Expression of dicistronic transcriptional units in transgenic tobacco.

Abstract

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...

Both sense and antisense RNAs are targets for the sense transgene-induced posttranscriptional silencing mechanism

“…Therefore, we assume that this variation is intrinsic to the composition of the chimeric constructs and unrelated to PTGS. Addition of a coding sequence to a gene construct has also been shown to decrease signi®cantly the steady-state levels of transcript accumulation (Angenon et al 1989;Van Blokland et al 1994). Here, we show that each gus construct with sequence homology to the nptII transgene yields signi®cantly lower GUS activities in nptII-silenced HOlo1 and HOloA leaves than in non-silenced HOlo2 leaves (Table 1; Fig.…”

Detection and assignment of proteins encoded by rice black streaked dwarf fijivirus S7, S8, S9 and S10.

“…The translational efficiency of the downstream ORF in a dicistronic construct is only 0n3-2n5 % of that in a monocistronic construct (Kaufman et al, 1987) when artificially engineered transcription units consisting of several normal-sized cistrons are introduced into mammalian cells. In plants, Angenon et al (1989) reported that a value 500-fold lower was obtained with a dicistronic unit.…”

Identification, characterization, and cell specificity of a human LINE-1 promoter.

“…Moreover, the plasmid sequences upstream of the LlHs construct are not necessary for LlHs transcription, although they are capable of modulating it (Fig. 9) (7,11,15,21,34,44,59). Like LlHs, the picornaviruses contain long 5' UTRs in which are found several AUG codons and short ORFs (34).…”