Gene stability
Dotaz
Zobrazit nápovědu
We investigated gene expression patterns in Lutzomyia and Phlebotomus sand fly vectors of leishmaniases. Using quantitative PCR, we assessed the expression stability of potential endogenous control genes commonly used in dipterans. We analyzed Lutzomyia longipalpis and Phlebotomus papatasi samples from L3 and L4 larval stages, adult sand flies of different sexes, diets, dsRNA injection, and Leishmania infection. Six genes were evaluated: actin, α-tubulin, GAPDH, 60 S ribosomal proteins L8 and L32 (RiboL8 and RiboL32), and elongation factor 1-α (EF1-α). EF1-α was among the most stably expressed along with RiboL8 in L. longipalpis larvae and RiboL32 in adults. In P. papatasi, EF1-α and RiboL32 were the top in larvae, while EF1-α and actin were the most stable in adults. RiboL8 and actin were the most stable genes in dissected tissues and infected guts. Additionally, five primer pairs designed for L. longipalpis or P. papatasi were effective in PCR with Lutzomyia migonei, Phlebotomus duboscqi, Phlebotomus perniciosus, and Sergentomyia schwetzi cDNA. Furthermore, L. longipalpis RiboL32 and P. papatasi α-tubulin primers were suitable for qPCR with cDNA from the other four species. Our research provides tools to enhance relative gene expression studies in sand flies, facilitating the selection of endogenous control for qPCR.
- Klíčová slova
- Lutzomyia, Phlebotomus, Endogenous control gene, Gene expression, Gene stability, Reference gene,
- MeSH
- esenciální geny * MeSH
- hmyz - vektory genetika MeSH
- hmyzí geny MeSH
- larva genetika MeSH
- Leishmania genetika MeSH
- Phlebotomus * genetika MeSH
- Psychodidae genetika MeSH
- stanovení celkové genové exprese metody MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The multidrug resistance 1 (MDR1) gene transfer to hematopoietic cells for protection against cytotoxic drugs has received considerable attention in gene therapy. However, ectopic expression of MDR1 from retroviral vectors has been hampered by its genetic instability resulting from cryptic splice sites within the cDNA. We have evaluated the efficiency of retroviral MDR1 vectors with introduced mutations of the MDR1 cryptic splice donor (cSD) located at nucleotide +339 and of the cryptic splice acceptor (cSA) at nucleotide +2319 of the cDNA. Sequence alterations of the cSD reduced the expression of MDR1 P-glycoprotein (P-gp), even when generated as silent mutations. A silent mutation of the cSA reduced the splicing activity shifting the splice acceptor site one base downstream; however, it significantly improved the expression of P-gp. The incidence of wild-type MDR1 pregenome splicing was markedly reduced when vectors were produced in human 293 packaging cells as opposed to murine PG13 and GP+envAm12. We conclude that complete splice correction of MDR1 in retroviral vectors may only be achieved with extensive alterations of the cDNA or neighboring vector sequences and that the splicing is significantly influenced by the choice of the packaging cells.
- MeSH
- buňky K562 MeSH
- exprese genu MeSH
- genetické vektory * MeSH
- geny MDR genetika MeSH
- komplementární DNA genetika MeSH
- lidé MeSH
- místa sestřihu RNA genetika MeSH
- molekulární sekvence - údaje MeSH
- mutageneze cílená * MeSH
- myši MeSH
- P-glykoprotein genetika metabolismus MeSH
- Retroviridae genetika MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- technika přenosu genů MeSH
- transdukce genetická MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- komplementární DNA MeSH
- místa sestřihu RNA MeSH
- P-glykoprotein MeSH
The determination of a suitable buffer environment for a protein of interest is not an easy task. The requirements of advanced techniques, the demands on the biological material and the researcher time needed for buffer optimization, as well as personal inflexibility, lead frequently to the use of sub-optimal buffers. Here, we demonstrate the design of a 48-condition buffer screen that can be used to determine an appropriate environment for downstream studies. By the combination of several techniques (differential scanning fluorimetry, dynamic light scattering, and bio-layer interferometry), we are able to assess the protein stability, homogeneity and binding activity across the screen with less than half a milligram of protein in 1 day. The application of this screen helps to avoid unsuitable conditions, to explain problems observed upon protein analysis and to choose the most suitable buffers for further research. The screen can be routinely used as a primary screen for buffer optimization in labs and facilities.
