K. Foss

Rapid site-specific mutagenesis in plasmids

A quick and simple method for introducing site-specific mutations into plasmids is described. The procedure involves restriction-enzyme digestion of the plasmid to give a linear fragment. A second preparation of the same plasmid is digested with other restriction enzymes to remove the targeted mutational region to give a gapped fragment. The linear fragment and the gapped fragment are mixed, then denatured and annealed in the presence of a short, synthetic oligodeoxynucleotide corresponding to the targeted region and containing the desired mutation. The mix is then transformed directly into cells where host enzymes fill single-stranded gaps to make a complete double-stranded, mutant plasmid.

A mutation of the wobble nucleotide of a bacteriophage T4 transfer RNA

We have shown previously that the psu2+ mutation in bacteriophage T4 represents a single nucleotide change in the anticodon of a T4 glutamine transfer RNA, that this mutation confers on the tRNA the ability to suppress UAG and UAA nonsense mutations, and that the activity of the suppressor tRNA is relatively weak (Comer et al., 1974; Seidman et al., 1974). In this paper we describe the effects of a second anticodon mutation in the glutamine tRNA. The new mutation is a change from N (a modified form of U) to C at the 34th nucleotide residue from the 5′ end of the molecule; hence, the mutation is designated C34. Whereas the wild-type glutamine tRNA had the anticodon NUG, and the Psu2+ single-mutant tRNA had the anticodon NUA, a psu2+-C34 double mutant has CUA. Several consequences of the C34 mutation are observed. There is a change in the nonsense condons that are suppressed; the psu2+-C34 strain can suppress UAG, but not UAA. The C34 mutation also increases the efficiency of suppression several-fold, as measured by the activity of lysozyme formed by suppression of a nonsense mutation in the lysozyme gene.

Hybrid transfer RNA genes in phage T4

We describe the isolation and characterization of two unusual amber suppressor forms of T4 tRNALeu. The sequences of the suppressor tRNAs can be described as hybrids of wild-type tRNALeu and suppressor tRNAGln molecules: the chain lengths and majority of the nucleotide residues corresponded to tRNALeu, but CUA anticodons flanked by 2-14 residues were identical to tRNAGln. The uncertainty as to the exact number of flanking residues correlated with tRNAGln is due to the similarity of the two tRNA sequences in this region. No evidence was found for changes in other T4 tRNAs. We propose that genes for the hybrid tRNAs were produced by mispairing of DNAs at anticodon segments of tRNALeu and tRNAGln with a double crossover flanking those segments.

Three suppressor forms of bacteriophage T4 leucine transfer RNA.

Abstract Three suppressor forms of bacteriophage T4 leucine transfer RNA were isolated and characterized. One suppresses U-A-G mutations, another suppresses U-A-G and U-A-A mutations, while the third suppresses U-G-A mutations. Each suppressor specifies a new anticodon sequence in leucine transfer RNA. Whereas wild-type leucine transfer RNA has the anticodon sequence N-A-A (N is a modified U), the suppressor forms have C-U-A, N-U-A or N-C-A, respectively.