Expression of Marker RNAs in Pseudomonas putida

D'Souza, Lisa M.; Willson, Richard C.; Fox, George E.

doi:10.1007/s002849910017

Cited by 4 publications

(4 citation statements)

References 16 publications

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“…A 5S rRNA-based system was established in previous work to produce artificial RNA chimeras (D'Souza et al, 2000. A variant of the artificial RNA system in which a ribosomal RNA promoter was replaced by a T7 promoter was used in this work.…”

Section: Artificial 5s Rrna Carrier Systemmentioning

confidence: 99%

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Engineered 5S ribosomal RNAs displaying aptamers recognizing vascular endothelial growth factor and malachite green

Xing

Potty

Jackson

et al. 2009

J of Molecular Recognition

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In previous work, Vibrio proteolyticus 5S rRNA was shown to stabilize 13-50 nucleotide "guest" RNA sequences for expression in Escherichia coli. The expressed chimeric RNAs accumulated to high levels in E. coli without being incorporated into ribosomes and without obvious effects on the host cells. In this work, we inserted sequences encoding known aptamers recognizing a protein and an organic dye into the 5S rRNA carrier and showed that aptamer function is preserved in the chimeras. A surface plasmon resonance competitive binding assay demonstrated that a vascular endothelial growth factor (VEGF) aptamer/5S rRNA chimera produced in vitro by transcriptional runoff could compete with a DNA aptamer for VEGF, implying binding of the growth factor by the VEGF "ribosomal RNA aptamer." Separately, a 5S rRNA chimera displaying an aptamer known to increase the fluorescence of malachite green (MG) also enhanced MG fluorescence. Closely related control rRNA molecules showed neither activity. The MG aptamer/5S rRNA chimera, like the original MG aptamer, also increased the fluorescence of other triphenyl methane (TPM) dyes such as crystal violet, methyl violet, and brilliant green, although less effectively than with MG. These results indicate that the molecular recognition properties of aptamers are not lost when they are expressed in the context of a stable 5S rRNA carrier. Inclusion of the aptamer in a carrier may facilitate production of large quantities of RNA aptamers, and may open an approach to screening aptamer libraries in vivo.

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Section: Artificial 5s Rrna Carrier Systemmentioning

confidence: 99%

“…Indeed, all members of random pools of 13-and 50-mers that were examined were stabilized to a significant extent . The system has also been extended to Pseudomonas (D'Souza et al, 2000). The expression system and a general model for expression of insert/5S rRNA chimeras are shown in Figure 1.…”

Section: Introductionmentioning

confidence: 99%

Engineered 5S ribosomal RNAs displaying aptamers recognizing vascular endothelial growth factor and malachite green

Xing

Potty

Jackson

et al. 2009

J of Molecular Recognition

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“…Bacterial rRNAs are comprised of three kinds of rRNAs 16S, 23S, and 5S [140]. Two consecutive studies from one research group showed that the so-called "tagged RNA molecules", which were constructed by a 17-nucleotide sequence embedded into the 5S rRNA of Vibrio proteolyticus or Pseudomonas putida, could be stably expressed and accumulated to relatively high levels [141,142]. Building on these findings, the 5S rRNA was developed as a carrier to accommodate multiple kinds of RNAs, including 13-nt or 50-nt random sequences, vascular endothelial growth factor (VEGF) aptamer, malachite green (MG) aptamer, all of which were highly expressed and showed some binding activities [132,143,144].…”

Section: Ribosomal Rna Scaffoldmentioning

confidence: 99%

Recent Advances in Novel Recombinant RNAs for Studying Post-transcriptional Gene Regulation in Drug Metabolism and Disposition

Jilek

2023

CDM

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Drug-metabolizing enzymes and transporters are major determinants of the absorption, disposition, metabolism, and excretion (ADME) of drugs, and changes in ADME gene expression or function may alter the pharmacokinetics/pharmacodynamics (PK/PD) and further influence drug safety and therapeutic outcomes. ADME gene functions are controlled by diverse factors, such as gene polymorphism, transcriptional regulation, and co-administered medications. MicroRNAs (miRNAs) are a superfamily of regulatory small noncoding RNAs that are transcribed from the genome to regulate target gene expression at the post-transcriptional level. The roles of miRNAs in controlling ADME gene expression have been demonstrated, and such miRNAs may consequently influence cellular drug metabolism and disposition capacity. Several types of miRNA mimics and small interfering RNA (siRNA) reagents have been developed and widely used for ADME research. In this review article, we first provide a brief introduction to the mechanistic actions of miRNAs in post-transcriptional gene regulation of drug-metabolizing enzymes, transporters, and transcription factors. After summarizing conventional small RNA production methods, we highlight the latest advances in novel recombinant RNA technologies and applications of the resultant bioengineered RNA (BioRNA) agents to ADME studies. BioRNAs produced in living cells are not only powerful tools for general biological and biomedical research but also potential therapeutic agents amenable to clinical investigations.

