@article{MTMT:34547384, title = {Conversion of the CG specific M.MpeI DNA methyltransferase into an enzyme predominantly methylating CCA and CCC sites}, url = {https://m2.mtmt.hu/api/publication/34547384}, author = {Albert, Pál and Varga, Bence and Ferenc, Györgyi and Kiss, Antal}, doi = {10.1093/nar/gkad1217}, journal-iso = {NUCLEIC ACIDS RES}, journal = {NUCLEIC ACIDS RESEARCH}, volume = {52}, unique-id = {34547384}, issn = {0305-1048}, abstract = {We used structure guided mutagenesis and directed enzyme evolution to alter the specificity of the CG specific bacterial DNA (cytosine-5) methyltransferase M.MpeI. Methylation specificity of the M.MpeI variants was characterized by digestions with methylation sensitive restriction enzymes and by measuring incorporation of tritiated methyl groups into double-stranded oligonucleotides containing single CC, CG, CA or CT sites. Site specific mutagenesis steps designed to disrupt the specific contacts between the enzyme and the non-substrate base pair of the target sequence (5 '-CG/5 '-CG) yielded M.MpeI variants with varying levels of CG specific and increasing levels of CA and CC specific MTase activity. Subsequent random mutagenesis of the target recognizing domain coupled with selection for non-CG specific methylation yielded a variant, which predominantly methylates CC dinucleotides, has very low activity on CG and CA sites, and no activity on CT sites. This M.MpeI variant contains a one amino acid deletion (Delta A323) and three substitutions (N324G, R326G and E305N) in the target recognition domain. The mutant enzyme has very strong preference for A and C in the 3 ' flanking position making it a CCA and CCC specific DNA methyltransferase. Graphical Abstract}, keywords = {BINDING; SEQUENCE; RESTRICTION; CLONING; ESCHERICHIA-COLI; DESIGN; EVOLUTION; RANDOM MUTAGENESIS; Recognition specificity}, year = {2024}, eissn = {1362-4962}, pages = {1896-1908}, orcid-numbers = {Ferenc, Györgyi/0000-0002-3456-319X} } @article{MTMT:34288968, title = {CRISPR/Cas9 Mutagenesis through Introducing a Nanoparticle Complex Made of a Cationic Polymer and Nucleic Acids into Maize Protoplasts}, url = {https://m2.mtmt.hu/api/publication/34288968}, author = {Nagy, Bettina and Öktem, Ayşegül and Ferenc, Györgyi and Ungor, Ditta Anita and Kalac, Aladina and Kelemen-Valkony, Ildikó and Ayaydin-Fodor, Elfrieda and Nagy, István and Dudits, Dénes and Ayaydin, Ferhan}, doi = {10.3390/ijms242216137}, journal-iso = {INT J MOL SCI}, journal = {INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES}, volume = {24}, unique-id = {34288968}, issn = {1661-6596}, abstract = {Presently, targeted gene mutagenesis attracts increasing attention both in plant research and crop improvement. In these approaches, successes are largely dependent on the efficiency of the delivery of gene editing components into plant cells. Here, we report the optimization of the cationic polymer poly(2-hydroxypropylene imine) (PHPI)-mediated delivery of plasmid DNAs, or single-stranded oligonucleotides labelled with Cyanine3 (Cy3) or 6-Carboxyfluorescein (6-FAM)-fluorescent dyes into maize protoplasts. Co-delivery of the GFP-expressing plasmid and the Cy3-conjugated oligonucleotides has resulted in the cytoplasmic and nuclear accumulation of the green fluorescent protein and a preferential nuclear localization of oligonucleotides. We show the application of nanoparticle complexes, i.e., “polyplexes” that comprise cationic polymers and nucleic acids, for CRISPR/Cas9 editing of maize cells. Knocking out the functional EGFP gene in transgenic maize protoplasts was achieved through the co-delivery of plasmids encoding components of the editing factors Cas9 (pFGC-pcoCas9) and gRNA (pZmU3-gRNA) after complexing with a cationic polymer (PHPI). Several edited microcalli were identified based on the lack of a GFP fluorescence signal. Multi-base and single-base deletions in the EGFP gene were confirmed using Sanger sequencing. The presented results support the use of the PHPI cationic polymer in plant protoplast-mediated genome editing approaches.