Sledovat ovlivnění či průběh genové exprese je v mnoha studiích velmi důležitou součástí výzkumu. V současné době dochází k rozvoji mnoha metod usnadňujících sledování regulace genové exprese, jedním z takových příkladů je využití tzv. genových reportérových testů (z angl. gene reporter assays). Tyto systémy představují rozsáhlý soubor nástrojů ke studiu regulačních sekvencí promotorů, zesilovačů a transkripčních faktorů. Existuje celá řada testů využívajících reportérové buňky pro stanovení biologické aktivity studovaných sloučenin. Cílem tohoto přehledového článku je představit přípravu reportérových plasmidů, což je vždy prvním krokem u testů využívajících reportérové geny. Následně budou popsány nejčastější druhy reportérových testů a představeny příklady jejich použití v testování in vitro.
Monitoring the influence or process of gene expression is a very important part of research in many studies. Currently, many methods are being developed to facilitate the monitoring of gene expression regulation, the use of gene reporter assays being one of the examples. These systems represent an extensive set of tools to study the regulatory sequences of promoters, enhancers, and transcription factors. There are several assays using reporter cells to determine the biological activity of the compounds studied. The aim of this review article is to present the preparation of reporter plasmids, which is always the first step in assays using reporter genes. Subsequently, the most common types of reporter test are described, and examples of their use in in vitro testing are presented.
The design of efficient and safe gene delivery vehicles remains a major challenge for the application of gene therapy. Of the many reported gene delivery systems, metal complexes with high affinity for nucleic acids are emerging as an attractive option. We have discovered that certain metallohelices-optically pure, self-assembling triple-stranded arrays of fully encapsulated Fe-act as nonviral DNA delivery vectors capable of mediating efficient gene transfection. They induce formation of globular DNA particles which protect the DNA from degradation by various restriction endonucleases, are of suitable size and electrostatic potential for efficient membrane transport and are successfully processed by cells. The activity is highly structure-dependent-compact and shorter metallohelix enantiomers are far less efficient than less compact and longer enantiomers.
- MeSH
- buněčné linie MeSH
- DNA chemie ultrastruktura MeSH
- exprese genu MeSH
- fluorescenční protilátková technika MeSH
- genetické vektory * chemie ultrastruktura MeSH
- kationty chemie MeSH
- kovové nanočástice chemie ultrastruktura MeSH
- lidé MeSH
- mikroskopie atomárních sil metody MeSH
- molekulární struktura MeSH
- průtoková cytometrie MeSH
- reportérové geny MeSH
- technika přenosu genů * MeSH
- transfekce MeSH
- viabilita buněk MeSH
- železnaté sloučeniny chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The initial activation step in the gating of ubiquitously expressed Orai1 calcium (Ca2+) ion channels represents the activation of the Ca2+-sensor protein STIM1 upon Ca2+ store depletion of the endoplasmic reticulum. Previous studies using constitutively active Orai1 mutants gave rise to, but did not directly test, the hypothesis that STIM1-mediated Orai1 pore opening is accompanied by a global conformational change of all Orai transmembrane domain (TM) helices within the channel complex. We prove that a local conformational change spreads omnidirectionally within the Orai1 complex. Our results demonstrate that these locally induced global, opening-permissive TM motions are indispensable for pore opening and require clearance of a series of Orai1 gating checkpoints. We discovered these gating checkpoints in the middle and cytosolic extended TM domain regions. Our findings are based on a library of double point mutants that contain each one loss-of-function with one gain-of-function point mutation in a series of possible combinations. We demonstrated that an array of loss-of-function mutations are dominant over most gain-of-function mutations within the same as well as of an adjacent Orai subunit. We further identified inter- and intramolecular salt-bridge interactions of Orai subunits as a core element of an opening-permissive Orai channel architecture. Collectively, clearance and synergistic action of all these gating checkpoints are required to allow STIM1 coupling and Orai1 pore opening. Our results unravel novel insights in the preconditions of the unique fingerprint of CRAC channel activation, provide a valuable source for future structural resolutions, and help to understand the molecular basis of disease-causing mutations.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- fosfatidylcholiny chemie metabolismus MeSH
