A positive feedback vector for identification of nucleotide sequences that enhance translation

Zhou, Wei; Edelman, Gerald M.; Mauro, Vincent P.

doi:10.1073/pnas.0409892102

Cited by 6 publications

(8 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For a different application, luciferase reporter can be substituted by the flg22 elicitor itself (Gómez-Gómez and Boller 2000; Zipfel and Felix 2005), or alternatively, by the resistance R protein genes known to induce the hypersensitive result which may lead to apoptosis (Glowacki et al 2010). Similar Gal4DBD-VP16-based transcriptional feedback loops were previously built by others for the expression of luciferase reporter in plants (Schwechheimer et al 2000), the amplification of cytotoxic diphtheria toxin A gene in mammalian cell lines (Imhof et al 2000), or in monitoring translation of the second coding region (feedback effector) within a dicistronic mRNA (Zhou et al 2005). Another commonly used transcriptional amplification system is the Gal4DBD-p65AD-based GeneSwitch System (Invitrogen), which uses the activation domain from NF-B transcription factor p65, rather than VP16.…”

Section: Resultsmentioning

confidence: 95%

A strategy for building an amplified transcriptional switch to detect bacterial contamination of plants

2011

View full text Add to dashboard Cite

We have designed and tested a transcriptional autofeedback loop that could be used to engineer plants to sense the presence of bacteria. The signal amplification circuit was built based on the biological switch responsive to the presence of bacterial flagellin. Several flagellin- and E. coli-inducible Arabidopsis promoters were cloned and tested in transient expression assays in Arabidopsis and lettuce protoplasts using a flagellin-based peptide. These were investigated either as direct drivers of a reporter gene, or as a component of a transcriptional autofeedback loop. Arabidopsis promoters from the xyloglucan endotransglucosylase/hydrolase 18 (ATXTH18) and cytochrome P450 family CYP82C3 monooxygenase worked well as biological switches. These promoters were incorporated into our feedback loop system for signal amplification. The inclusion of a transcriptional repressor reduced basal expression, thereby increasing fold-amplification of signal detection and fine-tuning the positive autofeedback loop regulation.

show abstract

Section: Resultsmentioning

confidence: 95%

A strategy for building an amplified transcriptional switch to detect bacterial contamination of plants

2011

View full text Add to dashboard Cite

show abstract

“…However, an IRES inserted into the ICS facilitates the translation of Gal4VP16. This triggers a positive feedback loop as the increased levels of Gal4VP16 bind to UAS sequences in the promoter and increase the transcription of this mRNA, resulting in more luciferase and Gal4VP16 (20).…”

Section: Resultsmentioning

confidence: 99%

“…To test SV40 sequences for IRES activity, we used p4xUAS ( Fig. 2A) (20). This plasmid encodes a dicistronic mRNA with the Renilla luciferase gene as the first cistron, followed by an intracistronic sequence (ICS), and the gene for the Gal4VP16 fusion protein as the second cistron.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

19S Late mRNAs of Simian Virus 40 Have an Internal Ribosome Entry Site Upstream of the Virion Structural Protein 3 Coding Sequence