- Klíčová slova
- Bio-layer interferometry, Buffer, Differential scanning fluorimetry, Dynamic light scattering, Protein stability, Screening,
- MeSH
- dynamický rozptyl světla MeSH
- fluorometrie MeSH
- proteiny MeSH
- pufry MeSH
- stabilita proteinů * MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- proteiny MeSH
- pufry MeSH
Nitrilases are highly conserved proteins with catabolic activity but much less understood functions in cell division and apoptosis. To elucidate the biological functions of Arabidopsis NITRILASE1, we characterized its molecular forms, cellular localization and involvement in cell proliferation and plant development. We performed biochemical and mass spectrometry analyses of NITRILASE1 complexes, electron microscopy of nitrilase polymers, imaging of developmental and cellular distribution, silencing and overexpression of nitrilases to study their functions. We found that NITRILASE1 has an intrinsic ability to form filaments. GFP-NITRILASE1 was abundant in proliferating cells, distributed in cytoplasm, in the perinuclear area and associated with microtubules. As cells exited proliferation and entered differentiation, GFP-NITRILASE1 became predominantly nuclear. Nitrilase silencing dose-dependently compromised plant growth, led to loss of tissue organization and sustained proliferation. Cytokinesis was frequently aborted, leading to enlarged polyploid cells. In reverse, independently transformed cell lines overexpressing GFP-NITRILASE1 showed slow growth and increased rate of programmed cell death. Altogether, our data suggest that NITRILASE1 homologues regulate the exit from cell cycle and entry into differentiation and simultaneously are required for cytokinesis. These functions are essential to maintain normal ploidy, genome stability and tissue organization.
- MeSH
- aminohydrolasy chemie genetika metabolismus ultrastruktura MeSH
- Arabidopsis cytologie genetika růst a vývoj MeSH
- buněčná diferenciace genetika MeSH
- buněčná smrt genetika MeSH
- buněčný cyklus genetika MeSH
- cytoplazma metabolismus MeSH
- cytoskelet genetika metabolismus MeSH
- hydrolasy působící na anhydridy kyselin genetika MeSH
- nádorové proteiny genetika MeSH
- nestabilita genomu * MeSH
- proliferace buněk MeSH
- regulace genové exprese u rostlin MeSH
- RNA interference MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- aminohydrolasy MeSH
- fragile histidine triad protein MeSH Prohlížeč
- hydrolasy působící na anhydridy kyselin MeSH
- nádorové proteiny MeSH
- nitrilase MeSH Prohlížeč
Rapid plant genome evolution is crucial to adapt to environmental changes. Chromosomal rearrangements and gene copy number variation (CNV) are two important tools for genome evolution and sources for the creation of new genes. However, their emergence takes many generations. In this study, we show that in Arabidopsis thaliana, a significant loss of ribosomal RNA (rRNA) genes with a past history of a mutation for the chromatin assembly factor 1 (CAF1) complex causes rapid changes in the genome structure. Using long-read sequencing and microscopic approaches, we have identified up to 15 independent large tandem duplications in direct orientation (TDDOs) ranging from 60 kb to 1.44 Mb. Our data suggest that these TDDOs appeared within a few generations, leading to the duplication of hundreds of genes. By subsequently focusing on a line only containing 20% of rRNA gene copies (20rDNA line), we investigated the impact of TDDOs on 3D genome organization, gene expression, and cytosine methylation. We found that duplicated genes often accumulate more transcripts. Among them, several are involved in plant-pathogen response, which could explain why the 20rDNA line is hyper-resistant to both bacterial and nematode infections. Finally, we show that the TDDOs create gene fusions and/or truncations and discuss their potential implications for the evolution of plant genomes.
