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The applicability and predictive properties of the linear solvent strength model and two nonlinear retention-time models, i.e., the quadratic model and the Neue model, were assessed for the separation of small molecules (phenol derivatives), peptides, and intact proteins. Retention-time measurements were conducted in isocratic mode and gradient mode applying different gradient times and elution-strength combinations. The quadratic model provided the most accurate retention-factor predictions for small molecules (average absolute prediction error of 1.5%) and peptides separations (with a prediction error of 2.3%). An advantage of the Neue model is that it can provide accurate predictions based on only three gradient scouting runs, making tedious isocratic retention-time measurements obsolete. For peptides, the use of gradient scouting runs in combination with the Neue model resulted in better prediction errors (<2.2%) compared to the use of isocratic runs. The applicability of the quadratic model is limited due to a complex combination of error and exponential functions. For protein separations, only a small elution window could be applied, which is due to the strong effect of the content of organic modifier on retention. Hence, the linear retention-time behavior of intact proteins is well described by the linear solvent strength model. Prediction errors using gradient scouting runs were significantly lower (2.2%) than when using isocratic scouting runs (3.2%).
- MeSH
- časové faktory MeSH
- chromatografie s reverzní fází * MeSH
- fenoly chemie izolace a purifikace MeSH
- molekulární modely MeSH
- molekulová hmotnost MeSH
- peptidy chemie izolace a purifikace MeSH
- proteiny chemie izolace a purifikace MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Accurate gene expression measurements are essential in studies of both crop and wild plants. Reverse transcription quantitative real-time PCR (RT-qPCR) has become a preferred tool for gene expression estimation. A selection of suitable reference genes for the normalization of transcript levels is an essential prerequisite of accurate RT-qPCR results. We evaluated the expression stability of eight candidate reference genes across roots, leaves, flower buds and pollen of Silene vulgaris (bladder campion), a model plant for the study of gynodioecy. As random priming of cDNA is recommended for the study of organellar transcripts and poly(A) selection is indicated for nuclear transcripts, we estimated gene expression with both random-primed and oligo(dT)-primed cDNA. Accordingly, we determined reference genes that perform well with oligo(dT)- and random-primed cDNA, making it possible to estimate levels of nucleus-derived transcripts in the same cDNA samples as used for organellar transcripts, a key benefit in studies of cyto-nuclear interactions. Gene expression variance was estimated by RefFinder, which integrates four different analytical tools. The SvACT and SvGAPDH genes were the most stable candidates across various organs of S. vulgaris, regardless of whether pollen was included or not.
- MeSH
- komplementární DNA genetika MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- polymerázová řetězová reakce s reverzní transkripcí metody MeSH
- rostlinné geny * MeSH
- sekvenční analýza RNA MeSH
- Silene genetika MeSH
- stanovení celkové genové exprese MeSH
- Publikační typ
- časopisecké články MeSH
- validační studie MeSH
Although it is generally accepted that signal transduction in plant mitogen-activated protein kinase signaling cascades is regulated via rapid posttranslational modifications, there are also several compelling examples of swift stress induced transcriptional activation of plant MAP kinase genes. A possible function of these fast and transient events is to compensate for protein losses caused by degradation of phosphorylated MAP kinases within stimulated pathways. Nevertheless, there is still need for additional evidence to precisely describe the regulatory role of plant MAP kinase transcriptional dynamics, especially in the context of whole stress stimulated pathways including also other signaling molecules and transcription factors. During the last two decades a reverse transcription quantitative real-time PCR became a golden choice for the accurate and fast quantification of the gene expression and gene expression dynamic. In here, we provide a robust, cost-effective SYBR Green-based RT-qPCR protocol that is suitable for the quantification of stress induced plant MAP kinase transcriptional dynamics in various plant species.
- MeSH
- Arabidopsis účinky léků genetika růst a vývoj fyziologie MeSH
- chlorid sodný farmakologie MeSH
- DNA primery genetika MeSH
- fyziologický stres genetika MeSH
- klonování DNA MeSH
- komplementární DNA biosyntéza genetika MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- mitogenem aktivované proteinkinasy genetika MeSH
- osmotický tlak účinky léků MeSH
- regulace genové exprese u rostlin * účinky léků MeSH
- reverzní transkripce * účinky léků MeSH
- RNA rostlin genetika izolace a purifikace MeSH
- semenáček růst a vývoj MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Ribosomal protein genes (RPGs) in Saccharomyces cerevisiae are a remarkable regulatory group that may serve as a model for understanding genetic redundancy in evolutionary adaptations. Most RPGs exist as pairs of highly conserved functional paralogs with divergent untranslated regions and introns. We examined the roles of introns in strains with various combinations of intron and gene deletions in RPL22, RPL2, RPL16, RPL37, RPL17, RPS0, and RPS18 paralog pairs. We found that introns inhibited the expression of their genes in the RPL22 pair, with the RPL22B intron conferring a much stronger effect. While the WT RPL22A/RPL22B mRNA ratio was 93/7, the rpl22aΔi/RPL22B and RPL22A/rpl22bΔi ratios were >99/<1 and 60/40, respectively. The intron in RPL2A stimulated the expression of its own gene, but the removal of the other introns had little effect on expression of the corresponding gene pair. Rpl22 protein abundances corresponded to changes in mRNAs. Using splicing reporters containing endogenous intron sequences, we demonstrated that these effects were due to the inhibition of splicing by Rpl22 proteins but not by their RNA-binding mutant versions. Indeed, only WT Rpl22A/Rpl22B proteins (but not the mutants) interacted in a yeast three-hybrid system with an RPL22B intronic region between bp 165 and 236. Transcriptome analysis showed that both the total level of Rpl22 and the A/B ratio were important for maintaining the WT phenotype. The data presented here support the contention that the Rpl22B protein has a paralog-specific role. The RPL22 singleton of Kluyveromyces lactis, which did not undergo whole genome duplication, also responded to Rpl22-mediated inhibition in K. lactis cells. Vice versa, the overproduction of the K. lactis protein reduced the expression of RPL22A/B in S. cerevisiae. The extraribosomal function of of the K. lactis Rpl22 suggests that the loop regulating RPL22 paralogs of S. cerevisiae evolved from autoregulation.
