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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    ferred to a medium containing the heavy precursor analogue, bromodeoxyuridine (BUdR), whose incorporation increases the density of DNA. After transformation, the activity of the donor DNA remained largely in the light part of the gradient; since it was not incorporated into regions containing BUdR, the competent cells could not be synthesizing DNA. In noncompetent cells, by contrast, preexisting recipient DNA enters the hybrid state as a result of replication. When cells are incubated with BUdR during and following transformation, the donor DNA con tinues to display little association with the hybrid label; newly trans formed cells also therefore fail to engage in DNA synthesis. These exper iments thus imply that competence is achieved only by nonreplicating cells and that this inactivity continues for some time after the entry of transforming DNA. That cessation of cell division is necessary for acqui sition of competence has been reported by Spencer and Herriott (1965) for H.influenzae also. Following entry of transforming DNA into cells of pneumococcus, integration takes place fairly rapidly and the transforming gene functions may be replicated as part of the host genome and expressed within a period measured in minutes. A much greater lag between the integration of donor DNA and expression of the transformed phenotype is shown in B.subtilis, however, in which transformants may become detectable only after some hours. In reextraction experiments when donor DNA carried a try + marker and recipient bacteria carried a his + gene, Stocker (1963) and Nester and Stocker (1963) found that try + his + recombinant DNA was formed within 30 minutes after uptake. Yet the transformed phenotype is not expressed (as measured by the synthesis of tryptophan synthetase) until some four hours after uptake. When the temperature is increased so that the cells grow more rapidly, the lag time is reduced, so that it seems to be related to the growth rate of the culture. Transformants remain resistant to penicillin killing for 3-5 hours after uptake of DNA; and no increase in the number of transformants is observed during this period. These results therefore suggest that failure to multiply explains the lag before the number of transformants rises and a consequence of this physiological state must be the delay in expression of donor genes. In other experiments, Chasin and Magasanik (1970) found a delay of only about 45 minutes before expression of the histidase gene following trans formation of B.subtilis. No wây to reconcile the difference between these sets of data is immediately apparent, but it remains clear that the lag period in B.subtilis is greater than in pneumococcus or H.influenzae. Early attempts to achieve transformation with E.coli all were unsuc cessful. However, Mandel and Higa (1970) found that treatment with CaCl 2 allowed E.coli cells to take up noninfective phage DNA; Cohen,