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PubMed1975

A simple method for the preparation of large quantities of pure plasmid DNA.

Polyethylene glycol quantitatively precipitates plasmid DNA of molecular weight 6-123-10-6, from cleared lysates of plasmid-carrying bacterial strains, After resuspension and density-gradient centrifugation of the precipitated DNA, it is unchanged in length and in transformation efficiency for Escherichia coli K12. Plasmid DNA can be easily prepared in large quantities by including a polyethylene glycol precipitation step in standard plasmid isolation procedures.

Anti-Bacterial Agents↗

PubMed1976

Characterization of a replication mutant of the bacteriocinogenic plasmid Clo DF13.

In a previous paper (Kool, A.J. and Nijkamp, H.J.J. (1974) J. Bacteriol. 120, 569--578) the isolation of a mutant of the bacteriocinogenic plasmid Clo DF13-Rep3, has been described. It was observed that cells harbouring the wild type plasmid synthesize more plasmid DNA cells harbouring the wild type plasmid. This paper deals with the characterization of the nature of this plasmid-specific mutation. The following properties of the Clo DF13-Rep3 mutant plasmid could be observed: 1. The plasmid-specific mutation did not lead to a significant change in the sedimentation value of Clo DF13 DNA. 2. The specific rate of Clo DF13-Rep3 DNA synthesis (expressed as the number of plasmid DNA molecules synthesized per min) is on average seven times the specific rate of wild type Clo DF13 DNA synthesis. 3. Also chromosomeless minicells, harbouring the Clo DF 13-Rep 3 plasmid, contain about seven times more plasmid DNA as wild type Clo DF13 harbouring minicells. 4. The replication time of the Clo DF13-Rep3 plasmid is approx. 90 s at 30 degrees C and does not differ significantly from the replication time of the wild type plasmid. 5. The Rep3 mutation did not alter the dependence of Clo DF13 plasmid replication on the dnaA and dnaC gene products. 6. The plasmid-specific mutation is cis-dominant over wild type. The data presented in this paper indicate that this mutant plasmid is not affected in the elongation but in the initiation of plasmid DNA replication.

Cell Division↗

PubMed1976

Specific excision of the inserted DNA segment from hybrid plasmids constructed by the poly(dA). poly (dT) method.

The hybrid plasmid PBETAG, consisting of plasmid PMB9 DNA with an insert of rabbit globin DNA (about 600 base pairs) flanked by poly(dA) poly(dT) regions (Maniatis, T., Kee, S.G., Efstratiadis, A. and Kafatos, F.C. (1976) Cell 8, 163-182), was cleaved into two fragments by endonuclease S1 under conditions of partial denaturation. Only the smaller fragment (575 base pairs) contained globin-specific sequences, showing that excision had occurred in the A-T-rich regions. This method of cleavage provides a useful procedure for assessing the length of inserts in hybrid plasmids prepared by the poly(dA)-POLY(DT) tail method, and allows the preparative recovery of the insert.

Animals↗

PubMed1977

Expression of a bacterial gene for guanine synthesis inserted into colicin E1 factor by an in vitro recombination.

Regulation of expression of a bacterial guaA gene inserted into colicin E1 DNA by an in vitro recombination was studied under various growth conditions. In Escherichia coli K-12 cells that carried this hybrid ColEl plasmid the level of guaA enzyme activity was not regulated by the concentration of guanine in the medium, but by the number of plasmid DNA copies. The optimal conditions for amplifying the guaA gene product by chloramphenicol treatment were determined. The level of guaA enzyme activity found under the optimal conditions was about 37 times that in extracts of wild-type E. coli cultured in guanine-free medium. The properties of the promoter for the guaA gene and applicability of this hybrid ColEl plasmid for amplification of various gene products were discussed.

Chloramphenicol↗

PubMed1977

Unidirectional replication of the P-group plasmid RK2.

The mode of replication of the broad host-range plasmid RK2 has been determined from examination of molecular replicative forms cleaved with the restriction endonucleases EcoRI and Hind III. Replication is unidirectional, and proceeds from a unique origin. The location of the origin and other evidence suggests that genes involved in plasmid maintenance are not tightly clustered.

DNA Replication↗

PubMed1977

Translation in Escherichia coli mini-cells containing hamster mitochondrial DNA-Co1E1 - Ampr recombinant plasmids.

Hamster mitochondrial DNA is cleaved into two fragments (4.2 and 11.4 kilobase pairs of DNA (kb)) by the restriction enzyme, Eco RI. Recombinant DNA molecules formed in vitro between an Escherichia coli plasmid, Co1E1 - Ampr, and Eco RI-digested hamster mitochondrial DNA were transformed into E. coli K12. The translation products of the parent plasmid, Co1E1 - Ampr, and recombinant plasmid DNAs containing (i) the 4.2 kb mitochondrial DNA fragment and (ii) the 11.4 kb fragment were characterized on sodium dodecyl sulfate-polyacrylamide gels using bacterial mini-cell lysates. The Co1E1 - Ampr plasmid specifies at least six polypeptides whose structural genes comprise 56% of the plasmid DNA. Insertion of hamster mitochondrial DNA at the Eco RI site of the plasmid alters the relative rate of synthesis of these six polypeptides and induces the occurrence of a new band on sodium dodecyl sulfate-polyacrylamide gels which is probably not specified by the inserted mitochondrial DNA sequences.

Ampicillin↗

PubMed1976

Changes in membrane proteins of Escherichia coli K12 mediated by bacteriophage IKe-specific plasmids.

At least 3 minor proteins have been detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the unseparated (31 000 daltons) and inner membranes (53 000 and 86 000 daltons) of a non-piliated strain of E. coli K12 bearing the phage IKe-specific resistance plasmid RM98 (Tra+); these proteins are lacking in its Tra- derivative and in the plasmidless host. In contrast, in another K12 host carrying each of different IKe-specific colicinogenic factors, an extra 30 000 dalton protein is observed in the outer membranes; a number of minor proteins seen in the inner membranes appear to be different from those observed in the RM98+ strain. The evidence suggests that the 30 000-31 000 dalton protein is transfer function specific.

Coliphages↗

PubMed1968