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In vitro system for the replication of the mini R1 factor Rsc11

✍ Scribed by G. S. Bezanson; W. Goebel

Publisher: Springer
Year: 1979
Tongue: English
Weight: 794 KB
Volume: 170
Category: Article
ISSN: 1617-4615
DOI: 10.1007/bf00268579

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✦ Synopsis

The in vitro synthesis of the mini R1-factor, Rsc11, was achieved using a soluble Escherichia coli cell-extract system. Triton X-100 lysates of the K12 strain 1101 (Rsc11) fractionated by Sephadex G25 chromatography supported the incorporation of labeled deoxyribonucleotides into covalently-closed circular (30S) and open-circular (25S) plasmid DNA as well as other molecules of various sizes. DNA synthesis required the presence of all four ribonucleotides and was rifampicin sensitive. Pulse-chase experiments indicated that this reaction is discontinuous. A dependence on ATP and sensitivity to nalidixic acid suggested this system capable of the replicative synthesis of Rsc11 DNA. Density-shift analysis confirmed this. In addition to hybrid, fully-heavy plasmid supercoils were synthesized indicating that more than one round of replication was completed. Approximately one-third of the molecules available to the system participate in this reaction.

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