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Restriction endonuclease mapping and mutagenesis of the F sex factor replication region
Abstract:
The plasmids pSC138 and pML31 each contain the EcoRI-generated f5 replicator fragment of the conjugative plasmid F in addition to an EcoRI fragment encoding antibiotic resistance: ampicillin resistance derived from Staphylococcus aureus in pSC138 and kanamycin resistance from Escherichia coli in pML31. We have mapped one HindIII and two BamHI restriction sites in the f5 region of these plasmids and one HindIII site in the antibiotic resistance region of each plasmid. The HindIII site in the Km region of pML31 occurs in the kan gene whereas the HindIII site in the Ap region of pSC138 appears to occur in an area important for the regulation of beta-lactamase production. By means of in vitro recombinant DNA manipulation of plasmids pML31 and pSC138, we have shown that approximately 1.9 X 10(6) daltons of the 6.0 X 10(6) dalton f5 fragment can be deleted without disrupting plasmid stability. In addition, we have used these same techniques to isolate a novel F-controlled Ap plasmid cloning vehicle which contains a single restriction site for each of the enzymes EcoRI, HindIII, and BamHI. This cloning vehicle has been linked via either its EcoRI or HindIII site to a ColE1 plasmid replicon to yield stable recombinants.
Insights
Researchers modified plasmids containing antibiotic resistance genes and the F plasmid replicator fragment. They successfully deleted a portion of the F replicator fragment without affecting plasmid stability, creating a new cloning vehicle.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Plasmids pSC138 and pML31 harbor the F plasmid's f5 replicator fragment and antibiotic resistance genes (ampicillin and kanamycin, respectively).
- Restriction site mapping was performed on these plasmids to identify key locations within the f5 and antibiotic resistance regions.
Purpose of the Study:
- To characterize restriction sites within the f5 replicator fragment and antibiotic resistance regions of pSC138 and pML31.
- To investigate the effects of deleting portions of the f5 fragment on plasmid stability.
- To develop a novel F-controlled ampicillin resistance plasmid cloning vehicle with unique restriction sites.
Main Methods:
- Restriction digestion using enzymes like EcoRI, HindIII, and BamHI.
- In vitro recombinant DNA manipulation techniques.
- Plasmid stability assays.
Main Results:
- Mapping identified specific HindIII and BamHI sites in the f5 region and HindIII sites in the antibiotic resistance regions.
- Deletion of approximately 1.9 x 10^6 daltons of the f5 fragment did not compromise plasmid stability.
- A new F-controlled ampicillin resistance plasmid cloning vehicle with single EcoRI, HindIII, and BamHI sites was constructed.
- This cloning vehicle was successfully linked to a ColE1 plasmid replicon, forming stable recombinants.
Conclusions:
- The f5 replicator fragment of the F plasmid can tolerate significant deletions while maintaining stability.
- A versatile F-controlled ampicillin resistance plasmid cloning vehicle was engineered, suitable for various molecular cloning applications.
- The developed cloning vehicle and its recombinants demonstrate potential for use in genetic engineering and biotechnology.