}, year = {2023}, eissn = {1422-0067}, orcid-numbers = {Öktem, Ayşegül/0000-0001-7828-999X; Ferenc, Györgyi/0000-0002-3456-319X; Ungor, Ditta Anita/0000-0002-7659-0428} } @article{MTMT:34201709, title = {Manifestation of Triploid Heterosis in the Root System after Crossing Diploid and Autotetraploid Energy Willow Plants}, url = {https://m2.mtmt.hu/api/publication/34201709}, author = {Dudits, Dénes and Cseri, András and Török, Katalin and Vankova, Radomira and Dobrev, Petre I. and Sass, László and Steinbach, Gábor and Kelemen-Valkony, Ildikó and Zombori, Zoltán and Ferenc, Györgyi and Ayaydin, Ferhan}, doi = {10.3390/genes14101929}, journal-iso = {GENES-BASEL}, journal = {GENES}, volume = {14}, unique-id = {34201709}, issn = {2073-4425}, abstract = {Successful use of woody species in reducing climatic and environmental risks of energy shortage and spreading pollution requires deeper understanding of the physiological functions controlling biomass productivity and phytoremediation efficiency. Targets in the breeding of energy willow include the size and the functionality of the root system. For the combination of polyploidy and heterosis, we have generated triploid hybrids (THs) of energy willow by crossing autotetraploid willow plants with leading cultivars (Tordis and Inger). These novel Salix genotypes (TH3/12, TH17/17, TH21/2) have provided a unique experimental material for characterization of Mid-Parent Heterosis (MPH) in various root traits. Using a root phenotyping platform, we detected heterosis (TH3/12: MPH 43.99%; TH21/2: MPH 26.93%) in the size of the root system in soil. Triploid heterosis was also recorded in the fresh root weights, but it was less pronounced (MPH%: 9.63–19.31). In agreement with root growth characteristics in soil, the TH3/12 hybrids showed considerable heterosis (MPH: 70.08%) under in vitro conditions. Confocal microscopy-based imaging and quantitative analysis of root parenchyma cells at the division–elongation transition zone showed increased average cell diameter as a sign of cellular heterosis in plants from TH17/17 and TH21/2 triploid lines. Analysis of the hormonal background revealed that the auxin level was seven times higher than the total cytokinin contents in root tips of parental Tordis plants. In triploid hybrids, the auxin–cytokinin ratios were considerably reduced in TH3/12 and TH17/17 roots. In particular, the contents of cytokinin precursor, such as isopentenyl adenosine monophosphate, were elevated in all three triploid hybrids. Heterosis was also recorded in the amounts of active gibberellin precursor, GA19, in roots of TH3/12 plants. The presented experimental findings highlight the physiological basics of triploid heterosis in energy willow roots.}, year = {2023}, eissn = {2073-4425}, orcid-numbers = {Vankova, Radomira/0000-0001-9101-8844; Dobrev, Petre I./0000-0001-7412-6982; Steinbach, Gábor/0000-0001-7137-7030; Ferenc, Györgyi/0000-0002-3456-319X} } @article{MTMT:33999425, title = {Expression of triploid heterosis in the biomass productivity of energy willow plants under salinity stress}, url = {https://m2.mtmt.hu/api/publication/33999425}, author = {Zombori, Zoltán and Török, Szabolcs and Nagy, Bettina and László, Nikolett and Sass, László and Jancsó, Mihály and Szabó, Gábor and Rádi, Feríz and Ferenc, Györgyi and Gyuricza, Csaba and Dudits, Dénes}, doi = {10.1016/j.biombioe.2023.106852}, journal-iso = {BIOMASS BIOENERGY}, journal = {BIOMASS & BIOENERGY}, volume = {174}, unique-id = {33999425}, issn = {0961-9534}, year = {2023}, eissn = {1873-2909}, orcid-numbers = {Jancsó, Mihály/0000-0003-1934-9686; Rádi, Feríz/0000-0002-4339-9256; Ferenc, Györgyi/0000-0002-3456-319X} } @article{MTMT:33697937, title = {Probing