To monitor apoptotic changes, we followed several parameters including cell viability, DNA integrity and telomerase activity.Preapoptotic phase has been achieved by exposure of cells to 50mM cadmium for 3 days.At that time the cell... more
To monitor apoptotic changes, we followed several parameters including cell viability, DNA integrity and telomerase activity.Preapoptotic phase has been achieved by exposure of cells to 50mM cadmium for 3 days.At that time the cell culture retains more than 95% viability although the majority of DNA is fragmented into 50-200 kb.Integrity of DNA is reestablished within two days after cadmium removal suggesting that an efficient DNA repair process has occurred.In parallel, a significant increase of telomerase activity in cell extracts is observed.The data suggest an involvement of telomerase in plant chromosome healing in response to damage caused by genotoxic agents.
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The assembly of ancient informational polymers from nucleotide precursors is the central challenge of life's origin on our planet. Among the possible solutions, dry polymerization of 3’,5’‐cyclic guanosine monophosphate (3’,5’‐cGMP)... more
The assembly of ancient informational polymers from nucleotide precursors is the central challenge of life's origin on our planet. Among the possible solutions, dry polymerization of 3’,5’‐cyclic guanosine monophosphate (3’,5’‐cGMP) has been proposed as a candidate to create oligonucleotides of 15–20 units in length. However, the reported sensitivity of the reaction to the presence of cations raised questions of whether this chemistry could be relevant in a geological context. The experiments in this study show that the presence of cations is not restrictive as long as the reaction is conducted in an acidic environment, in contrast to previous reports that suggested optimal conditions at pH 9.
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Trabajo presentado en la International Conference on Polyploidy, Hybridization and Biodiversity, celebrada en Rovinj (Croacia) del 11 al 14 de mayo de 2016
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Research Interests: Chemistry, Biology, Epigenetics, DNA, DNA methylation, and 6 moreDNA demethylation, Gene, Germination, Glycine, demethylation, and Ethephon
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A simple method of revealing proteins on nitrocellulose blots is described. It is based on high affinity of colloidal silver for proteins. The sensitivity is about the same as that of colloidal gold and it appears to be about 20 times... more
A simple method of revealing proteins on nitrocellulose blots is described. It is based on high affinity of colloidal silver for proteins. The sensitivity is about the same as that of colloidal gold and it appears to be about 20 times higher than the presently used staining methods. The whole procedure is very simple and does not take more than 30 min.
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Research Interests: Genetics, Plant Biology, Biology, Plant, Genome, and 3 moreChromosome, Gymnosperm, and Short Tandem Repeat
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During our initial phylogenetic study of the monocot genus Erythronium (Liliaceae), we observed peculiar eudicot-type internal transcribed spacer (ITS) sequences in a dataset derived from genomic DNA of Erythronium dens-canis. This raised... more
During our initial phylogenetic study of the monocot genus Erythronium (Liliaceae), we observed peculiar eudicot-type internal transcribed spacer (ITS) sequences in a dataset derived from genomic DNA of Erythronium dens-canis. This raised the possibility horizontal transfer of a eudicot alien ribosomal DNA (rDNA) into the Erythronium genome. In this work we aimed to support the hypothesis by carrying out genomic, molecular and cytogenetic analyses. Genome skimming coupled by PacBio HiFi sequencing of a flow-sorted bacterial artificial chromosome (BAC) clone was used to characterise the alien 45S rDNA. Integration of alien rDNA in the recipient genome was further proved by Southern blotting and fluorescence in situ hybridisation (FISH) using specific probes. Alien rDNA, nested among Potentilla species in phylogenetic analysis, likely entered Erythronium lineage in the common ancestor of E. dens-canis and E. caucasicum. Transferred eudicot-type rDNA preserved its tandemly arrayed feature on a single chromosome and was found to be transcribed in the monocot host albeit much less efficiently than the native counterpart. This study adds a new example to the rarely documented nuclear-to-nuclear jumps of DNA between eudicots and monocots while holding the scientific community continually in suspense about the mode of DNA transfer.
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Nucleolar dominance (ND) is an epigenetic, developmentally regulated phenomenon that describes the selective inactivation of 35S rDNA loci derived from one progenitor of a hybrid or allopolyploid. The presence of ND was documented in an... more
Nucleolar dominance (ND) is an epigenetic, developmentally regulated phenomenon that describes the selective inactivation of 35S rDNA loci derived from one progenitor of a hybrid or allopolyploid. The presence of ND was documented in an allotetraploid grass, Brachypodium hybridum (genome composition DDSS), which is a polyphyletic species that arose from crosses between two putative ancestors that resembled the modern B. distachyon (DD) and B. stacei (SS). In this work, we investigated the developmental stability of ND in B. hybridum genotype 3-7-2 and compared it with the reference genotype ABR113. We addressed the question of whether the ND is established in generative tissues such as pollen mother cells (PMC). We examined condensation of rDNA chromatin by fluorescence in situ hybridization employing state-of-art confocal microscopy. The transcription of rDNA homeologs was determined by reverse-transcription cleaved amplified polymorphic sequence analysis. In ABR113, the ND was sta...
