Introduction Early Studies of Plasmids

The brilliant early studies of bacterial genetics (see Hayes, 1964, for a review) were helped by good fortune in Joshua Lederberg's choice of Escherichia coli K-12 as the strain for investigation, because this strain happened to have a genetic determinant, termed the F-factor, that permitted genes to be transferred between strains, albeit at a low frequency (< 10-6). By the mid-1950s it had been deduced that all the genetic markers of E. coli were arranged on a single linkage group, i.e. there was a single chromosome, with the exception of F itself: its transfer was not associated with the transfer of particular chromosomal regions. F was also fairly easily lost, and such F- variants could readily be converted to F+ by growth in contact with a differently marked F+ strain, at frequencies many times higher than the transfer of markers on the chromosome. Thus, F was a plasmid—a genetic element able to replicate separately from the chromosome. However, F+ cultures contained rare variants that showed a much higher frequency of chromosomal recombination in crosses with F- strains. In these Hfr (high frequency of recombination) variants, the F-factor was no longer easily lost, and it showed genetic linkage to chromosomal genes, a result which meant that the factor had integrated into the chromosome.

It was subsequently found that the integrated F-factor could sometimes excise from the chromosome of Hfr strains together with adjacent chromosomal DNA, to give rise to F-prime (F;) factors, which provided tools for geneticists to carry out functional tests, such as dominance and complementation. These observations provided early evidence of the kinds of molecular exchange that might be contributing to the evolution of bacterial chromosomes. Moreover, the E. coli chromosome was shown both genetically and physically to be cir cular, so the fact that the F-factor could integrate into it and excise from it suggested that F was also circular, with a single, reversible recombination event between it and the chromosome accounting for integration. Such single crossover integration events became known as "Campbell integration", after the first clear proposal by Campbell that they could account for the ability of the DNA of bacteriophage lambda to integrate into, and excise from, the E. coli chromosome.

Within a few years, Watanabe found that other transmissible plasmids (R-factors) were the agents of the spread of multiple antibiotic resistance among enteric bacteria, and subsequently numerous different phenotypic traits also turned out to be encoded by plasmids. It became obvious that plasmids could be found in bacteria of almost any taxonomic group. Clearly, such elements were contributing in various ways to host evolution and adaptation. This, coupled with the possibility that they might provide model systems for studies of DNA-related physiological questions, made it important to isolate and characterise them physically. The circularity of known plasmids gave rise to the first method for plasmid purification (Clewell and Helin-ski 1970). The method depends on the intercalating dye ethidium bromide, which is taken up to a reduced extent by covalently closed circular (CCC) plasmid DNA compared to open-circular (nicked) plasmid DNA, or linear DNA fragments such as those inevitably generated by shearing chromosomal DNA during its isolation. The CCC plasmids therefore undergo a smaller decrease in buoyant density than linear DNA during high-speed centrifuga-tion in a CsCl-ethidium bromide solution, and form a separate band below chromosomal DNA when the density gradient reaches equilibrium. Material separated in this way, coupled with other advances such as the development by Kleinschmidt of a method for displaying DNA in the electron microscope, led quickly to a much greater understanding of the genetic and physical organisation of plasmids, and allowed their first use as vectors for gene cloning in the early 1970s. At that time, there was no reason to expect exceptions to the rule that plasmids were circular DNA molecules, and further rapid and convenient methods for their purification, all depending on their CCC nature, became available (e.g. Birnboim and Doly 1979).

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