telomeric-like G4 structures with full or partial 2′-deoxy-5-hydroxyuridine substitutions}, url = {https://m2.mtmt.hu/api/publication/33697937}, author = {Szeltner, Zoltán and Ferenc, Györgyi and Juhász, Tünde and Kupihár, Zoltán and Váradi, Zoltán and Szüts, Dávid and Kovács, Lajos}, doi = {10.1016/j.biochi.2023.01.009}, journal-iso = {BIOCHIMIE}, journal = {BIOCHIMIE}, volume = {214}, unique-id = {33697937}, issn = {0300-9084}, abstract = {Guanine quadruplexes (G4s) are stable four-stranded secondary DNA structures held together by noncanonical G-G base tetrads. We synthesised the nucleoside analogue 2′-deoxy-5-hydroxyuridine (H) and inserted its phosphoramidite into telomeric repeat-type model oligonucleotides. Full and partial substitutions were made, replacing all guanines in all the three tetrads of a three-tier G4 structure, or only in the putative upper, central, or lower tetrads. We characterised these modified structures using CD, UV absorbance spectroscopy, native gel studies, and a capture oligo-based G4 disruption kinetic assay. The strand separation activity of BLM helicase on these substituted structures was also investigated. Two of the partially H-substituted constructs adopted G4-like structures, but displayed lower thermal stabilities compared to unsubstituted G4. The construct modified in its central tetrad remained mostly denatured, but the possibility of a special structure for the fully replaced variant remained open. H substitutions did not interfere with the G4-resolving activity of BLM helicase, but its efficiency was highly influenced by construct topology and even more by the G4 ligand PhenDC3. Our results suggest that the H modification can be incorporated into G quadruplexes, but only at certain positions to maintain G4 stability. The destabilizing effect observed for 2′-deoxy-5-hydroxyuridine indicates that the cytosine deamination product 5-hydroxyuracil and its nucleoside counterpart in RNA (5-hydroxyuridine), might also be destabilizing in cellular DNA and RNA quadruplexes. The kinetic assay employed in this study can be generally employed for a fast comparison of the stabilities of various G4s either in their free or ligand-bound states. © 2023 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM)}, keywords = {CD spectroscopy; G-QUADRUPLEX DNA; Oligonucleotides; HELICASES; 2′-deoxy-5-hydroxyuridine; Kinetic assays}, year = {2023}, eissn = {1638-6183}, pages = {33-44}, orcid-numbers = {Ferenc, Györgyi/0000-0002-3456-319X; Kupihár, Zoltán/0000-0001-5499-7617; Kovács, Lajos/0000-0002-0331-3980} } @article{MTMT:33597958, title = {Improved Metal-Free Approach for the Synthesis of Protected Thiol Containing Thymidine Nucleoside Phosphoramidite and Its Application for the Synthesis of Ligatable Oligonucleotide Conjugates}, url = {https://m2.mtmt.hu/api/publication/33597958}, author = {Kupihár, Zoltán and Ferenc, Györgyi and Petrovicz, Vencel László and Fáy, Viktória R. and Kovács, Lajos and Martinek, Tamás and Hegedüs, Zsófia}, doi = {10.3390/pharmaceutics15010248}, journal-iso = {PHARMACEUTICS}, journal = {PHARMACEUTICS}, volume = {15}, unique-id = {33597958}, issn = {1999-4923}, abstract = {Oligonucleotide conjugates are versatile scaffolds that can be applied in DNA-based screening platforms and ligand display or as therapeutics. Several different chemical approaches are available for functionalizing oligonucleotides, which are often carried out on the 5′ or 3′ end. Modifying oligonucleotides in the middle of the sequence opens the possibility to ligate the conjugates and create DNA strands bearing multiple different ligands. Our goal was to establish a complete workflow that can be applied for such purposes from monomer synthesis to templated ligation. To achieve this, a monomer is required with an orthogonal functional group that can be incorporated internally into the oligonucleotide sequence. This is followed by conjugation with different molecules and ligation with the help of a complementary template. Here, we show the synthesis and the application of a thiol-modified thymidine nucleoside phosphoramidite to prepare ligatable oligonucleotide conjugates. The conjugations were performed both in solution and on solid phase, resulting in conjugates that can be assembled into multivalent oligonucleotides decorated with tissue-targeting peptides using templated ligation.