Abstract
5′d(TGATTGATTGA)3′ We have chemically synthesized an oligonucleotide 3′d(ACTAACTAACT)5′ that encogdes the translation termination codon TGA in all three reading frames. After ligation of appropriate restriction endonuclease linkers to the ends, the double-stranded oligonucleotide (STOP-oligonucleotide) was joined to the plasmid pBR322 between the EcoRI and BamHI, or HindIII and BamHI sites, and the hybrid plasmids were transformed into Escherichia coli HB101. Four different constructions were obtained: (i) EcoRI-STOP- BamHI (STOP-oligonucleotide flanked by EcoRI and BamHI linkers; pKTH606), (ii) HindIII-STOP- BamHI (pKTH601), (iii) BamHI-STOP- HindIII (pKTH604), and (iv) HindIII-STOP-POTS- BamHI (two STOP-oligonucleotides in opposite orientation; pKTH605). The inserts in pKTH606 and pKTH601 were excised and transferred to a modified plasmid constructed previously for the expression and secretion of foreign gene products from Bacillus subtilis. The resulting secretion plasmids now contain the promoter/signal sequence region of the α-amylase gene from Bacillus amyloliquefaciens joined to the STOP-oligonucleotide by EcoRI or HindIII linkers. Foreign genes can be
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