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.

    241/952 (page 221)
    was shown by measuring the proportion of DNA found in covalently closed circles; this increased from the wild type equivalent of about one copy of plasmid DNA per bacterial chromosome to a level of 2-4 copies per chromosome. Uhlin and Nordstrom (1975) noted that some copy mutants decrease incompatibility while others increase it; this might be explained if the first class has a mutation reducing the affinity of a repressor for its operator (which should increase the total number of copies of the plasmid able to exist in the cell), while the second class represents a mutation reducing the affinity of the operator for the re pressor (which increases the number of copies of the mutant plasmid but also increases the concentration of repressor, which therefore is more active upon other, wild type copies of the plasmid introduced into the cell). Of course, while this speculation supports a repressor model, the characterization of putative regulator protein and operator mutants by determining their dominance with wild type alleles is necessary before a model can be constructed. Copy number mutants of R100 have been isolated by Morris et al. (1974). Although incompatibility is a plasmid function, it may rely upon the utilization of certain host functions, those concerned with replication. Thus Palchoudhury and Iyer (1971) found that both surface exclusion and plasmid incompatibility were reduced between two F' factors when E.coli cells temperature sensitive in DNA replication were incubated at high temperature. And Davis and Vapnek (1976) have isolated a bacterial mutant that has an increased resistance to antibiotics because of a chromosomal mutation that increases the copy number of R53S-\drd from 5 to 13 copies per host chromosome origin. An obvious question about R factor replication is whether the RTF or r component is responsible for controlling initiation of the complete factor. Since transfer from donor to recipient is a property of the RTF, it seems clear that this replicón must function in the transfer mode during conjuga tion. Analogy with the sex factor suggests that this requires the introduc tion of a nick at the origin for transfer, followed by rolling circle replica tion to provide the strand that is transferred to the recipient (see Figure 2.29). In F-like R factors, the RTF component shares considerable homology with the sex factor and may therefore utilize the same transfer origin. For vegetative replication, however, it is not clear how the two component replicons interact, although it has generally been supposed that one is active and the other is suppressed. There is no a priori reason to suggest which component fills which role. One experimental approach is to determine by visualization of replicat ing molecules where the origin is located. Perlman and Rownd (1976) have obtained replicating DNA of R100 from Proteus, using conditions in