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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    DNA CLONING AND PLASMID BIOLOGY 39 ability of divalent cations such as calcium chloride (24) or baritmi chloride (25) to alter membrane permeability of bac¬ teria — thereby enabling them to take up plasmid DNA mole¬ cules (23). When the entering plasmid carries a replication system capable of functioning in the recipient cell, the plas¬ mid can propagate itself and other DNA fragments that are attached to it. Bacteriophage vectors are also taken up by appropriately treated cells by a process called transfection, but in this case cloning of inserted DNA fragments does not require survival of recipient cells — such cells need only yield viable viral particles consisting of the phage replicón plus the foreign DNA segment. Introduction of phage chimeras into bacterial cells potentially can be accomplished using one of several transfection procedures that employ calcium chlor¬ ide treatment (23), spheroplast production (26), or a helper phage assay (27). Separate antibiotic resistance genes carried by both the plasmid cloning vehicle and by the inserted DNA fragments were initially used to select for bacterial cells that had acquired the chimeric molecule (1). Other phenotypic markers such as colicin immimity or metabolic characteristics have since been used (6,16,28). As noted above, certain plasmid genes contain restriction endonuclease cleavage sites within them, and inactivation of such genes by insertion of a foreign DNA frag¬ ment can be employed to select for chimeric plasmids (10). Certain plasmid vectors have been designed especially to utilize insertional inactivation; the pAClSl plasmid (Figure 2) is one of these. pAClSl was formed by linking the replica¬ tion functions of the mini-circular plasmid from ]E. coli strain 15 to the chloramphenicol resistance gene of R6-5 and the tetracycline resistance gene of pSClOl. The resulting plasmid has an EcoRI cleavage site within the chloramphenicol resistance gene, and insertion of DNA fragments within Hindlll, BamI, or Sail cleavage sites of the plasmid inactivate the tetracycline resistance gene. Inactivation of the resistance markers is easily detected by plating procedures, or selected using modifications of a penicillin-selection procedure (29). In addition to being suitable for use with the insertional inactivation procedure for EcoRI, Hindlll, BamI, or Sail DNA fragments, the pAClSl plasmid is multicopy, is amplifiable, and is non-conjugative (Chang and Cohen, manuscript in prepara¬ tion) .
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