Cloning of the MspI modification enzyme. The site of modification and its effects on cleavage by MspI and HpaII
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Abstract:
The gene for the MspI modification enzyme from Moraxella was cloned in Escherichia coli using the plasmid vector pBR322. Selection of transformants carrying the gene was based on the resistance of the modified plasmid encoding the enzyme to cleavage by MspI. Both chromosomal and plasmid DNA were modified in the selected clones. None of the clones obtained produced the cognate restriction enzyme which suggests that in this system the genes for the restriction enzyme and methylase are not closely linked. Crude cell extracts prepared from the recombinant strains, but not the host (E. coli HB101), contain an S-adenosylmethionine-dependent methyltransferase specific for the MspI recognition site, CCGG. Production of the enzyme is 3-4-fold greater in the transformants than in the original Moraxella strain. 5-Methylcytosine was identified as the product of the reaction chromatographically. The outer cytosine of the recognition sequence, *CCGG, was shown to be the site of methylation by DNA-sequencing methods. This modification blocks cleavage by both MspI and its isoschizomer HpaII. HpaII, but not MspI, is able to cleave the unmethylated strand of a hemimethylated substrate. The relevance of these results to the use of MspI and HpaII to analyze patterns of methylation in genomic DNA is discussed.
Insights
The Moraxella MspI methylase gene was cloned into E. coli, producing a highly active enzyme that methylates the CCGG sequence. This methylation prevents cleavage by MspI and HpaII restriction enzymes, impacting genomic DNA analysis.
Area of Science:
- Molecular Biology
- Enzymology
- Genetics
Background:
- Restriction enzymes and DNA methyltransferases are crucial tools in molecular biology.
- Understanding DNA methylation patterns is vital for studying gene regulation and genome stability.
Purpose of the Study:
- To clone and express the MspI methylase gene from Moraxella in Escherichia coli.
- To characterize the MspI methyltransferase activity and its effect on DNA methylation.
- To investigate the implications for using MspI and HpaII in genomic DNA methylation analysis.
Main Methods:
- Gene cloning using plasmid vector pBR322 in E. coli.
- Selection of transformants based on resistance to MspI cleavage.
- Analysis of DNA modification in recombinant E. coli strains.
- Characterization of methyltransferase activity in cell extracts.
- Identification of methylation products and sites using chromatography and DNA sequencing.
Main Results:
- Successful cloning and expression of the MspI methylase gene in E. coli.
- Recombinant E. coli produced a highly active S-adenosylmethionine-dependent methyltransferase specific for the CCGG recognition site.
- Methylation occurred at the outer cytosine of the CCGG sequence, producing 5-methylcytosine.
- This methylation rendered DNA resistant to cleavage by both MspI and HpaII restriction enzymes.
- The genes for the restriction and modification enzymes are not closely linked in this system.
Conclusions:
- The MspI methylase can be efficiently produced in E. coli.
- MspI methylation at the CCGG site blocks cleavage by MspI and HpaII, providing insights into DNA methylation analysis.
- The differential cleavage of hemimethylated DNA by HpaII has implications for studying DNA methylation patterns.