Open AccessReplication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans
Author(s) -
David H. Figurski,
Donald R. Helinski
Publication year - 1979
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.76.4.1648
Subject(s) - cole1 , plasmid , biology , t dna binary system , origin of replication , dna replication , genetics , replicon , microbiology and biotechnology , transformation (genetics) , escherichia coli , dna , gene , recombinant dna , vector (molecular biology)
pRK212.2, a derivative of the broad host range plasmid RK2, contains twoEco RI cleavage fragments, A and B, neither of which can replicate by itself inEscherichia coli . Fragment A (41.7 kilobases), but not fragment B (14.4 kilobases), can be cloned by insertion into the unrelated plasmids mini-F and ColE1. Fragment B contains the origin of replication and the ampicillin-resistance determinant of RK2. Transformation ofE. coli cells containing the mini-F-fragment A hybrid plasmid with fragment B DNA results in the recircularization and replication of fragment B as a nonmobilizable plasmid (pRK2067) with the copy number and incompatibility properties of RK2. Fragment B cannot be cloned in the absence of fragment A because the latter fragment suppresses a function, specified by fragment B, that results in loss of host cell viability. A small segment (2.4 kilobases) of fragment B that contains the RK2 origin of replication but no longer affects host cell growth in the absence of fragment A had been cloned previously by insertion into a ColE1 plasmid. This hybrid plasmid, designated pRK256, will replicate inE. coli polA mutants only when a fragment A-bearing helper plasmid is present. These results demonstrate that the potentially lethal function specified by fragment B of RK2 is not necessary for replication and that at least onetrans -acting function is directly involved in RK2 replication.