We investigated whether the two cistrons of a dicistronic mRNA can be translated in plants to yield both gene products. The coding sequences of various reporter genes were combined in dicistronic units, and their expression was analyzed in stably transformed tobacco plants at the RNA and protein levels. The presence of an upstream cistron resulted in all cases in a drastically reduced expression of the downstream cistron. The translational efficiency of the gene located downstream in the dicistronic units was 500-to 1,500-fold lower than that in a monocistronic control; a 500-fold lower value was obtained with a dicistronic unit in which both cistrons were separated by 30 nucleotides, whereas a 1,500-fold lower value was obtained with a dicistronic unit in which the stop codon of the upstream cistron and the start codon of the downstream cistron overlapped. As a strategy to select indirectly for transformants with enhanced levels of expression of a gene which is by itself nonselectable, the gene of interest can be cloned upstream from a selectable marker in a dicistronic configuration. This strategy can be used provided that the amount of dicistronic mRNA is high. If, on the other hand, the expression of the dicistronic unit is too low, selection of the downstream cistron will primarily give clones with rearranged dicistronic units.The most consistent model to describe initiation of translation in eucaryotes is the modified scanning model (27), which states that 40S ribosomal subunits bind at or near the 5' cap structure of the mRNA and migrate downstream along the mRNA until the first AUG codon is reached. If this AUG codon occurs in a favorable context for initiation, the 40S ribosomal subunits are halted, the 60S subunits associate with them, and translation is started; if the context around the first AUG codon is less favorable, some or most of the 40S subunits proceed to the next AUG. The favorable contexts for initiation, as established by nucleotide sequence analysis and site-directed mutagenesis, are AACAAIUGGC for plant mRNAs (23,25,36,45) and (GCC)GCC'CCAUG for vertebrate mRNAs (29-31). The scanning model is in accordance with the finding that nucleus-encoded genes in eucaryotes are normally organized in monocistronic transcriptional units.The concept of reinitiation has been included in the scanning model to account for the observation that an upstream AUG codon, even in an optimal context, still allows initiation of translation at a downstream AUG codon provided that a termination codon in-frame with the upstream AUG codon precedes the downstream AUG codon. Indeed, in several reports it has been demonstrated that in mammalian cells a "minicistron" inserted upstream from the main cistron does not abolish translation of the main cistron (22,28,35). After translation of the minicistron, reinitiation of translation seems to occur at the start codon of the main cistron on the same mRNA. Similar processes have been described in barley protoplasts (16). Kozak (32) showed that the efficiency of reinitiation...