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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    in an action that succeeds the generation of duplex breaks. On the other hand, it remains possible that the endonuclease must initiate this process but that some other enzyme accomplishes the removal of the unwanted strand. Presumably the oligonucleotide fragments released into the medium are derived from the strand that is degraded, while the other enters the cell. Cells of B.subtilis are able to degrade exogenous DNA both endonu- cleolytically and exonucleolytically. Dubnau and Cirigliano (1972a) noted that duplex DNA added to B.subtilis cells suffers endonucleolytic breaks during uptake, so that the final length of integrated donor DNA is some what less than the size of the molecules originally added to the cells. Joenje and Venema (1975) found that when competent and noncompetent cells are separated by their different buoyant densities, transforming DNA may be inactivated by exposure to either culture, more effectively by the competent bacteria. The weight of the duplex DNA preparation decreased on exposure to either set of cells; that is both can make endonucleolytic breaks. In addition the competent cells degraded some DNA exonucleolytically, a capacity not possessed by the noncompetent cells. Exposure to EDTA inhibited both transformation and exonucleoly- tic attack, suggesting that the breakdown may be part of the transforma tion process. The behavior of B.subtilis cells towards exogenous DNA thus appears to be broadly similar to, although not necessarily identical with, that of pneumococcal cells. Before transforming DNA can become part of the recipient genome it must be transported from the membrane where it enters the cell to the nucleoid body that represents the compactly folded bacterial chromo some. Vermeulan and Venema (1974a,b) followed the uptake of 3 H labeled DNA in B.subtilis by incubating the transforming preparation with competent cells for a short time and then localizing the label by electron microscope autoradiography after various intervals. Over a one hour period DNA migrates from the cell periphery to the nucleoid bodies, association being completed within 15-60 minutes. The transforming DNA appears to be preferentially associated with the mesosomes during its movement. Mesosomes are convoluted invaginations of the cell mem brane and two types can be distinguished in these cells: some are con nected only to the plasma membrane; but others are connected also to the nuclear bodies. The number of nuclear mesosomes is increased in compe tent cells, consistent with the idea that they may provide the mechanism by which DNA is transported to the nucleoids. Another observation that may be relevant to this concept was noted by Joenje, Konings and Venema (1975): isolated membrane vesicles can bind DNA.