Tobacco ribosomal DNA spacer element stimulates amplification and expression of heterologous genes

Abstract

Here we show that the cis-acting genetic element aps (amplification-promoting sequence), isolated from the nontranscribed spacer region of tobacco ribosomal DNA (rDNA), increases the level of expression of recombinant proteins. Transgenic tobacco plants, transformed with expression cassettes containing the herbicide-resistant acetolactate synthase (hr-ALS) gene or the green fluorescent protein (GFP) gene fused to the aps sequence, had greater levels of corresponding messenger RNAs (mRNAs) and proteins compared to transformants lacking aps. Analysis of transgenic plants showed that aps increased the copy number and transcription of the adjacent heterologous genes and, in the case of hr-ALS, enhanced the herbicide resistance phenotype. Both the increased transgene copy number and enhanced expression were stably inherited. These data provide the first evidence that the aps sequence can be used for gene amplification in transgenic plants and possibly other multicellular organisms.

High-level expression of modified gene encoding human adiponectin in transgenic rice

“…1C). It was previously reported that the presence of aps could be associated with 2-5-fold increase in the copy number of transgenic genes (Borisjuk et al, 2000). Similarly, as revealed in our study by comparative southern hybridization, the transformants with aps (line RM3, RM7 and RM8) seemed to contain many more multiple copies.…”

“…Although the gene copy number is not always proportional to the mRNA expression level due to positional affect, methylation, and partial gene silencing, etc (Meyer and Saedler, 1996;Stam et al, 1998), our results from Southern blot and RT-PCR analysis suggested that aps is likely to promote transgenic expression by increasing both the target gene copy number and the transcription level in rice, similar to its effects previously reported in tobacco and tomato. Moreover, Borisjuk et al (2000) demonstrated the effects of aps on the increase in the transgene copy number and the enhanced expression could be stably inherited in the progeny of the transgenic tobacco. Further experiments in our future study are still needed to observe if the offspring of transgenic rice carrying multiple copies of the Acrp30 gene would stably retain a high level of expression.…”

“…Moreover, Fig. 2 it was reported that an average 2.5 or 2-5 fold increase in mRNA expression levels, further resulting in distinct increase in recombinant protein by introducing aps into tomato and tobacco, respectively (Borisjuk et al, 2000;Yakoby et al, 2006). Therefore the aps was also inserted upstream of the Ubi promoter in the modifi cation.…”

“…The maize ubiquitin promoter (Ubi) was cloned into the HindIII-BamHI site, and the nopaline synthase terminator (T-nos) was ligated into the SacI-EcoRI site. The aps found to increase transcriptional activity of recombinant protein in plants (Borisjuk et al, 2000) was introduced into the HindIII site, upstream of the Ubi promoter. The binary plant vector containing the modifi ed gene with the insertion of aps, under the control of Ubi promoter and T-nos, was named pM-aps-ADPN.…”

Geographically localised bursts of ribosomal DNA mobility in the grasshopper Podisma pedestris

“…Damage to rDNA might therefore be particularly prone to set off a repair cascade. Whatever the proximate trigger, different genetic elements have been identified which seem to facilitate rapid amplification either in cis (Borisjuk et al, 2000) or in trans (Lohe and Roberts, 1990). This sort of expansion could potentially lead to very rapid changes in rDNA sequence composition within populations.…”