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  • Title: [The plasmid carrying the temperature-sensitive mutation in the DNa-methylase gene of the PStI system: effect on host cells at nonpermissive temperature].
    Author: Danilevich VN, Livshits VA.
    Journal: Genetika; 1999 May; 35(5):574-86. PubMed ID: 10495945.
    Abstract:
    Temperature-sensitive (ts) derivatives of plasmid pRMP1, the derivative of PBR322 containing restriction and modification (RM) genes of the PstI system, were obtained using hydroxylamine mutagenesis. One of the isolated plasmids responsible for the inhibition of Escherichia coli cell growth at 42 degrees C, pRMPts, was analyzed in this work. Cells of Rec+ strains carrying this plasmid were unable to divide at 42 degrees C and formed long non-septated filaments that died upon prolonged cultivation. Cells of the RecA- strains carrying pRMPts did not form filaments at 42 degrees C and rapidly disappeared. On agar media with or without ampicillin, Rec+ and RecA- strains with this plasmid formed colonies of temperature-resistant (tr) derivatives with frequencies ranging from 1.5 x 10(-4) to 4 x 10(-6) in independent clones. The structure of plasmids from cells of tr-derivatives of Rec+ and RecA- strains carrying plasmid pRMPts was analyzed by the set of restriction enzymes. Reversions to the temperature-resistant phenotype were shown to result from the following events: (1) the insertional inactivation of the PstI restriction enzyme gene in pRMPts (the insertion of the IS1 element); (2) deletions in plasmid DNA fragments that partially or completely cover the restriction enzyme gene; (3) point mutations; and (4) others. The effect of the chromosomal sulA mutation on the maintenance of the ts-plasmid in bacterial cells was studied at 42 degrees C. High efficiency loss of the plasmid was detected in pRMPts-carrying Rec+ cells with the sulA::Tn5 mutation grown in liquid and solid nutrient media at this temperature. Under similar conditions, plasmid loss was not detected in SulA+ cells. On the basis of the data obtained, it is concluded that the ts-mutation is located in the DNA-methylase gene of plasmid pRMPts. Mutant DNA methylase was unable to methylate all sites in the chromosomal DNA at 42 degrees C. Some of the unmethylated sites can be digested with the PstI enzyme, which leads to the induction of SOS response in Rec+ cells or to total mortality in cells with the recA phenotype.
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