.
- Phage DNA replicated in modified hosts was resistant to DpnII but sensitive to DpnI restriction.
- Absence of DpnII endonuclease activity in the cloned strains.
Conclusions:
- The cloned gene successfully encodes functional pneumococcal DNA adenine methylase.
- Expression of the dam gene leads to significant DNA methylation and altered phage restriction patterns.
- The cloned fragment does not contain the DpnII endonuclease gene, ruling out latent gene activation.