Replication of the thermosensitive drug resistance factor Rtsl was studied at the nonpermissive temperature (42°C). It was concluded from the following observations that replication of this plasmid takes place at 420C without involving the covalently closed circular (CCC) form of deoxyribonucleic acid (DNA). (i) DNA-DNA reassociation kinetics studies with purified Rtsl DNA showed that Rtsl DNA increased several-fold during cell growth at 420C while very little, if any, CCC DNA was synthesized.

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... ii) When Escherichia coli 20SO(Rtsl) was labeled with [3H]thymidine at 420C, a significant amount of radioactive DNA hybridizable to Rtsl DNA was formed. This DNA was found in a fraction where DNA other than CCC DNA was expected in alkaline sucrose density gradient centrifugation analysis. WhenE. coli 20SO(Rtsl) was labeled at 32°C, the labeled CCC DNA did not disappear during a chase period at 4200. This indicates that preformed CCC DNA does not participate in replication at the nonpermissive temperature. These results are consistent with the hypothesis that there are two modes of replication of Rtsl DNA, one involving a CCC molecule and the other not involving this form, and that only the latter mode takes place at the nonpermissive temperature. Recent studies suggest that these multiple thermosensitive characteristics may not be functions of one single plasmid gene. For example, the thermosensitive nature of plasmid transfer has been shown to be unrelated to its thermosensitive effect on the host (35, 42). Temperature-dependent restriction of T-even phages by Rtsl may be unrelated to the effect on host growth, although it may be controlled by the same gene as that for plasmid transfer (42). However, the thermosensitive nature of plasmid replication may be closely related to its influence on host growth. The close relationship between the effect on host growth and the 90 on May 9, 2020 by guest