Recent years have seen a dramatic improvement in protein-design methodology. Nevertheless, most methods demand expert intervention, limiting their widespread adoption. By contrast, the PROSS algorithm for improving protein stability and heterologous expression levels has been successfully applied to a range of challenging enzymes and binding proteins. Here, we benchmark the application of PROSS as a stand-alone tool for protein scientists with no or limited experience in modeling. Twelve laboratories from the Protein Production and Purification Partnership in Europe (P4EU) challenged the PROSS algorithm with 14 unrelated protein targets without support from the PROSS developers. For each target, up to six designs were evaluated for expression levels and in some cases, for thermal stability and activity. In nine targets, designs exhibited increased heterologous expression levels either in prokaryotic and/or eukaryotic expression systems under experimental conditions that were tailored for each target protein. Furthermore, we observed increased thermal stability in nine of ten tested targets. In two prime examples, the human Stem Cell Factor (hSCF) and human Cadherin-Like Domain (CLD12) from the RET receptor, the wild type proteins were not expressible as soluble proteins in E. coli, yet the PROSS designs exhibited high expression levels in E. coli and HEK293 cells, respectively, and improved thermal stability. We conclude that PROSS may improve stability and expressibility in diverse cases, and that improvement typically requires target-specific expression conditions. This study demonstrates the strengths of community-wide efforts to probe the generality of new methods and recommends areas for future research to advance practically useful algorithms for protein science.
- Klíčová slova
- PROSS, Protein expression, Protein stability, Recombinant proteins, Rosetta,
- MeSH
- algoritmy * MeSH
- dánio pruhované MeSH
- Escherichia coli metabolismus MeSH
- HEK293 buňky MeSH
- lidé MeSH
- molekulární modely MeSH
- proteiny chemie metabolismus MeSH
- rozpustnost MeSH
- rychlé screeningové testy MeSH
- stabilita proteinů * MeSH
- teplota MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- proteiny MeSH
The ribosome, owing to its exceptional conservation, harbours a remarkable molecular fossil known as the protoribosome. It surrounds the peptidyl transferase center (PTC), responsible for peptide bond formation. While previous studies have demonstrated the PTC activity in RNA alone, our investigation reveals the intricate roles of the ribosomal protein fragments (rPeptides) within the ribosomal core. This research highlights the significance of rPeptides in stability and coacervation of two distinct protoribosomal evolutionary stages. The 617nt 'big' protoribosome model, which associates with rPeptides specifically, exhibits a structurally defined and rigid nature, further stabilized by the peptides. In contrast, the 136nt 'small' model, previously linked to peptidyltransferase activity, displays greater structural flexibility. While this construct interacts with rPeptides with lower specificity, they induce coacervation of the 'small' protoribosome across a wide concentration range, which is concomitantly dependent on the RNA sequence and structure. Moreover, these conditions protect RNA from degradation. This phenomenon suggests a significant evolutionary advantage in the RNA-protein interaction at the early stages of ribosome evolution. The distinct properties of the two protoribosomal stages suggest that rPeptides initially provided compartmentalization and prevented RNA degradation, preceding the emergence of specific RNA-protein interactions crucial for the ribosomal structural integrity.
- MeSH
- konformace nukleové kyseliny MeSH
- molekulární modely MeSH
- peptidy chemie metabolismus MeSH
- peptidyltransferasy metabolismus chemie MeSH
- ribozomální proteiny * metabolismus chemie MeSH
- ribozomy * metabolismus MeSH
- RNA metabolismus chemie MeSH
- stabilita RNA MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- peptidy MeSH
- peptidyltransferasy MeSH
- ribozomální proteiny * MeSH
- RNA MeSH
The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. Here, we identify PHD-finger protein 3 (PHF3) as a regulator of transcription and mRNA stability that docks onto Pol II CTD through its SPOC domain. We characterize SPOC as a CTD reader domain that preferentially binds two phosphorylated Serine-2 marks in adjacent CTD repeats. PHF3 drives liquid-liquid phase separation of phosphorylated Pol II, colocalizes with Pol II clusters and tracks with Pol II across the length of genes. PHF3 knock-out or SPOC deletion in human cells results in increased Pol II stalling, reduced elongation rate and an increase in mRNA stability, with marked derepression of neuronal genes. Key neuronal genes are aberrantly expressed in Phf3 knock-out mouse embryonic stem cells, resulting in impaired neuronal differentiation. Our data suggest that PHF3 acts as a prominent effector of neuronal gene regulation by bridging transcription with mRNA decay.
