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.

    205/952 (page 185)
    Since ability to transfer between cells lies with the RTF component, this procedure selects for transfer of the entire R factor (that is RTF + r), for the RTF does not carry the selected drug resistance determinants and the r component cannot transfer between cells. (We shall see later that the RTF component in fact carries the tetracycline marker, but that all other markers appear to be carried on the r determinant; thus this statement remains true for selection for all drug resistance markers except that for tetracycline.) Cohen and Miller ( 1970b) argued that if R + Proteus cells are used as donors and the recipients are not selected for drug resistance, then some should gain the RTF alone. These cells can be characterized by their drug sensitivity, in spite of which they possess circles of DNA. By using several successive matings between resistant Proteus and sensitive E.coli, to increase the probability of transfer, they were able to identify drug-sensitive cells carrying circular molecules of buoyant density 1.709 g/cm 3 and length 28 /xm. This suggests that the 28 /xm molecule represents the RTF component and the 6 /xm molecule represents the r component. Another approach for isolating the RTF was developed by Silver and Falkow ( 1970a,b), who made use of derepressed R factors that are able to transfer between cells at much increased frequencies (see later). By using the labeling technique originally developed by Freifelder for the sex factor, they were able to identify closed circles of factor R1 in E.coli, with a density of 1.711 g/cm 3 and a size of 65 x 10 6 daltons. They then isolated from this strain of E.coli a variant that was sensitive to all of the drugs against which R1 confers resistance, but which remained sensitive to the phage MS2 (this RNA phage is able to lyse cells carrying either the F factor or certain R factors, including Rl). The idea that this variant had lost the drug resistance determinant r but had retained the RTF compo nent was supported by the observation that it possessed a DNA fraction of buoyant density 1.709 g/cm 3 and size 55 x 10 6 daltons. These experiments therefore suggest that the Rl factor can exist as a circular molecule of about 65 x 10 6 daltons (roughly 100,000 base pairs). In Proteus it may be obtained in the form of an RTF component of about 55 x 10 6 daltons (82,000 base pairs), which alone remains infectious, and an r component of about 12 x 10 6 daltons (18,000 base pairs), which although not infectious can be maintained in the cells. Dissociation of the R factor into RTF and r components does not take place often in E.coli, but may occur upon occasion. In terms of gene numbers, the RTF could carry about 85 genes of average length 1000 base pairs and the r compo nent could include about 15 genes of this size. Analysis of variants of R100 suggests that deletions may on occasion be responsible for the loss of drug resistance markers. The factor R100, then described as the isolate 222, was one of the early Japanese factors carry-