}, year = {2023}, eissn = {1999-4923}, orcid-numbers = {Kupihár, Zoltán/0000-0001-5499-7617; Ferenc, Györgyi/0000-0002-3456-319X; Petrovicz, Vencel László/0000-0002-5437-2462; Kovács, Lajos/0000-0002-0331-3980; Martinek, Tamás/0000-0003-3168-8066; Hegedüs, Zsófia/0000-0002-5546-8167} } @article{MTMT:32832922, title = {Viable protoplast formation of the coral endosymbiont alga Symbiodinium spp. in a microfluidics platform}, url = {https://m2.mtmt.hu/api/publication/32832922}, author = {Bashir, Faiza and Kovács, Sándor and Ábrahám, Ágnes and Nagy, Krisztina and Ayaydin, Ferhan and Kelemen-Valkony, Ildikó and Ferenc, Györgyi and Galajda, Péter and Tóth, Szilvia Zita and Sass, László and Kós, Péter and Vass, Imre and Szabó, Milán}, doi = {10.1039/D2LC00130F}, journal-iso = {LAB CHIP}, journal = {LAB ON A CHIP}, volume = {22}, unique-id = {32832922}, issn = {1473-0197}, abstract = {Symbiodiniaceae is an important dinoflagellate family which lives in endosymbiosis with reef invertebrates, including coral polyps, making them central to the holobiont. With coral reefs currently under extreme threat from climate change, there is a pressing need to improve our understanding on the stress tolerance and stress avoidance mechanisms of Symbiodinium spp. Reactive oxygen species (ROS) such as singlet oxygen are central players in mediating various stress responses; however, the detection of ROS using specific dyes is still far from definitive in intact Symbiodinium cells due to the hindrance of uptake of certain fluorescent dyes because of the presence of the cell wall. Protoplast technology provides a promising platform for studying oxidative stress with the main advantage of removed cell wall, however the preparation of viable protoplasts remains a significant challenge. Previous studies have successfully applied cellulose-based protoplast preparation in Symbiodiniaceae; however, the protoplast formation and regeneration process was found to be suboptimal. Here, we present a microfluidics-based platform which allowed protoplast isolation from individually trapped Symbiodinium cells, by using a precisely adjusted flow of cell wall digestion enzymes (cellulase and macerozyme). Trapped single cells exhibited characteristic changes in their morphology, cessation of cell division and a slight decrease in photosynthetic activity during protoplast formation. Following digestion and transfer to regeneration medium, protoplasts remained photosynthetically active, regrew cell walls, regained motility, and entered exponential growth. Elevated flow rates in the microfluidic chambers resulted in somewhat faster protoplast formation; however, cell wall digestion at higher flow rates partially compromised photosynthetic activity. Physiologically competent protoplasts prepared from trapped cells in microfluidic chambers allowed for the first time the visualization of the intracellular localization of singlet oxygen (using Singlet Oxygen Sensor Green dye) in Symbiodiniaceae, potentially opening new avenues for studying oxidative stress.