- MeSH
- dospělí MeSH
- lidé MeSH
- reakční čas MeSH
- zraková percepce MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
Accuracy and transparency of scientific data are becoming more and more relevant with the increasing concern regarding the evaluation of data reproducibility in many research areas. This concern is also true for quantifying coding and noncoding RNAs, with the remarkable increase in publications reporting RNA profiling and sequencing studies. To address the problem, we propose the following recommendations: (a) accurate documentation of experimental procedures in Materials and methods (and not only in the supplementary information, as many journals have a strict mandate for making Materials and methods as visible as possible in the main text); (b) submission of RT-qPCR raw data for all experiments reported; and (c) adoption of a unified, simple format for submitted RT-qPCR raw data. The Real-time PCR Data Essential Spreadsheet Format (RDES) was created for this purpose.
The single-celled parasite Giardia intestinalis (Diplomonadida) has two equally sized nuclei in one cell. The nuclei have been considered identical. We have previously shown that they contain different chromosomal sets and proceed through the cell cycle with some asynchrony. Here, we demonstrate by fluorescence in situ hybridization that several genes from chromosome 5 are lost in one of the two nuclei of the WBc6 Giardia line. The missing segment stretches over at least 50kb near the 5' chromosome end. In both WB and WBc6 Giardia cell lines, chromosome 5 is trisomic in one nucleus and monosomic in the other nucleus. The described chromosomal deletion has always been observed at the monosomic chromosome in WBc6; however, the deletion was not detected in the parent line WB. The chromosomal segment was thus initially lost after biological cloning of WB, which gave rise to clone WBc6. We show that Giardia is capable of carrying out gene expression from only one nucleus. The two nuclei display a certain level of diversity, making each of them irreplaceable. The doubled karyomastigonts of diplomonads likely have separate functions both in the mastigont/flagellar organization and in chromosomal and gene content. To our knowledge, our results offer the first methodical approach to differentiating the two, so far indistinguishable nuclei.
- MeSH
- buněčné jádro genetika MeSH
- časové faktory MeSH
- chromozomální delece MeSH
- delece genu MeSH
- Giardia lamblia * genetika ultrastruktura MeSH
- hybridizace in situ fluorescenční normy MeSH
- komplementární DNA MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- mitóza MeSH
- monozomie * MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- regulace genové exprese fyziologie MeSH
- reverzní transkripce MeSH
- RNA protozoální genetika izolace a purifikace MeSH
- signální transdukce MeSH
- trizomie genetika MeSH
The single-celled parasite Giardia intestinalis (Diplomonadida) has two equally sized nuclei in one cell. The nuclei have been considered identical. We have previously shown that they contain different chromosomal sets and proceed through the cell cycle with some asynchrony. Here, we demonstrate by fluorescence in situ hybridization that several genes from chromosome 5 are lost in one of the two nuclei of the WBc6 Giardia line. The missing segment stretches over at least 50 kb near the 5' chromosome end. In both WB and WBc6 Giardia cell lines, chromosome 5 is trisomic in one nucleus and monosomic in the other nucleus. The described chromosomal deletion has always been observed at the monosomic chromosome in WBc6; however, the deletion was not detected in the parent line WB. The chromosomal segment was thus initially lost after biological cloning of WB, which gave rise to clone WBc6. We show that Giardia is capable of carrying out gene expression from only one nucleus. The two nuclei display a certain level of diversity, making each of them irreplaceable. The doubled karyomastigonts of diplomonads likely have separate functions both in the mastigont/flagellar organization and in chromosomal and gene content. To our knowledge, our results offer the first methodical approach to differentiating the two, so far indistinguishable nuclei.
- MeSH
- buněčné jádro genetika MeSH
- časové faktory MeSH
- chromozomální delece MeSH
- delece genu MeSH
- Giardia lamblia genetika ultrastruktura MeSH
- hybridizace in situ fluorescenční normy MeSH
- komplementární DNA genetika MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- mitóza MeSH
- monozomie * genetika MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- regulace genové exprese fyziologie MeSH
- reverzní transkripce MeSH
- RNA protozoální genetika izolace a purifikace MeSH
- signální transdukce MeSH
- trizomie * genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
