Abstract
We describe a method for creating a population of randomly digested, blunt-ended DNA fragments with 5′-TG… 3′-AC… (TG) at their termini. When these fragments were ligated to a blunt-ended vector that contains …CATA-5′ …GTAT-3′ at its termini, as high as 84% of the clones obtained after transformation contained plasmids with a reconstructed NdeI site …CATATG… …GTATAC…. When the DNA vector is prepared from an appropriate plasmid [Gross et al., Mol. Cell. Biol. 5 (1985) 1015–1024; Kotewicz et al., Gene 35 (1985) 249–258], the ATG within the restriction site corresponds to a start codon positioned downstream from a strong ribosome-binding site and controllable promoter. If a gene has been digested to the TG contained within its authentic initiation codon, the endogenous translation-initiation control sequences are deleted and expression can be controlled using the plasmid-derived promoter. In addition, a gene digested to other in-frame TGs can potentially express proteins with altered N termini. Using this method, we have placed the structural gene of SP6 RNA polymerase, trimmed precisely to its authentic start codon, under the control of the tac promoter.
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