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Published on: November 12, 2012
Isolation of Escherichia coli mutants lacking methylcytosine-dependent restriction systems for cloning extensively
1Department of Biological Sciences, Western Michigan University, Kalamazoo 49008.
Abstract:
Many bacterial strains possess methylation-dependent restriction systems (MDRS) that demonstrate methylcytosine-dependent restriction endonuclease activity for the dinucleotide sequence, dCpdG. This makes these strains unsuitable for cloning methylated DNA. Some commercially available bacterial cells are recommended for cloning DNA fragments with methylated cytosines and adenines, e.g., Escherichia coli DH5-alpha MCR. Our attempts to clone frog virus 3 (FV3) DNA, which has the highest degree of cytosine methylation ever reported, using DH5-alpha MCR cells, were not successful. This and other observations suggested the existence of additional MDRS that have not yet been eliminated from DH5-alpha MCR cells. In order to isolate a mutant from this bacterial strain that is suitable to clone highly methylated FV3 DNA, we transformed these cells with a recombinant pUC19 plasmid containing a methylated 1.4-kb genomic DNA fragment from FV3, and selected for ampicillin (Ap) resistance. Three such attempts yielded only one colony that contained a fully methylated 1.4-kb FV3 genomic DNA fragment. Furthermore, plasmid-cured Ap-sensitive colonies originating from this clone were isolated and have been successfully employed to clone the highly methylated FV3 genomic DNA fragment.
Insights
Researchers developed a new bacterial strain capable of cloning highly methylated frog virus 3 (FV3) DNA. This overcomes limitations of existing methylation-dependent restriction systems (MDRS) for cloning challenging DNA.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- Bacterial methylation-dependent restriction systems (MDRS) restrict methylated DNA, hindering cloning of certain DNA fragments.
- Standard cloning strains like Escherichia coli DH5-alpha MCR are unsuitable for highly methylated DNA, such as frog virus 3 (FV3) DNA.
Purpose of the Study:
- To isolate a bacterial mutant strain capable of cloning highly methylated FV3 DNA.
- To overcome limitations imposed by MDRS in cloning highly methylated genomic DNA.
Main Methods:
- Transformation of DH5-alpha MCR cells with a recombinant pUC19 plasmid containing methylated FV3 DNA.
- Selection for ampicillin resistance to identify potential transformants.
- Isolation and characterization of plasmid-cured, ampicillin-sensitive colonies.
Main Results:
- One colony successfully retained a fully methylated FV3 DNA fragment after transformation and selection.
- Plasmid-cured derivatives of this clone were isolated and confirmed to be suitable for cloning highly methylated FV3 DNA.
Conclusions:
- A novel bacterial strain has been successfully developed for cloning highly methylated DNA.
- This new strain overcomes the restrictions posed by MDRS, enabling the cloning of previously unmanageable DNA sources like FV3.