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We demonstrate that apoptotic process can be fully reversed if cadmium is removed from cell culture after three days of incubation.To monitor apoptotic changes,we followed several parameters including cell viability,DNA integrity and... more
We demonstrate that apoptotic process can be fully reversed if cadmium is removed from cell culture after three days of incubation.To monitor apoptotic changes,we followed several parameters including cell viability,DNA integrity and telomerase activity.
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Poster presentado al International Conference on Polyploidy, Hybridization and Biodiversity, celebrado en Rovinj, Croacia del 11 al 14 de mayo de 2016
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Magnetic hypercrosslinked poly(styrene-co-divinylbenzene) microspheres (mgt.HPS1-NH2 and mgt.HPS2-NH2) containing different contents of amino groups were prepared and characterized in this study. The microspheres were used for the capture... more
Magnetic hypercrosslinked poly(styrene-co-divinylbenzene) microspheres (mgt.HPS1-NH2 and mgt.HPS2-NH2) containing different contents of amino groups were prepared and characterized in this study. The microspheres were used for the capture of uncompacted and compacted bacterial and calf thymus DNAs in the presence of different PEG 6000 and NaCl concentrations. Magnetic macroporous poly(glycidyl methacrylate-co-[2-(methacryloyloxy)ethoxy]acetic acid-co-ethylene dimethacrylate) microspheres containing amino and carboxyl groups [mgt.P(GMA-MOEAA-EDMA)-NH2] and magnetic non-porous carboxyl group-functionalized poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) [mgt.P(HEMA-GMA)-COOH] microspheres were used as a control. The size changes of uncompacted and compacted bacterial and calf thymus DNAs were measured in the range of concentrations 0–16% PEG 6000 and 0.5–2.0 M NaCl by dynamic light scattering (DLS). The highest capture of compacted bacterial and calf thymus DNAs was achieved with mgt.HPS1-NH2, mgt.HPS2-NH2 microspheres using 8% PEG 6000 and 2.0 M NaCl and with mgt.P(GMA-MOEAA-EDMA)-NH2 microspheres using 8% and 16% PEG 6000 and 2.0 M NaCl. Developed microspheres were used for DNA isolation from real vegetable (broccoli flowering head) samples. DNA was in quality suitable for PCR.
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Sex determination and sex chromosomes have been extensively studied due to their importance in evolutionary biology. Species of dioecious plants, which may have sex chromosomes, offer us unique insights into the history of this phenomenon... more
Sex determination and sex chromosomes have been extensively studied due to their importance in evolutionary biology. Species of dioecious plants, which may have sex chromosomes, offer us unique insights into the history of this phenomenon across the Tree of Life. Here, we present Sex-chrom: a database on plant sex chromosomes (www.sexchrom.csic.es), which aims to provide an easily accessible and organized information source for scientists and a general audience interested in this field. Basic data such as complete taxonomic classification of the species, chromosome number, genome size (2C), ploidy level, sex determination mechanism, presence of homomorphic or heteromorphic sex chromosomes and their corresponding sources are easily available for 178 species of 84 genera and 65 families. Besides, the database contains specific sections for ten selected model systems in plant sex chromosome research such as Silene latifolia and Rumex acetosa. In these sections, more detailed information is also available, including data on sex-linked genes, transposable elements or tandemly repeated DNA present in sex chromosomes. Data have been extracted from 431 sources published between 1919 and 2020.
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Cellular senescence, induced by genotoxic or replication stress, is accompanied by defects in nuclear morphology and nuclear membrane-heterochromatin disruption. In this work, we analyzed cytological and molecular changes in the linker of... more
Cellular senescence, induced by genotoxic or replication stress, is accompanied by defects in nuclear morphology and nuclear membrane-heterochromatin disruption. In this work, we analyzed cytological and molecular changes in the linker of nucleoskeleton and cytoskeleton (LINC) complex proteins in senescence triggered by γ-irradiation. We used human mammary carcinoma and osteosarcoma cell lines, both original and shRNA knockdown clones targeting lamin B receptor (LBR) and leading to LBR and lamin B (LB1) reduction. The expression status and integrity of LINC complex proteins (nesprin-1, SUN1, SUN2), lamin A/C, and emerin were analyzed by immunodetection using confocal microscopy and Western blot. The results show frequent mislocalization of these proteins from the nuclear membrane to cytoplasm and micronuclei and, in some cases, their fragmentation and amplification. The timing of these changes clearly preceded the onset of senescence. The LBR deficiency triggered neither senescence ...
