Fig. 1. Schematic representation of the chimeric gene constructs used for the in vitro transcription/translation experiments and functional in vitro analysis of the translation initiation context in the vicinity of the two potential in-frame AUG start codons in the thi1 mRNA. (A) The THI64-GUS construct retains both 5' thi1 in-frame ATGs intact codons. THI641-GUS contains a mutated first codon and an intact second codon. THI642-GUS presents an intact first ATG codon and a mutated second codon. THI64-1c-GUS contains a poor context introduced around the first ATG start codon. THI64-2c-GUS presents an optimum context inserted in the vicinity of the second in-frame ATG. In THI64-12c-GUS both initiation codons were altered (same modifications as described before). (B) Translational products from the first and second in-frame AUGs, 83 kDa and 75 kDa, respectively, of the chimeric gene constructions presented above (lanes 1 to 6) and SK+ GUS (67 kDa) used as a control. (C) Quantification of the translational products of the chimeric gene constructs. The GUS translation initiation efficiency was assumed to be similar in all situations. The intensity of the translational products is given as the ratio of the translation initiation in each of the two in-frame AUG start codons to that observed at the GUS initiation codon.