HYPER-REPLICATION OF THE AMPICILLIN-RESISTANCE GENE ON R FACTOR AND THEIR STABLE INTEGRATION IN EPISOME
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There has not been elucidated the conclusive mechanism of high resistance mutant on R factor. In this studies, we have examined that the demonstration of R factor mutant capable of hypersynthesis of penicillinase and genetic studies of the mutant.<BR>When the strains of <I>E. coli</I> W3630 harboring R<SUB>M201</SUB> factor (TC.CM.SM.SA.APC) were inoculated on plates containing various concentration of APC, mutants carrying various levels of APC resistance were due to a quantitative increase in the formation of penicillinase. By conjugation experiments and transduction analysis, the mutation was found to affect the gene (<I>amp</I>) governing APC resistance on the R factor. The R-factor mutnats carrying high and stable APC resistance were conjugally transferred at the same frequency as their parent R factors, and the level of their resistance to drugs other than APC was not distinct from that conferred by their parents. Such R-factor mutants could easily be obtained from wild-type R factors carrying low APC resistance. The hypersynthesis of penicillinase by such R-factor mutants was considered to be due to the replication of the <I>amp</I> gene on the R factor at hyper-rates and the integration of multiple copies of the <I>amp</I> gene (<I>amp</I>-hyper) in the R-factor genome.<BR>The physical characteristics of a mutant R<SUB>M201-2</SUB> capable of conferring high and stable APC-resistance was analysed. The R<SUB>M201-2</SUB> and its parent R factor DNA could be isolated as an extrachromosomal and covalently closed circular form, their buoyant densities both 1.712 g/cm<SUP>3</SUP> and the molecular weight were about 8.2× 10<SUP>7</SUP> and 6.4 × 10<SUP>7</SUP> daltons respectively when measured by CsCl and sucrose density gradient analyses. The contour lengths under electromicroscope were 35.9±0.6μm and 31±0.6μm, respectively. Using the extracted R factor DNA, the mutant and parent characters were transformable to another <I>E. coli</I> strain, respectively. The mutant R factor showed an increased amount of DNA even after conjugaltransfer to <I>Protues</I>. Increase in the size of R factor DNA was thus considered to be the cause for the high level of APC resistance.
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