- gating iontového kanálu genetika MeSH
- genetické vektory chemie metabolismus MeSH
- HEK293 buňky MeSH
- interakční proteinové domény a motivy MeSH
- konformace proteinů, alfa-helix MeSH
- konformace proteinů, beta-řetězec MeSH
- lidé MeSH
- liposomy chemie metabolismus MeSH
- luminescentní proteiny genetika metabolismus MeSH
- metoda terčíkového zámku MeSH
- mutace MeSH
- nádorové proteiny chemie genetika metabolismus MeSH
- protein ORAI1 chemie genetika metabolismus MeSH
- protein STIM1 chemie genetika metabolismus MeSH
- regulace genové exprese MeSH
- rekombinantní proteiny chemie genetika metabolismus MeSH
- reportérové geny MeSH
- simulace molekulární dynamiky MeSH
- substituce aminokyselin MeSH
- vápník metabolismus MeSH
- vápníková signalizace * MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- zelené fluorescenční proteiny genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Split reporter protein-based genetic section systems are widely used to identify and characterize protein-protein interactions (PPI). The assembly of split markers that antagonize toxins, rather than required for synthesis of missing metabolites, facilitates the seeding of high density of cells and selective growth. Here we present a newly developed split chloramphenicol acetyltransferase (split-CAT) -based genetic selection system. The N terminus fragment of CAT is fused downstream of the protein of interest and the C terminus fragment is tethered upstream to its postulated partner. We demonstrate the system's advantages for the study of PPIs. Moreover, we show that co-expression of a functional ubiquitylation cascade where the target and ubiquitin are tethered to the split-CAT fragments results in ubiquitylation-dependent selective growth. Since proteins do not have to be purified from the bacteria and due to the high sensitivity of the split-CAT reporter, detection of challenging protein cascades and post-translation modifications is enabled. In addition, we demonstrate that the split-CAT system responds to small molecule inhibitors and molecular glues (GLUTACs). The absence of ubiquitylation-dependent degradation and deubiquitylation in E. coli significantly simplify the interpretation of the results. We harnessed the developed system to demonstrate that like NEDD4, UBE3B also undergoes self-ubiquitylation-dependent inactivation. We show that self-ubiquitylation of UBE3B on K665 induces oligomerization and inactivation in yeast and mammalian cells respectively. Finally, we showcase the advantages of split-CAT in the study of human diseases by demonstrating that mutations in UBE3B that cause Kaufman oculocerebrofacial syndrome exhibit clear E. coli growth phenotypes.
- MeSH
- aktivace enzymů MeSH
- biotest metody MeSH
- chloramfenikol-O-acetyltransferasa genetika metabolismus MeSH
- Escherichia coli genetika metabolismus MeSH
- exprese genu * MeSH
- posttranslační úpravy proteinů MeSH
- proteolýza MeSH
- reportérové geny * MeSH
- ubikvitinace * MeSH
- ubikvitinligasy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Firefly luciferase is susceptible to inhibition and stabilization by compounds under investigation for biological activity and toxicity. This can lead to false-positive results in in vitro cell-based assays. However, firefly luciferase remains one of the most commonly used reporter genes. Here, we evaluated isoflavonoids for inhibition of firefly luciferase. These natural compounds are often studied using luciferase reporter-gene assays. We used a quantitative structure-activity relationship (QSAR) model to compare the results of in silico predictions with a newly developed in vitro assay that enables concomitant detection of inhibition of firefly and Renilla luciferases. The QSAR model predicted a moderate to high likelihood of firefly luciferase inhibition for all of the 11 isoflavonoids investigated, and the in vitro assays confirmed this for seven of them: daidzein, genistein, glycitein, prunetin, biochanin A, calycosin, and formononetin. In contrast, none of the 11 isoflavonoids inhibited Renilla luciferase. Molecular docking calculations indicated that isoflavonoids interact favorably with the D-luciferin binding pocket of firefly luciferase. These data demonstrate the importance of reporter-enzyme inhibition when studying the effects of such compounds and suggest that this in vitro assay can be used to exclude false-positives due to firefly or Renilla luciferase inhibition, and to thus define the most appropriate reporter gene.
