Effects of promoter strengths and growth conditions on copy number of transcription-fusion vectors

Abstract

Promoters with widely different transcriptional activities have been fused to the galactokinase gene contained in a multicopy plasmid ( McKenny , K., Shimatake , H., Court, D., Schmeissner , U., Brady, C., and Rosenberg, M. (1982) in Gene Amplification and Analysis: Analysis of Nucleic Acids ( Chirkjian , J. G., and Papas , T., eds) pp. 383-415, Elsevier /North-Holland Biomedical Press, Netherlands). Assay methods which allow determination of galactokinase-specific activity (nanomoles of galactose 1-phosphate/min/mg of protein) and plasmid-specific number (femtomoles of plasmid/mg of protein) in the same sonicated cellular extract are described. These methods provide a way to accurately measure and compare the promoter activities (nanomoles of galactose 1-phosphate/min/fmol of plasmid) of different plasmid constructions which exhibit different in vivo plasmid copy numbers. It is demonstrated that in vivo, copy number fluctuations are correlated with such parameters as promoter strength and cellular growth conditions. The ability to account for these uncontrolled in vivo copy number variations when comparing the transcriptional activities of different DNA inserts in multicopy transcription-fusion plasmids greatly facilitates the utility of these systems.