Volume 1

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.

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    types of nonsense mutant are distinct, they must be related. A third class of nonsense mutant (which lacks any agreed exotic name) was isolated by Sambrook, Fan and Brenner (1967) and by Zipser (1967); its suppressors do not act on either amber or ochre codons. The nucleotide sequences of the amber and ochre codons were first deduced by Brenner, Stretton and Kaplan (1965) by experiments in inducing and reverting nonsense codons in the rll cistron with the chemical mutagens hydroxylamine and 2-aminopurine. Hydroxylamine causes the replacement of G/C base pairs by A/T base pairs and 2-aminopurine causes both this reaction and the reverse replacement of A/T by G/C. The patterns of production of the mutants show that each nonsense codon must contain at least one A/T base pair in DNA and that the ochre must contain another which is matched by a G/C pair at the same site in amber. The orientation of the base pairs on the DNA double helix can be distin guished by making use of the need of the phage to express the rll function before replication. This means that the phage cannot grow on K12 bacteria, on which rll function is essential, if the coding strand of DNA specifies a nonsense codon; but mutations to the anticoding strand do not inhibit growth until a later generation of phage, when replication has transferred the mutation to the daughter coding strand. As a control, phage can be grown on B type bacteria, where rll nonsense mutants do grow to show an altered phenotype. The characteristics of the phages induced by hydroxylamine imply that amber triplets must contain two bases derived from A/T base pairs in opposite orientation in the DNA, that is one A and one U on the messenger. This led to the conclusion that the nucleotide compositions of these codons must be: amber U, A, G/C ochre U, A, A/U A second type of experiment was used to resolve the sequences of the nucleo tides within the codons. Studies on mutants in which a purine is replaced only by the other purine, or a pyrimidine only by the other pyrimidine, showed that in T4 head protein, ambers are derived only from glutamine codons (CAA or CAG) or tryptophan (UGG). This suggests that the amber codon is UAG; the ochre must therefore be UAA. (These experiments have been discussed in more detail by Stretton, 1965 and Lewin, 1970). Studies on the suppression of nonsense triplets have confirmed that a transfer RNA may be mutated so that it recognises certain nonsense codon(s) instead of—or in addition to—the codons to which it usually responds. There are many different suppressor strains and the various amino acids which they insert are always related to the appropriate nonsense codons by a single base change, just as nonsense mutants are always derived by mutations of amino acids which are specified by codons differing in only one base from UAA, UAG or UGA.