- MeSH
- isoflavony chemie metabolismus MeSH
- luciferasy renil chemie metabolismus MeSH
- reportérové geny genetika fyziologie MeSH
- sekundární struktura proteinů MeSH
- světluškovití MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Regulation of translation via stop codon readthrough (SC-RT) expands not only tissue-specific but also viral proteomes in humans and, therefore, represents an important subject of study. Understanding this mechanism and all involved players is critical also from a point of view of prospective medical therapies of hereditary diseases caused by a premature termination codon. tRNAs were considered for a long time to be just passive players delivering amino acid residues according to the genetic code to ribosomes without any active regulatory roles. In contrast, our recent yeast work identified several endogenous tRNAs implicated in the regulation of SC-RT. Swiftly emerging studies of human tRNA-ome also advocate that tRNAs have unprecedented regulatory potential. Here, we developed a universal U6 promotor-based system expressing various human endogenous tRNA iso-decoders to study consequences of their increased dosage on SC-RT employing various reporter systems in vivo. This system combined with siRNA-mediated downregulations of selected aminoacyl-tRNA synthetases demonstrated that changing levels of human tryptophan and tyrosine tRNAs do modulate efficiency of SC-RT. Overall, our results suggest that tissue-to-tissue specific levels of selected near-cognate tRNAs may have a vital potential to fine-tune the final landscape of the human proteome, as well as that of its viral pathogens.
- MeSH
- buněčné linie MeSH
- lidé MeSH
- mutace MeSH
- nádorový supresorový protein p53 biosyntéza genetika MeSH
- plazmidy genetika MeSH
- promotorové oblasti (genetika) MeSH
- proteiny genetika MeSH
- proteosyntéza * MeSH
- reportérové geny MeSH
- RNA malá jaderná genetika MeSH
- RNA transferová Trp genetika metabolismus MeSH
- RNA transferová Tyr genetika metabolismus MeSH
- terminační kodon * MeSH
- tryptofan-tRNA-ligasa genetika MeSH
- tyrosin-tRNA-ligasa genetika MeSH
- virové proteiny genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The constitutive androstane receptor (CAR) is the essential regulator of genes involved both in xenobiotic and endobiotic metabolism. Diazepam has been shown as a potent stimulator of CAR nuclear translocation and is assumed as an indirect CAR activator not interacting with the CAR cavity. In this study, we sought to determine if diazepam is a ligand directly interacting with the CAR ligand binding domain (LBD) and if it regulates its target genes in a therapeutically relevant concentration. We used different CAR constructs in translocation and luciferase reporter assays, recombinant CAR-LBD in a TR-FRET assay, and target genes induction studied in primary human hepatocytes (PHHs), HepaRG cells, and in CAR humanized mice. We also used in silico docking and CAR-LBD mutants to characterize the interaction of diazepam and its metabolites with the CAR cavity. Diazepam and its metabolites such as nordazepam, temazepam, and oxazepam are activators of CAR+Ala in translocation and two-hybrid assays and fit the CAR cavity in docking experiments. In gene reporter assays with CAR3 and in the TR-FRET assay, only diazepam significantly interacts with CAR-LBD. Diazepam also promotes up-regulation of CYP2B6 in PHHs and in HepaRG cells. However, in humanized CAR mice, diazepam significantly induces neither CYP2B6 nor Cyp2b10 genes nor does it regulate critical genes involved in glucose and lipids metabolism and liver proliferation. Thus, we demonstrate that diazepam interacts with human CAR-LBD as a weak ligand, but it does not significantly affect expression of tested CAR target genes in CAR humanized mice.
- MeSH
- buněčné linie MeSH
- diazepam farmakologie MeSH
- dospělí MeSH
- hepatocyty účinky léků MeSH
- játra účinky léků MeSH
- lidé středního věku MeSH
- lidé MeSH
- ligandy MeSH
- myši MeSH
- proliferace buněk účinky léků MeSH
- proteinové domény účinky léků MeSH
- receptory cytoplazmatické a nukleární metabolismus MeSH
- reportérové geny účinky léků genetika MeSH
- transport proteinů účinky léků MeSH
- zvířata MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In plants, posttranscriptional gene silencing (PTGS) is induced by small RNAs (sRNAs) generated from various dsRNA precursors. To assess the impact of dsRNA origin, we compared downregulation of GFP expression triggered by inverted repeat (IR), antisense (AS) and unterminated sense (UT) transcripts transiently expressed from the estradiol-inducible promoter. The use of homogeneously responding tobacco BY-2 cell lines allowed monitoring the onset of silencing and its reversibility. In this system, IR induced the strongest and fastest silencing accompanied by dense DNA methylation. At low induction, silencing in individual cells was binary (either strong or missing), suggesting that a certain threshold sRNA level had to be exceeded. The AS variant specifically showed a deviated sRNA-strand ratio shifted in favor of antisense orientation. In AS lines and weakly induced IR lines, only the silencer DNA was methylated, but the same target GFP sequence was not, showing that DNA methylation accompanying PTGS was influenced both by the level and origin of sRNAs, and possibly also by the epigenetic state of the locus. UT silencing appeared to be the least effective and resembled classical sense PTGS. The best responding UT lines behaved relatively heterogeneously possibly due to complexly arranged T-DNA insertions. Unlike IR and AS variants that fully restored GFP expression upon removal of the inducer, only partial reactivation was observed in some UT lines. Our results pointed out several not yet described phenomena and differences between the long-known silencer variants that may direct further research and affect selection of proper silencer variants for specific applications.
