Molecular cloning of recombinant DNA : proceedings of the Miami winter symposia, January 1977 / edited by W.A. Scott, R. Werner ; sponsored by the Department of Biochemistry, University of Miami, School of Medicine, Miami, Florida.

Date:
1977
Catalogue details

Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: Molecular cloning of recombinant DNA : proceedings of the Miami winter symposia, January 1977 / edited by W.A. Scott, R. Werner ; sponsored by the Department of Biochemistry, University of Miami, School of Medicine, Miami, Florida. Source: Wellcome Collection.

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    52 S. N. COHEN eí а/. Plasmids even in recA~ bacteria (Chang and Cohen, unpublished data). The reassortment/rearrangement of DNA sequences may occur more frequently with chimeric plasmids, but it also seem to occur in naturally occurring coli plasmids. During some of the earliest cloning experiments, an EcoRl-generated fragment of R6-5 carrying resistance to sulfonamide, but not to streptomycin, was cloned. In later experiments, in which the EcoRI-generated fragments of the R6-5 plasmid were each indivi¬ dually cloned into a ColEl-derived vector (Timmis, Cabello and Cohen, in preparation), an EcoRI-generated sulfonamide-resis- tance fragment was isolated but this fragment additionally carried a resistance gene to streptomycin. In other studies, (Timmis, Cabello, and Cohen, in preparation) variability in the length of corresponding fragments cloned from different prepara¬ tions of the R6-5 plasmid has been noted, and even a difference in the restriction endonuclease cleavage pattern has been observed with different preparations of this plasmid. These findings are analogous to the loss or gain of the restriction endonuclease sites observed in prokaryotic-eukaryotic chimeric plasmids, as noted above. Several years ago, at the time of construction of the first Interspecies chimeric plasmids (37), it was observed that the pSC112 plasmid, which was formed by insertion of an Ap-resis- tance segment of the staphylococcal pl258 plasmid into the EcoRI cleavage site of pSClOl, expressed tetracycline resistance at the same level as the pSClOl parent. However, an analogous plasmid (pSC113) which had two other segments of pI258 inserted into the EcoRI cleavage site of pSClOl, expressed Tc resistance at a very much lower level. A tetracycline resistance plasmid having physical and biological properties (including Tc-resis- tance levels) indistinguishable from those of pSClOl could be recovered after transformation of coli by an EcoRI digested pSC113 DNA. Although it had been shown earlier that inclusion of a DNA fragment into the single EcoRI cleavage site of pSClOl did not interrupt the Tc-resistance gene of the plasmid (1), it appeared from these experiments that in at least some instances insertion of a fragment at this site would affect the level of expression of Tc-resistance (37, 41). Subsequent experiments (Tait et al., submitted for publication; Cabello et al., manuscript in preparation) have provided more direct evidence that insertion of DNA into the EcoRI cleavage site of pSClOl influences the level of expression of tetracycline- resistance by the plasmid. Whereas Tc-resistance of pSClOl is inducible, insertion of certain DNA fragments into this site