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  • Title: Establishing a markerless genetic exchange system for Methanosarcina mazei strain Gö1 for constructing chromosomal mutants of small RNA genes.
    Author: Ehlers C, Jäger D, Schmitz RA.
    Journal: Archaea; 2011; 2011():439608. PubMed ID: 21941461.
    Abstract:
    A markerless genetic exchange system was successfully established in Methanosarcina mazei strain Gö1 using the hpt gene coding for hypoxanthine phosphoribosyltransferase. First, a chromosomal deletion mutant of the hpt gene was generated conferring resistance to the purine analog 8-aza-2,6-diaminopurine (8-ADP). The nonreplicating allelic exchange vector (pRS345) carrying the pac-resistance cassette for direct selection of chromosomal integration, and the hpt gene for counterselection was introduced into this strain. By a pop-in and ultimately pop-out event of the plasmid from the chromosome, allelic exchange is enabled. Using this system, we successfully generated a M. mazei deletion mutant of the gene encoding the regulatory non-coding RNA sRNA(154). Characterizing M. mazei ΔsRNA(154) under nitrogen limiting conditions demonstrated differential expression of at least three cytoplasmic proteins and reduced growth strongly arguing for a prominent role of sRNA(154) in regulation of nitrogen fixation by posttranscriptional regulation.
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