- MeSH
- DNA bakterií MeSH
- dvouvláknová RNA genetika MeSH
- metylace DNA MeSH
- posttranskripční úpravy RNA * MeSH
- regulace genové exprese u rostlin * MeSH
- reportérové geny MeSH
- RNA interference * MeSH
- RNA rostlin genetika MeSH
- tabák genetika MeSH
- umlčovací elementy transkripční MeSH
- umlčování genů * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cyanobacteria routinely release potentially harmful bioactive compounds into the aquatic environment. Several recent studies suggested a potential link between the teratogenicity of effects caused by cyanobacteria and production of retinoids. To investigate this relationship, we analysed the teratogenicity of field-collected cyanobacterial bloom samples by means of an in vivo zebrafish embryo test, an in vitro reporter gene bioassay and by the chemical analysis of retinoids. Extracts of biomass from cyanobacterial blooms with the dominance of Microcystis aeruginosa and Aphanizomenon klebahnii were collected from water bodies in the Czech Republic and showed significant retinoid-like activity in vitro, as well as high degrees of teratogenicity in vivo. Chemical analysis was then used to identify a set of retinoids in ng per gram of dry weight concentration range. Subsequent fractionation and bioassay-based characterization identified two fractions with significant in vitro retinoid-like activity. Moreover, in most of the retinoids eluted from these fractions, teratogenicity with malformations typical for retinoid signalling disruption was observed in zebrafish embryos after exposure to the total extracts and these in vitro effective fractions. The zebrafish embryo test proved to be a sensitive toxicity indicator of the biomass extracts, as the teratogenic effects occurred at even lower concentrations than those expected from the activity detected in vitro. In fact, teratogenicity with retinoid-like activity was detected at concentrations that are commonly found in biomasses and even in bulk water surrounding cyanobacterial blooms. Overall, these results provide evidence of a link between retinoid-like activity, teratogenicity and the retinoids produced by cyanobacterial water blooms in the surrounding environment.
- MeSH
- Aphanizomenon patogenita MeSH
- dánio pruhované embryologie genetika MeSH
- embryo nesavčí účinky léků MeSH
- Microcystis patogenita MeSH
- reportérové geny MeSH
- retinoidy biosyntéza toxicita MeSH
- sinice chemie patogenita MeSH
- teratogeny toxicita MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
The interplays between the metabolic products of intestinal microbiota and the host signaling through xenobiotic receptors, including pregnane X receptor (PXR), are of growing interest, in the context of intestinal health and disease. A distinct class of microbial catabolites is formed from dietary tryptophan, having the indole scaffold in their core structure, which is a biologically active entity. In the current study, we examined a series of ten tryptophan microbial catabolites for their interactions with PXR signaling. Utilizing a reporter gene assay, we identified indole (IND) and indole-3-acetamide (IAD) as PXR agonists. IND and IAD induced PXR-regulated genes CYP3A4 and MDR1 in human intestinal cancer cells. Using time-resolved fluorescence resonance energy transfer, we show that IND (IC50 292 μM) and IAD (IC50 10 μM) are orthosteric ligands of PXR. Binding of PXR in its DNA response elements was enhanced by IND and IAD, as revealed by chromatin immunoprecipitation assay. We demonstrate that tryptophan microbial intestinal metabolites IND and IAD are ligands and agonists of human PXR. These findings are of particular importance in understanding the roles of microbial catabolites in human physiology and pathophysiology. Furthermore, these results are seminal in expanding potential drug repertoire through microbial metabolic mimicry.
- MeSH
- cytochrom P-450 CYP3A genetika metabolismus MeSH
- indoly metabolismus MeSH
- kultivované buňky MeSH
- kyseliny indoloctové metabolismus MeSH
- lidé MeSH
- ligandy MeSH
- nádorové buněčné linie MeSH
- P-glykoproteiny genetika metabolismus MeSH
- pregnanový X receptor agonisté genetika MeSH
- reportérové geny MeSH
- střevní mikroflóra * MeSH
- střevní sliznice * metabolismus mikrobiologie MeSH
- transfekce MeSH
- tryptofan metabolismus MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