}, year = {2022}, eissn = {1473-0189}, pages = {2986-2999}, orcid-numbers = {Ferenc, Györgyi/0000-0002-3456-319X} } @article{MTMT:32722153, title = {Triploid Hybrid Vigor in Above-Ground Growth and Methane Fermentation Efficiency of Energy Willow}, url = {https://m2.mtmt.hu/api/publication/32722153}, author = {Dudits, Dénes and Cseri, András and Török, Katalin and Sass, László and Zombori, Zoltán and Ferenc, Györgyi and Poór, Péter and Borbély, Péter Gábor and Czékus, Zalán and Vankova, Radomira and Dobrev, Petre and Szántó, Judit and Bagi, Zoltán and Kovács, Kornél Lajos}, doi = {10.3389/fpls.2022.770284}, journal-iso = {FRONT PLANT SCI}, journal = {FRONTIERS IN PLANT SCIENCE}, volume = {13}, unique-id = {32722153}, issn = {1664-462X}, year = {2022}, eissn = {1664-462X}, orcid-numbers = {Ferenc, Györgyi/0000-0002-3456-319X; Poór, Péter/0000-0002-4539-6358; Bagi, Zoltán/0000-0001-7795-2024; Kovács, Kornél Lajos/0000-0002-3926-0497} } @article{MTMT:32575627, title = {A Triple Combination of Targeting Ligands Increases the Penetration of Nanoparticles across a Blood-Brain Barrier Culture Model}, url = {https://m2.mtmt.hu/api/publication/32575627}, author = {Veszelka, Szilvia and Mészáros, Mária and Porkoláb, Gergő and Szecskó, Anikó and Kondor, Nóra and Ferenc, Györgyi and Polgár, Tamás Ferenc and Katona, Gábor and Kóta, Zoltán and Kelemen, Lóránd and Páli, Tibor and Vigh, Judit Piroska and Walter, Fruzsina and Bolognin, Silvia and Schwamborn, Jens C. and Jan, Jeng-Shiung and Deli, Mária Anna}, doi = {10.3390/pharmaceutics14010086}, journal-iso = {PHARMACEUTICS}, journal = {PHARMACEUTICS}, volume = {14}, unique-id = {32575627}, issn = {1999-4923}, year = {2022}, eissn = {1999-4923}, orcid-numbers = {Ferenc, Györgyi/0000-0002-3456-319X; Katona, Gábor/0000-0003-1564-4813; Kóta, Zoltán/0000-0003-2420-8773; Kelemen, Lóránd/0000-0001-7772-2165; Páli, Tibor/0000-0003-1649-1097; Walter, Fruzsina/0000-0001-8145-2823; Bolognin, Silvia/0000-0002-1399-2999; Deli, Mária Anna/0000-0001-6084-6524} } @article{MTMT:32557484, title = {The Arabidopsis Rho of Plants GTPase ROP1 Is a Potential Calcium-Dependent Protein Kinase (CDPK) Substrate}, url = {https://m2.mtmt.hu/api/publication/32557484}, author = {Ménesi, Dalma and Klement, Éva and Ferenc, Györgyi and Fehér, Attila}, doi = {10.3390/plants10102053}, journal-iso = {PLANTS-BASEL}, journal = {PLANTS-BASEL}, volume = {10}, unique-id = {32557484}, abstract = {Plant Rho-type GTPases (ROPs) are versatile molecular switches involved in a number of signal transduction pathways. Although it is well known that they are indirectly linked to protein kinases, our knowledge about their direct functional interaction with upstream or downstream protein kinases is scarce. It is reasonable to suppose that similarly to their animal counterparts, ROPs might also be regulated by phosphorylation. There is only, however, very limited experimental evidence to support this view. Here, we present the analysis of two potential phosphorylation sites of AtROP1 and two types of potential ROP-kinases. The S74 site of AtROP1 has been previously shown to potentially regulate AtROP1 activation dependent on its phosphorylation state. However, the kinase phosphorylating this evolutionarily conserved site could not be identified: we show here that despite of the appropriate phosphorylation site consensus sequences around S74 neither the selected AGC nor CPK kinases phosphorylate S74 of AtROP1 in vitro. However, we identified several phosphorylation sites other than S74 for the CPK17 and 34 kinases in AtROP1. One of these sites, S97, was tested for biological relevance. Although the mutation of S97 to alanine (which cannot be phosphorylated) or glutamic acid (which mimics phosphorylation) somewhat altered the protein interaction strength of AtROP1 in yeast cells, the mutant proteins did not modify pollen tube growth in an in vivo test.}, keywords = {PHOSPHORYLATION; PHOSPHORYLATION; BINDING; RESISTANCE; ACTIVATION; FAMILY; Powdery mildew; SWITCH; Arabidopsis thaliana; AGC kinase; calcium-dependent protein kinase; effectors; CDPK; Tip growth; GTP-BINDING PROTEIN; post-translational modifica-tion}, year = {2021}, eissn = {2223-7747}, orcid-numbers = {Ferenc, Györgyi/0000-0002-3456-319X; Fehér, Attila/0000-0002-4183-3696} }