Temperature-sensitive copy number mutants of CoIE1 are located in an untranslated region of the plasmid genome.

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Abstract
Two mutant plasmid derivatives of ColE1 that exhibit temperature-sensitive replication properties were isolated. Both mutants have a normal copy number at 30.degree. C but increase their copy number 30-to 40-fold after a shift in temperature to 42.degree. C. A plasmid-encoded enzmye, .beta.-lactamase (penicillinase, EC 3.5.2.6), undergoes a 30-to 40-fold increase in specific activity concomitant with the increase in plasmid copy number. The copts phenotype of these mutants is not due to the synthesis of a temperature-sensitive polypeptide. Both mutations are located in an untranslated region of the plasmid genome encoding 2 overlapping transcripts involved in plasmid replication: a small transcript known as RNA I that acts as a negative control element in replication and a large transcript that has been characterized as the replication primer in vitro. The mutations alter the sequence encoding the primer but lie immediately 5'' to the initiating nucleotide of RNA I, in the RNA I promoter region. The possibility that the temperature-dependent plasmid DNA amplification is a consequence of a temperature-sensitive RNA I promoter was tested by inserting the RNA I promoters from the wild-type and mutant plasmids into a plasmid in which galactokinase expression is dependent on an exogenous promoter. The mutant promoters are evidently not temperature-sensitive. Rather, the mutations may affect the secondary structure of the replication primer in a region important for RNA I interaction.