The pri gene locus of the conjugative broad host range plasmid RP4 maps between coordinates 40.3 and 43.5 and encodes two antigenically related forms of a DNA primase with a molecular mass of 118 and 80 kDa (kilodalton). Genesis of these two products has been examined using Pri +-recombinant plasmids. As shown by deletion analysis, the primase polypeptides are two separate translation products which arise from an in-phase overlapping gene arrangement. It is suggested that transcription of a set of RP4 genes including the pri gene starts at a promoter site within the Tral region. In vivo, RP4 mutant primase can apparently substitute for Escherichia coli primase as demonstrated by measuring suppression of the dnaG3(ts) mutant.
Methods
Bacterial strain and plasmids. E. coli strain BW86 dnaG3 leu thyA deoB rpsL A (chlA-uvrB) cir (Boulnois and Wilkins 1979) was obtained from B.M. Wilkins. The plasmids used in this study are listed in Table 1.
Media. TY-medium and TY-agar plates (1.5% agar) contained 1% Tryptone (Oxoid, England), 0.5% yeast extract (Merck, Darmstadt, FRG), 0.5% NaC1, 0.1% glucose supplemented with thymine 25 gg/ml, thiamine. HC1 25 gg/ml and buffered with 50 mM 3-[N-morpholino]propane-sulfonic acid, sodium salt (pH 8.0). Antibiotics were added to the following concentrations: Ampicillin (sodium salt)