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“…The aRNA product is expressed at high levels from an rRNA promoter and accumulates in the cell without an obvious effect on growth rate (2). Although it resembles 5S rRNA, the aRNA is inactive and not integrated into the E. coli ribosomes (2,11,14,21). The high accumulation level of the aRNA allows its presence to be readily detected by many routine molecular methods, including RNA size, hybridization, fluorescence, and immunohybridization (15,16,22).…”

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confidence: 99%

Effect of an Artificial RNA Marker on Gene Expression in Escherichia coli

Tucker¹,

Karouia²,

Wang³

et al. 2005

Appl Environ Microbiol

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Transcriptional analysis was used to examine the effect of a genomically encoded artificial RNA on Escherichia coli in rich and minimal media. Only the expression of a single gene, deoC, was unequivocally affected under both conditions. E. coli marker strains of this type may be useful in monitoring the fate and transport of bacteria in various applications.A previously constructed Escherichia coli tracking strain known as PCPHR expresses a genomic gene encoding an artificial RNA (aRNA) which contains a unique 17-nucleotide identifier sequence (2). The aRNA gene was originally derived from Vibrio proteolyticus 5S rRNA by deleting a key functional region and replacing it with the identifier sequence (21). The aRNA product is expressed at high levels from an rRNA promoter and accumulates in the cell without an obvious effect on growth rate (2). Although it resembles 5S rRNA, the aRNA is inactive and not integrated into the E. coli ribosomes (2,11,14,21). The high accumulation level of the aRNA allows its presence to be readily detected by many routine molecular methods, including RNA size, hybridization, fluorescence, and immunohybridization (15,16,22). Large numbers of alternative but equally unique identifier sequences could be introduced into the aRNA (12), thereby making it possible to simultaneously differentiate between multiple sources of E. coli. These aRNA strains are, however, genetically modified organisms whose use in field applications, such as source tracking or bioremediation, would require regulatory approval.The issue, which was addressed here, is whether or not the presence of the aRNA tracking gene will significantly modify the behavior of the cells carrying it. E. coli has eight genes for 5S rRNA that are found in seven operons. The synthesis of the rRNAs is strongly linked with cell growth and subject to sophisticated regulation (9). For example, it has been shown that if the levels of 16S and 23S rRNA are artificially reduced by deletions in one or more rRNA operons, the cell will upregulate its remaining rRNA operons to compensate for the loss (9). In contrast, the successive deletion of 5S rRNA genes revealed that there is apparently no ability to directly compensate for underexpression of 5S rRNA (3). These results suggest that the addition of an artificial RNA to the genome would not have a significant impact on other cellular processes. Nevertheless, the safety and utility of tracking strains expressing aRNAs that resemble 5S rRNA will depend on whether their presence affects other components of the cellular machinery in a significant or unexpected way.In order to address this issue, arrays of PCR products targeting each known open reading frame in the E. coli genome were used to determine what transcriptional changes are induced in the PCPHR strain compared to the wild-type (WT) E. coli EMG2 strain. Such arrays have been widely employed in the study of E. coli gene expression with considerable success (5-7, 17, 23-25). These experiments have generated extensive lists of genes that respo...

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Expression of Marker RNAs in Pseudomonas putida

Cited by 4 publications

References 16 publications

Engineered 5S ribosomal RNAs displaying aptamers recognizing vascular endothelial growth factor and malachite green

Engineered 5S ribosomal RNAs displaying aptamers recognizing vascular endothelial growth factor and malachite green

Recent Advances in Novel Recombinant RNAs for Studying Post-transcriptional Gene Regulation in Drug Metabolism and Disposition

Effect of an Artificial RNA Marker on Gene Expression in Escherichia coli

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