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Enzymatic cleavage of a bacterial chromosome at a transposon-inserted rare site
1California Institute of Biological Research, La Jolla 92037.
Abstract:
The sequential use of the methylase M.Xbal (5'.TCTAGm6A) and the methylation-dependent endonuclease Dpnl (5'-Gm6A decreases TC) results in cleavage at 5'.TCTAGA decreases TCTAGA. This recognition sequence was introduced into a transposon derived from the Mu bacteriophage and transposed into the genome of the bacterium Salmonella typhimurium. M.Xbal methylation was provided in vivo by a plasmid containing the M.Xbal gene and the S. typhimurium genome was cleaved to completion by Dpnl at one or more sites, depending on the number of transposon insertions. The resulting genomic fragments were resolved by pulsed-field electrophoresis. The potential use of single M.Xbal/Dpnl cleavage sites as reference positions to map rare restriction sites is discussed.
Insights
Researchers used methylase M.Xbal and endonuclease DpnI to cleave bacterial DNA. This novel method, utilizing transposon technology in Salmonella typhimurium, allows for genomic mapping of rare restriction sites.
Area of Science:
- Molecular Biology
- Genomics
- Bacteriology
Background:
- The methylase M.Xbal and methylation-dependent endonuclease DpnI recognize and cleave specific DNA sequences.
- Restriction enzyme digestion is crucial for DNA analysis and genome mapping.
Purpose of the Study:
- To introduce a specific M.Xbal/DpnI recognition site into the Salmonella typhimurium genome via a Mu bacteriophage-derived transposon.
- To evaluate the utility of this engineered site for mapping rare restriction sites using pulsed-field electrophoresis.
Main Methods:
- In vivo methylation of the Salmonella typhimurium genome using the M.Xbal methylase.
- Genomic DNA cleavage using the methylation-dependent endonuclease DpnI.
- Analysis of resulting DNA fragments by pulsed-field gel electrophoresis.
Main Results:
- Successful introduction and methylation of the M.Xbal/DpnI recognition site into the bacterial genome.
- Completion of genomic cleavage by DpnI, with the number of cleavage sites correlating to transposon insertions.
- Generation of genomic fragments suitable for analysis by pulsed-field electrophoresis.
Conclusions:
- The sequential action of M.Xbal and DpnI creates a unique cleavage site for genomic manipulation.
- This method provides a potential reference system for mapping rare restriction sites within bacterial genomes.
- Transposon-mediated site-specific integration offers a versatile tool for genomic engineering and analysis.