CHARACTERIZATION OF COPY NUMBER MUTANTS OF PLASMID-PSC101

We have characterized three copy number mutants of the plasmid pSC101. These mutations caused single amino acid substitutions at the 46th, 8 3rd and 115th codons in the rep gene and an increase in the copy numbe r by 4- to 8-fold. Although the in vivo and in vitro repressor activit ies of these mutated Rep proteins were quite different from each other , the intracellular concentrations of the proteins were maintained at higher levels than the wild-type protein. It has been reported that ex cess amounts of Rep inhibit pSC101 replication (Ingmer and Cohen, 1993 ). This inhibitory activity of Rep was markedly decreased in all three mutants. When both the wild-type and one of the mutated rep genes wer e retained in the same plasmids, the copy number of these plasmids was decreased compared with plasmids retaining a single mutated rep gene. These results support the theory that the inhibitory activity of Rep for its own replication plays an important role in copy number regulat ion.