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.

    48/952 (page 28)
    related to transformation; for example, additional nucleases may be pres ent whose degradation of DNA is not related to its incorporation. To dissect transformation and determine the step at which the generation of single strands becomes necessary may therefore require the isolation of mutants defective at this stage. Utilization of only a single donor strand is common to transformation in pneumococcus, H.influenzae, and B.subtilis. But the timing of events in these three bacterial species is different. In pneumococcus, a short eclipse period separates the entry of donor DNA from the recovery of its ability to transform, which is more or less simultaneous with the expres sion of the transformed phenotype; during the eclipse period the donor DNA can be retrieved in the single stranded state. An eclipse period also has been reported in B.subtilis, but competent cells are in a different physiological state from noncompetent cells and the transformed phenotype is not expressed until quite some time after uptake; the state of the donor DNA following entry has not been followed with the same clarity as in pneumococcus, but it is possible that conversion to single strands may occur during this time. No eclipse period has been detected in Hemophilus, and until recently single strands could not be detected immediately following uptake, but only later as part of the recipient genome; now short single stranded regions have been detected in donor DNA, but it remains true that no general conversion of donor material into this state can be detected until after recombination with the recipient genome. The generation of single stranded DNA may therefore precede association with the recipient genome in pneumococcus and Bacillus but may accompany it in Hemophilus. Upon binding to pneumococcal cells, donor DNA is degraded. The initial binding reaction can be distinguished from the subsequent stages of transformation by adding the donor DNA to competent recipients in the presence of EDTA. In this case the DNA adsorbs to the bacteria but no further reaction occurs. Seto and Tomasz (1974) found that when divalent cations then are added to release the block, two events occur; donor DNA enters the cells in a DNAase-resistant state, concomitant with a rise in the number of transformants; and DNA is released into the medium in the form of small (acid soluble) oligonucleotides. Similar conclusions were reported previously by Morrison and Guild (1972b, 1973), who found that DNA bound to cells (in the DNAase-sensitive state) can be eluted with guanidium hydrochloride; the eluted DNA is smaller than the original DNA preparation due to the introduction of single strand breaks. These results suggest that cleavage of the DNA takes place during its uptake. The effect of EDTA may be to inhibit nucleases that depend on divalent cations; the inhibition of transformation caused by the EDTA then would