Alwine

2006

J Virol

View full text Add to dashboard Cite

The late mRNAs of simian virus 40 (SV40) are polycistronic. The 19S mRNAs encode primarily the virion structural proteins VP2 and VP3. The VP2 and VP3 coding sequences are located in the same reading frame, and the VP3 AUG is an internal AUG for VP2. We tested whether an internal ribosome entry site (IRES) might be located upstream of the VP3 AUG that would facilitate its utilization, especially late in infection when cap-dependent translation is reduced (19). Using dicistronic reporter systems for IRES detection, we detected IRES activity within SV40 nucleotides (nts) 565 to 916, the region between the VP2 and VP3 AUGs. Nuclease protection analysis and primer extension analysis indicate no aberrant transcription or splicing that could account for false prediction of an IRES. Deletion analysis of the region indicates the presence of two functional IRESs, one within nts 661 to 830 and the other within nts 771 to 915. These two regions, each containing one IRES, have essentially the same IRES activity as the entire region spanning nts 616 to 915, which contains both IRESs. This suggests that potential secondary structures in the overlapping regions spanning nts 661 to 830 and nts 771 to 915 may be in dynamic equilibrium, such that there is only one functional IRES at any one time. These data strongly suggest that an IRES can be utilized for VP3 translation from the SV40 19S late mRNAs.Eukaryotic translation can be initiated by either cap-dependent or cap-independent mechanisms. In some mRNAs, capindependent translation is facilitated by internal ribosome entry sites (IRESs), first discovered in the uncapped picornavirus RNA genome. These sites enable ribosomes to initiate on highly structured regions located within untranslated regions 5Ј of the initiator AUG (9, 12, 13). Subsequently, many IRESs were identified in both RNA and DNA viral genomes (1,6,7,9,12,13,16) and in a broad range of cellular mRNAs from mammals, insects, and Saccharomyces cerevisiae (references 2, 8, and 17 and references therein).The late coding region of the simian virus 40 (SV40) genome encodes four proteins, the virion structural proteins VP1, VP2, VP3 and the agnoprotein. The differential splicing of late transcripts produces two classes of mRNA, 16S and 19S late mRNAs (Fig. 1). Each class has a number of members due to heterogeneity in start sites and utilization of splice sites (15). However, the 5Ј end that is most utilized, the major cap site (Fig. 1), is at SV40 nucleotide (nt) 325, and the majority of transcripts display the splicing patterns shown in Fig. 1. The 16S mRNAs can encode the agnoprotein and the major virion structural protein VP1, and the start codons for VP2 and VP3 are spliced out. A ribosome entering at the cap would scan to the agnoprotein AUG (nt 335) and initiate and translate to the stop codon at nt 523, where it would terminate. We have predicted that ribosomes can then scan on for about 40 nucle-

show abstract

“…Many of the previous reports where transcriptional amplification was used utilized a fusion of a viral VP16 protein with GAL4 binding domain. 16,18,27 In our previous studies we successfully used a mammalian transactivation domain p65. At this point it is unclear whether each of the two chimera has any particular advantage in terms of degree of enhancement or potential side effects or longevity of gene expression.…”

Section: Lentiviral Vectors For Targeting Serotonergic Neurons K Benzmentioning

confidence: 99%

Targeting central serotonergic neurons with lentiviral vectors based on a transcriptional amplification strategy

et al. 2009

View full text Add to dashboard Cite

Numerous pathological processes have been linked to disorders of the central 5-hydroxytryptamine (5HT) system but the possibility of gene therapy through modulation of 5HT system remained unexplored due to the lack of the specific targeting vectors. We explored the sequences upstream of the tryptophan hydroxylase-2 (TPH-2) gene, the key enzyme in neuronal 5HT synthesis and generated lentiviral vectors, which were then tested in vivo using microinjections into the rat raphe. All fragments longer than 1 kb (called 2TPH, 3.6TPH and 6.7TPH) drove highly specific expression in 5HT neurons which was, however too weak to be detectable without immunostaining. To enhance the level of expression, a two-step transcriptional amplification strategy was employed whereby the activity of a TPH-2 promoter fragment was potentiated by a chimeric enhancer GAL4|p65. Surprisingly, previously published implementations of this strategy compromised the specificity of expression. We therefore used a new approach where both, the transgene and GAL4|p65 are co-expressed using an internal ribosomal entry site and GAL4|p65 operates in a positive feedback manner to amplify the expression. Thus, we have generated new vectors, which are both, highly specific for 5HT neurons and sufficiently powerful. They open a range of new opportunities for enquiries into genetic correction of 5HT-related disorders.

show abstract

A positive feedback vector for identification of nucleotide sequences that enhance translation

Cited by 6 publications

References 30 publications

A strategy for building an amplified transcriptional switch to detect bacterial contamination of plants

A strategy for building an amplified transcriptional switch to detect bacterial contamination of plants

19S Late mRNAs of Simian Virus 40 Have an Internal Ribosome Entry Site Upstream of the Virion Structural Protein 3 Coding Sequence

Targeting central serotonergic neurons with lentiviral vectors based on a transcriptional amplification strategy

Contact Info

Product

Resources

About