Volume 3

Gene expression / Benjamin Lewin.

  • Lewin, Benjamin.
Date:
1974-1977
Catalogue details

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

Credit: Gene expression / Benjamin Lewin. Source: Wellcome Collection.

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    Chang and Hsu (1972) found that under these conditions E.coli cells become effective recipients for plasmid DNA. The CaCl 2 probably causes changes in the structure of the cell wall that are necessary for uptake of DNA. In both these sets of experiments, the donor DNA molecules were autonomous replicating units that then were maintained in this form; they did not undergo recombination with the bacterial genome. Using the CaCl 2 treatment, Cosley and Oishi (1973) were able to transform E.coli cells for several nutritional markers; they found that it is necessary to use recipients that are defective in the recBC enzyme sys tem. These genes code for a nuclease that is part of a recombination- repair pathway; presumably in the recBC + cells the transforming DNA is degraded before it can recombine with the resident genome. By applying the CaCl 2 treatment to recBC~ strains, it may therefore be possible to investigate transformation in E.coli. This may make it possible to investi gate which genes are necessary for integration of transforming DNA and how they are related to the genes involved in other forms of genetic exchange that have been characterized in E.coli. We may ask what significance the acquisition of competence and the transformation with donor DNA that it makes possible has for the normal life cycle of bacteria. An indication that such transfer of genetic informa tion may occur with pneumococcus in the wild is provided by the obser vation of Ottolenghi and Hotchkiss (1962) that pneumococcal cultures release DNA into the medium, thus making material available to act as donor molecules. On the other hand, Steinhart and Herriot (1968) ob served that cells of H.influenzae do not release DNA; transforming activ ity equivalent to no more than 0.1% of the cellular DNA could be found in the medium. The physiological significance of transformation therefore remains to be established. Uptake of DNA Transformation is initiated by random collisions between bacteria and donor DNA, the reaction depending upon the concentrations of compe tent cells and added DNA. Donor DNA is first bound reversibly and then is converted into an irreversibly bound state. The initial interaction prob ably occurs by an ionic binding; most of the DNA initially bound can be released if the subsequent irreversible binding is inhibited (for review see Tomasz, 1969). The donor DNA enters the cell as a linear molecule; this is converted to a single strand which is utilized in transformation and the subsequent stages of the reaction are those concerned with its integration. The usual protocol for transformation is to add duplex DNA to compe tent cells, allow a suitable period of time for uptake, and then to end the