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Prior art
Please see references 2 and 3 for reviews of the principles behind the experiment and reference 4 for a discussion of the experimental system used, the rII gene of bacteriophage T4.
Key elements of the experimental system
- On E. coli strain B, wild-type bacteriophage T4 produces small plaques
- On E. coli strain B, derivatives of phage T4 defective in any r gene (rI, rIIA, or rIIB) produces large plaques.
- On E. coli strain K12, wild-type bacteriophage T4 forms plaques as on strain B.
- On E. coli strain K12, mutant phages defective in an r gene do not form plaques at all.
These characteristics make it possible to screen for mutant phages, looking for large plaques on strain B, and to select for wild-type recombinant phages, looking for plaques on strain K12.
Brief synopsis
In brief, the
experiment made use of the ability of the mutagens acridine yellow and
proflavine to add or remove nucleotides from DNA [5], as follows.
- A proflavine-induced mutant (called
FC0) in the rIIB gene of bacteriophage T4 was further mutagenized with
acridine yellow to produce mutants that have the wild-type ability to
produce infection on E. coli K12. These mutants carried two mutations:
the original (the same as in FC0) and a second mutation that suppressed
the first.
- The two mutations were separated, and FC0 was arbitrarily
labeled a (+) mutation and the suppressors (-) mutations, under the
theory that a (+) and a (-) mutation together could suppress each
other.
- By a similar means, (+) suppressors of the (-) suppressors
were obtained and combined to form strains with two or three (+)
mutations.
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