Cultures from each were grown overnight in LB broth containing the appropriate antibiotics. After overnight growth at 37 C, zones of growth inhibition were detected around antibiotic discs. Zones of growth inhibition indicate sensitivity to chloramphenicol. The chloramphenicol resistance phenotype is dependent upon both the specific activity of the mutant enzyme and the stability of that mutant in the cell. Western analysis indicates that some variability is observed in enzyme levels.
Specific activity is a better measure of the effect of the substitution.
Mutant CAT was purified from each of the suppressor strains and the enzymatic activity determined. The purification consists of an affinity binding step to a chloramphenicol-coupled resin. The results of the specific activity determination are shown in Table 2 . These results indicate that the substitutions can be divided among several types. In this case, it appears that the substitution at position 25 alters the substrate binding pocket sufficiently to prevent binding of the chloramphenicol.