Applications of Temperature-sensitive Suppressors to the Study of Cellular Biochemistry and Physiology

Abstract

The value of suppressors as biochemical and physiological research tools was first demonstrated in the study of bacteriophage replication (Epstein et al. 1964; Wood et al. 1968; Kozak and Nathans 1972). Phages are ideally suited for investigation with this system because they can be freely moved from one bacterial strain to another. This feature permits ready identification of phage strains that carry an amber mutation in an essential gene because they can form plaques on suppressor-containing (Su⁺) strains but not on Su⁻ strains. The amber gene product, which cannot be made in Su⁻ strains, can be identified by comparing the phage proteins produced in infections of Su⁺ and Su⁻ cells. The role of the amber gene product in the phage replication cycle is established by determining the step at which development is arrested in Su⁻ cells.

The application of this approach to cellular processes was not possible until the isolation of temperature-sensitive suppressor strains, with which, depending upon the temperature, the same cell can be either Su⁺ or Su⁻. A bacterial strain carrying a temperature-sensitive suppressor and an amber mutation in an essential gene is viable, but only at permissive temperatures, and can be recognized by its temperature-sensitive phenotype. In such a strain, temperature affects synthesis, rather than specific activity, of the protein encoded by the amber gene. As a result, the number of molecules of a selected protein can be systematically varied within the intact cell. The ability to titrate protein levels in vivo with amber mutant temperature-sensitive suppressor...