Figure 1.

Oligonucleotides used in the lacZ constructions in this study. Pairs of complementary oligonucleotides with sticky HindIII and BamHI overhangs were ligated into the multiple cloning site in the lacZ gene on pEK-0F which had been simultaneously cut with HindIII and BamHI. The noncoding strands are shown for only the top two oligonucleotides. Symbols representing sequences in L-16 (TTT) shared by the other constructs are ∗, sequences before the slide; −, bypassed regions; and +, sequences after the slide. pEK-0F (pMLB1115EK-0F; ref. 9) is a pBR322 derivative carrying the lacIZ genes. Its lacZ gene contains a modified pUC 9 multiple cloning site, into which we inserted the coding sequence for 18 amino acids ending in the enterokinase cleavage site (Asp-Asp-Asp-Asp-Lys) just upstream of the pUC HindIII site. Enterokinase cleaves the resulting β-galactosidase after the Lys, producing an N terminus of Val-Ser followed by our sequence of interest, with the result that Edman sequence data are greatly improved (9). The left-shift reporter, named LefShf here, has an ATA isoleucine codon following the “shifty” quadruplet T TTC and is identical to that reported in figure 3 of ref. 9. The family of leap reporters have similar sequences, but the TTT triplet two codons before the isoleucine ATA was changed to TAT so that there would be only one phenylalanine codon, to avoid ambiguity in protein sequence. The zero frame control, L-0f, is identical to L-16 except that the blocking TAG terminator in the 0 reading frame has been changed to a sense TAC and a T two nucleotides further downstream has been eliminated, thus continuing the 0 frame into the β-galactosidase coding sequence.