- MeSH
- buněčné linie MeSH
- fosforylace MeSH
- genetická transkripce MeSH
- genový knockdown MeSH
- lidé MeSH
- myši knockoutované MeSH
- myši MeSH
- neurony chemie metabolismus MeSH
- posttranskripční úpravy RNA MeSH
- proteinové domény MeSH
- regulace genové exprese MeSH
- RNA-polymerasa II chemie genetika metabolismus MeSH
- RNA * chemie genetika metabolismus MeSH
- stabilita RNA MeSH
- transkripční faktory genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- PHF3 protein, human MeSH Prohlížeč
- RNA-polymerasa II MeSH
- RNA * MeSH
- transkripční faktory MeSH
Two Bacillus cereus feather-degrading strains (23/1 and 6/2) were transformed using a recombinant plasmid p5.2 carrying the alkaline proteinase gene (aprE). A high level of the aprE gene expression was observed when the recombinant strains were grown on sporulation medium. The expression of the aprE gene proceeded during the early stationary phase and the p5.2 plasmid was segregationally and structurally stable in both strains. The two recombinant strains grown on a mineral medium with 1 % chicken feather as source of energy, carbon and nitrogen exhibited higher proteolytic activity ( approximately 6-fold and 2.4-fold higher for strains 23/1 (p5.2) and 6/2 (p5.2), respectively. Keratinolytic activity increased approximately 3.5-fold and 4.15-fold, respectively. The keratinolytic activity further increased when an optimized medium with yeast extract and corn oil was used. Considerable amounts of free amino acids were obtained after the biodegradation of feather which makes the new strains promising for application in feather-waste treatment to, e.g., transformation to animal feedstuff.
- MeSH
- Bacillus cereus enzymologie metabolismus MeSH
- bakteriální proteiny biosyntéza MeSH
- bakteriální transformace MeSH
- endopeptidasy biosyntéza MeSH
- exprese genu * MeSH
- geny hub MeSH
- keratiny metabolismus MeSH
- kultivační média chemie MeSH
- kur domácí MeSH
- nestabilita genomu MeSH
- peří metabolismus MeSH
- plazmidy MeSH
- rekombinantní proteiny biosyntéza MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- alkaline protease MeSH Prohlížeč
- bakteriální proteiny MeSH
- endopeptidasy MeSH
- keratiny MeSH
- kultivační média MeSH
- rekombinantní proteiny MeSH
The RNA chaperone Hfq plays crucial roles in bacterial gene expression and is a major facilitator of small regulatory RNA (sRNA) action. The toroidal architecture of the Hfq hexamer presents three well-characterized surfaces that allow it to bind sRNAs to stabilize them and engage target transcripts. Hfq-interacting sRNAs are categorized into two classes based on the surfaces they use to bind Hfq. By characterizing a systematic alanine mutant library of Hfq to identify amino acid residues that impact survival of Escherichia coli experiencing nitrogen (N) starvation, we corroborated the important role of the three RNA-binding surfaces for Hfq function. We uncovered two, previously uncharacterized, conserved residues, V22 and G34, in the hydrophobic core of Hfq, to have a profound impact on Hfq's RNA-binding activity in vivo. Transcriptome-scale analysis revealed that V22A and G34A Hfq mutants cause widespread destabilization of both sRNA classes, to the same extent as seen in bacteria devoid of Hfq. However, the alanine substitutions at these residues resulted in only modest alteration in stability and structure of Hfq. We propose that V22 and G34 have impact on Hfq function, especially critical under cellular conditions when there is an increased demand for Hfq, such as N starvation.
- MeSH
- bakteriální RNA * metabolismus genetika MeSH
- dusík metabolismus MeSH
- Escherichia coli genetika metabolismus MeSH
- hydrofobní a hydrofilní interakce MeSH
- konzervovaná sekvence MeSH
- malá nekódující RNA * metabolismus genetika MeSH
- molekulární chaperony genetika metabolismus chemie MeSH
- mutace MeSH
- protein hostitelského faktoru 1 * genetika chemie metabolismus MeSH
- proteiny z Escherichia coli * genetika chemie metabolismus MeSH
- regulace genové exprese u bakterií MeSH
- stabilita RNA * MeSH
- stanovení celkové genové exprese MeSH
- transkriptom MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- bakteriální RNA * MeSH
- dusík MeSH
- Hfq protein, E coli MeSH Prohlížeč
- malá nekódující RNA * MeSH
- molekulární chaperony MeSH
- protein hostitelského faktoru 1 * MeSH
- proteiny z Escherichia coli * MeSH
