The gene of an archaeal a-l-fucosidase is expressed by translational frameshifting

Cobucci-Ponzano, Beatrice; Conte, Fiorella; Benelli, Dario; Londei, Paola; Flagiello, Angela; Monti, Maria; Pucci, Piero; Rossi, Mosè; Moracci, Marco

Genomic DNA from S.solfataricus P2 strain was prepared as described previously (24) . A DNA fragment of 1538 nt containing the complete fucA1 gene, was prepared by PCR, by using the following synthetic oligonucleotides (Genenco, Florence, Italy): FucA1-fwd, 5 0 -CTGGAGGCGCGCTAA-TACGACTCACTATAGGTCAGTTAAATGTCACAAAA-TTCT-3 0 ; FucA1-rev, 5 0 -GACTTGGCGCGCCTATCTAT-AATCTAGGATAACCCTTAT-3 0 , in which the sequence corresponding to the genome of S.solfataricus is underlined. In the FucA1-fwd primer, the sequence of the promoter of the T7 RNA polymerase is in boldface and the sequence of the BssHII site is shown in italics. The PCR amplification was performed as described previously (24) and the amplification products were cloned in the BssHII site of the plasmid pBluescript II KS+. The fucA1 gene was completely re-sequenced to check if undesired mutations were introduced by PCR and the recombinant vector obtained, named pBlu-FucA1, was used for translation in vitro experiments.