TY - JOUR
T1 - RepC is rate limiting for pT181 plasmid replication
AU - Manch-Citron, J. N.
AU - Gennaro, M. L.
AU - Majumder, S.
AU - Novick, R. P.
N1 - Funding Information:
We thank Natalie Jacobs for excellentt echnicala sis-tance.T his work was supportedb y grant NP-505 from theA mericanC ancerS ocietyt o R.P.N. andg rantA I 20472 from the National Instituteso f Health to J.N.M.-C.
PY - 1986/9
Y1 - 1986/9
N2 - The effect on pT181 plasmid replication of the concentration of the plasmid-coded initiator protein, RepC, has been analyzed. In one type of experiment, plasmid replication was found to stop immediately after the addition of an inhibitory concentration of chloramphenicol (Cm) to growing cultures. Chromosomal replication showed the slow turnoff that is usual for Cm inhibition. Because plasmid replication rate is determined autogenously, no host factor can be rate limiting, suggesting that the specific factor affected is Rep C. In another type of experiment, we constructed a translational fusion between the repC coding sequence and a translationally inducible Cm-acetylase gene, cat-86, using pUB110 as the carrier replicon. The fusion plasmid showed an eightfold amplification of its own copy number and a similar amplification of a co-resident pT181 plasmid upon Cm induction. The amplified plasmids did not show autocatalytic runaway replication but rather established stable elevated copy numbers, indicating the existence of a secondary level of regulation. These results suggest that RepC is rate limiting for pT181 replication and support the hypothesis that pT181 replication is regulated at the level of RepC synthesis. The nature of the secondary regulation is unknown.
AB - The effect on pT181 plasmid replication of the concentration of the plasmid-coded initiator protein, RepC, has been analyzed. In one type of experiment, plasmid replication was found to stop immediately after the addition of an inhibitory concentration of chloramphenicol (Cm) to growing cultures. Chromosomal replication showed the slow turnoff that is usual for Cm inhibition. Because plasmid replication rate is determined autogenously, no host factor can be rate limiting, suggesting that the specific factor affected is Rep C. In another type of experiment, we constructed a translational fusion between the repC coding sequence and a translationally inducible Cm-acetylase gene, cat-86, using pUB110 as the carrier replicon. The fusion plasmid showed an eightfold amplification of its own copy number and a similar amplification of a co-resident pT181 plasmid upon Cm induction. The amplified plasmids did not show autocatalytic runaway replication but rather established stable elevated copy numbers, indicating the existence of a secondary level of regulation. These results suggest that RepC is rate limiting for pT181 replication and support the hypothesis that pT181 replication is regulated at the level of RepC synthesis. The nature of the secondary regulation is unknown.
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U2 - 10.1016/0147-619X(86)90069-7
DO - 10.1016/0147-619X(86)90069-7
M3 - Article
C2 - 3462754
AN - SCOPUS:0022776726
SN - 0147-619X
VL - 16
SP - 108
EP - 115
JO - Plasmid
JF - Plasmid
IS - 2
ER -