Cloning of the replication and incompatibility regions of a plasmid derived from Rts1

doi:10.1016/0147-619X(82)90061-0

Plasmid

Volume 8, Issue 3, November 1982, Pages 232-243

https://doi.org/10.1016/0147-619X(82)90061-0 Get rights and content

Abstract

A replication region, consisting of a 1.1-megadalton (Md) EcoRI/HindIII fragment, was isolated from an Rts1 derivative plasmid. This 1.1-Md fragment, designated as mini-Rts1, was ligated to either pBR322 or a nonreplicating DNA fragment specifying a drug resistance, and its replication properties were investigated. The mini-Rts1 plasmid was cured at a high frequency at 42 °C, while it was maintained stably at 37 °C despite it existed in low copy number. These behaviors are quite similar to those of Rts1. By dissecting the pBR322:mini-Rts1 chimeric plasmid with AccI endonuclease, an inc region of 0.34 Md in size was cloned, which expressed incompatibility toward Rts1. Proteins encoded on the mini-Rts1 genome were examined in the minicell system, and one specific product of 35,000 daltons in molecular weight was identified. Any polypeptides specific for the 0.34-Md inc⁺ region within mini-Rts1 were not detected.

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A new proteic killer gene system,hig,was identified on the plasmid Rts1. Thehiglocus consisting ofhigAandhigBis directly related to the temperature sensitive host cell growth conferred by Rts1. We proved thathigBencoding presumably a 92-amino-acid polypeptide inhibited segregation of plasmid free cells, andhigAencoding a 104-amino-acid polypeptide suppressed the HigB function both incisand intrans.
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Mini-Rts1 was found to be unable to replicate in a dnaA-null mutant. However, a mini-Rts1 derivative lacking entire tandem DnaA boxes in the replication origin retained the replication ability in a dnaA⁺ host although its copy number was about half that of the mini-Rts1 having complete DnaA boxes. Mini-Rts1cop1 that contains a high copy number mutation in repA was found to replicate more efficiently than mini-Rts1 of wild repA when DnaA boxes were deleted. In addition, the copy number of mini-Rts1cop1 without DnaA boxes increased 1.5-fold upon removal of incI iterons, whereas that of mini-Rts1 without DnaA boxes did not increase after the iterons were deleted. These indicate that the RepAcop1 protein can initiate the replication of mini-Rts1 efficiently even when DnaA boxes are absent from the origin of replication.
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Regular articleCloning of the replication and incompatibility regions of a plasmid derived from Rts1

Abstract

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Gene

J. Biol. Chem

Plasmid

Plasmid

Plasmid

Biochem. Biophys. Res. Commun

Plasmid

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Regular article
Cloning of the replication and incompatibility regions of a plasmid derived from Rts1