A copy-number mutant of plasmid pSC101.
Copy-number mutants of plasmid pSC101 were isolated by u.v. mutagenesis and selection for elevated expression of ampicillin resistance. Three independent mutations were identical and mapped in codon 93 of the initiation protein RepA. The mutated plasmids were maintained at a level four to five times higher than that of the wild type. For one of them, it was determined that: (i) the mRNA of the autoregulated repA gene, cloned onto a pUC19 plasmid under the control of its own promoter, was expressed at a level 1.7 times higher than that of the wild type; (ii) the RepA protein, under the same conditions, was expressed at a similarly higher level; (iii) the affinity of the mutated protein for three repeated sequences in the origin region of the plasmid was, on average, 3.4 times higher than that of the wild-type protein. We postulate that the copy-number effect is due to a combination of these two effects, i.e. higher protein concentration and increased affinity of the protein for the repeated sequences.
Xia
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GX
Department of Molecular Biology, University of Geneva, Switzerland.
Manen
D
D
Goebel
T
T
Linder
P
P
Churchward
G
G
Caro
L
L
D000668:Ampicillin Resistance; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D004265:DNA Helicases; D004261:DNA Replication; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011506:Proteins; D012329:RNA, Bacterial / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D012475:Salmonella / Q000235:genetics; D015534:Trans-Activators
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D011506:Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases