Plasmid encoded DNA primases.
thesisposted on 19.11.2015, 08:53 by Brian Paul. Dalrymple
Plasmid ColIb-P9 of the l? incompatibility group encodes a DNA primase that acts in conjugal transfer of the plasmid and can substitute for mutant dnaG gene product in the vegetative replication of the Escherichia coli chromosome. The relevant genetic determinant (sog) has been cloned into a multicopy vector plasmid. Prototype IncB plasmid R16 also suppresses host dnaG mutations. The equivalent gene(s) (pri) of R16 have been cloned into plasmid pBR325 and shown by Southern transfer hybridisation to be different from the ColIb-P9 primase gene(s). The cloned fragment carrying the pri determinant encodes two polypeptides with apparent molecular weights 240,000 and 180,000 which can initiate DNA synthesis in vitro on single stranded phage M13 template, but which are antigenically distinct from ColIb-P9 primase. The cloned primase genes were used as probes in colony hybridisation screening of strains carrying plasmids of the IncI complex and IncB group, which specify serologically similar conjugation pili. Plasmids R64drdll, R144drd3 (IncI?), R621a (IncI?), RIP72 and R864a (IncB) contain nucleotide sequences honologous with the cloned ColIb-P9 sog gene(s). Plasmids R805a (IncI?), R724 (Incl?), TP125 and pLG101 (IncB) showed sequence homology with the R16 pri determinant. R387 (IncK) is also a member of the I-complex and encodes a primase which is genetically and serologically distinct from ColIb-P9 primase, but which is genetically similar to R16 primase. The gene(s) has been cloned into plasmid pBR328 and the fragment carrying the pri determinant encodes two polypeptides of 270,000 and 200,000 daltons. The hybridisation and dnaG suppression screens were extended to include representatives of all of the E.coli incompatibility groups. R40a (IncC) and RA3 (IncU) were shown to suppress the dnaG3 mutation and to encode DNA primases. The IncC primase encoded by R40a is genetically and antigenically distinct from ColIb-P9 primase.