Amber supression

Experimental isolation & identification of amber (UAG) & Amber suppressor mutants

    When T4 phage is added at very low concentration to a continuous lawn of E. coli bacteria [grey background], individual phages attack single bacteria, lyse them, and produce more phage that go on to lyse other bacteria. This produces a clear, circular plaque on the bacterial lawn [white circles].

    Rare mutants in a suspension of T4 phage include an amber stop mutation (UAG) that terminates polypeptide growth of a protein necessary for lysis of E. coli. Addition of the suspension to a lawn of E. coli (strain CR63) produces a number of plaques: when the plaques are replica-plated onto a second dish with wild-type bacteria, two of the plaques do not grow. (These regions are circled on the wild-type plate: compare locations with the two amber colonies on the CR63 plate). Transfer of the two plaques on the CR63 plate onto ordered "pick & patch" CR63 and wild-type dishes again shows that they grow on the former but not the latter. The CR63 experiment is a control, which shows that the phage is viable. The wild-type dish shows that the phage mutant is conditional, that is, it has different phenotypes (growth or no growth) on different bacterial strains.

    The CR63 bacterial strain turns out to have a serendipitously pre-existing amber suppressor tDNA mutation. tRNA molecules produced by the E. coli are able to "read through" the amber mutant in the phage DNA, thus producing a functional protein and "suppressing" the effect of the phage mutant. This allows the mutant phage to attack and lyse the mutated bacteria, producing the two exceptional, conditional plaques on CR63.

    [Amber does not refer to the color of the colonies. A graduate student offered to help with the "pick and patch" experiment, on the condition that any mutant found would be named after his mother. When the experiment worked, the UAG phage mutant was accordingly designated "amber," the English word for the substance called Bernstein in Yiddish. The Opel and Ochre stop codons were subsequently named by analogy with Amber, but do not refer either to colors or proper names].


All figure &  text material ©2015 by Steven M. Carr