Replication of the streptococcal plasmid pMV158 and derivatives in cell-free extracts of Escherichia coli

Molecular & General Genetics : MGG
G del SolarM Espinosa

Abstract

pMV158 is a 5.4 kb broad host range multicopy plasmid specifying tetracycline resistance. This plasmid and two of its derivatives, pLS1 and pLS5, are stably maintained and express their genetic information in gram-positive and gram-negative hosts. The in vitro replication of plasmid pMV158 and its derivatives was studied in extracts prepared from plasmid-free Escherichia coli cells and the replicative characteristics of the streptococcal plasmids were compared to those of the E. coli replicons, ColE1 and the mini-R1 derivative pKN182. The optimal replicative activity of the E. coli extracts was found at a cellular phase of growth that corresponded to 2 g wet weight of cells per litre. Maximal synthesis of streptococcal plasmid DNA occurred after 90 min of incubation and at a temperature of 30 degrees C. The optimal concentration of template DNA was 40 micrograms/ml. Higher plasmid DNA concentrations resulted in a decrease in the incorporation of dTMP, indicating that competition of specific replication factor(s) for functional plasmid origins may occur. In vitro replication of plasmid pMV158 and its derivatives required the host RNA polymerase and de novo protein synthesis. The final products of the streptococcal plasmid DNAs r...Continue Reading

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Citations

Dec 23, 2003·Journal of Molecular Biology·Carmen de AntonioManuel Espinosa
Oct 11, 1988·Nucleic Acids Research·J Pérez-MartínM Espinosa
Feb 8, 2011·Nucleic Acids Research·Fabián Lorenzo-DíazManuel Espinosa
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