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An amber replication mutant of F plasmid mapped in the minimal replication region

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Summary

We have constructed a mini-F derivative (pKP1013) consisting of a 5.4 kilobase pairs (kb) segment (44.0 to 49.4 kb) of mini-F and fragments carrying the chloramphenicol and spectinomycin resistance genes that originated from the R plasmid NR1. The plasmid pKP1013 replicates autonomously in a manner indistinguishable from that of the parental mini-F. An amber mutant defective in replication has been isolated from pKP1013 by localized mutagenesis using N-methyl-N′-nitro-N-nitrosoguanidine. The virtual absence of incorporation of [3H]-thymidine into the plasmid DNA as well as the kinetics of appearance of plasmid-free segregants suggest that plasmid DNA synthesis is primarily affected under nonpermissive conditions. The amber mutation has been mapped within the 530 base pairs (bp) region that extends from 45.25 (XmaI) to 45.78 Kb (PstI) by extensive analysis of in vitro recombinants constructed from rep + and rep − plasmids.

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Abbreviations

NG:

N-methyl-N′-nitro-N-nitrosoguanidine

Cm:

chloramphenicol

Ap:

ampicillin

Sp:

spectinomycin

Sm:

streptomycin

Tc:

tetracycline

kb:

kilobase pairs

bp:

base pair

kd:

kilodaltons

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Authors and Affiliations

  1. Faculty of Pharmaceutical Sciences, Kyushu University, Maidashi, Higashi-ku, 812, Fukuoka, Japan

    Satoko Maki, Minoru Kuribayashi, Takeyoshi Miki & Tadao Horiuchi

Authors
  1. Satoko Maki
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  2. Minoru Kuribayashi
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  3. Takeyoshi Miki
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  4. Tadao Horiuchi
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Communicated by T. Yura

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Maki, S., Kuribayashi, M., Miki, T. et al. An amber replication mutant of F plasmid mapped in the minimal replication region. Mol Gen Genet 191, 231–237 (1983). https://doi.org/10.1007/BF00334819

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  • DOI: https://doi.org/10.1007/BF00334819

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