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The classical model of concerted evolution states that hundreds to thousands of ribosomal DNA (rDNA) units undergo homogenization, making the multiple copies of the individual units more uniform across the genome than would be expected... more
The classical model of concerted evolution states that hundreds to thousands of ribosomal DNA (rDNA) units undergo homogenization, making the multiple copies of the individual units more uniform across the genome than would be expected given mutation frequencies and gene redundancy. While the universality of this over 50-year-old model has been confirmed in a range of organisms, advanced high throughput sequencing techniques have also revealed that rDNA homogenization in many organisms is partial and, in rare cases, even apparently failing. The potential underpinning processes leading to unexpected intragenomic variation have been discussed in a number of studies, but a comprehensive understanding remains to be determined. In this work, we summarize information on variation or polymorphisms in rDNAs across a wide range of taxa amongst animals, fungi, plants, and protists. We discuss the definition and description of concerted evolution and describe whether incomplete concerted evolu...
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Three out of four RNA components of ribosomes are encoded by 45S ribosomal DNA (rDNA) loci, which are organized as long head‐to‐tail tandem arrays of nearly identical units, spanning several megabases of sequence. Due to this structure,... more
Three out of four RNA components of ribosomes are encoded by 45S ribosomal DNA (rDNA) loci, which are organized as long head‐to‐tail tandem arrays of nearly identical units, spanning several megabases of sequence. Due to this structure, the rDNA loci are the major sources of gaps in genome assemblies, and gene copy number, sequence composition, and expression status of particular arrays remain elusive, especially in complex genomes harboring multiple loci. Here we conducted a multi‐omics study to decipher the 45S rDNA loci in hexaploid bread wheat. Coupling chromosomal genomics with optical mapping, we reconstructed individual rDNA arrays, enabling locus‐specific analyses of transcription activity and methylation status from RNA‐ and bisulfite‐sequencing data. We estimated a total of 6,650 rDNA units in the bread wheat genome, with approximately 2,321, 3,910, 253, and 50 gene copies located in short arms of chromosomes 1B, 6B, 5D, and 1A, respectively. Only 1B and 6B loci contributed substantially to rRNA transcription at a roughly 2:1 ratio. The ratio varied among five tissues analyzed (embryo, coleoptile, root tip, primary leaf, mature leaf), being the highest (2.64:1) in mature leaf and lowest (1.72:1) in coleoptile. Cytosine methylation was considerably higher in CHG context in the silenced 5D locus as compared with the active 1B and 6B loci. In conclusion, a fine genomic organization and tissue‐specific expression of rDNA loci were deciphered, for the first time, in a complex polyploid species. The results are discussed in the context of wheat evolution and transcription regulation.
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The 5'-sequences flanking the human MUC1 gene have been analyzed for their ability to direct expression of a reporter gene (the chloramphenicol acetyltransferase gene (CAT)) in cell lines that normally express or... more
The 5'-sequences flanking the human MUC1 gene have been analyzed for their ability to direct expression of a reporter gene (the chloramphenicol acetyltransferase gene (CAT)) in cell lines that normally express or do not express the MUC1 gene. A construct containing 2.9 kilobase pairs of MUC1 5'-flanking sequence sequence showed expression of CAT in breast and pancreatic cell lines but not in the non-epithelial cell lines HT-1080, SK23, and HTB96. Deletion analysis showed that maximum expression was obtained in ZR-75 (breast cancer line) and HPAF (pancreatic cancer line) with only 743 base pairs of 5'-flanking sequence. Sequences within 1.6 kilobase pairs of the transcriptional start site showed enhancing activity in a vector carrying an enhancerless SV40 promoter. Analysis of proximal 5'-sequences in a promoterless CAT vector carrying the SV40 enhancer showed that sequences between -60 and -150 were crucial for tissue-specific expression. An Sp1 site at -99/-90 and an E box (E-MUC1) at -84/-64 in this region were shown by mutational analysis to play a role in the regulation of transcription. Gel shift analysis with oligonucleotides and nuclear extracts of ZR-75 showed protein binding to both of these sites. Sp1 binding activity was similar in ZR-75 and HT1080 cells, whereas binding of factors to the E-MUC1 oligonucleotide revealed quantitative and qualitative differences between epithelial and non-epithelial cells.
Research Interests: Kinetics, Biology, Biological Chemistry, Medicine, Biological Sciences, and 15 moreCell line, Humans, Pancreas, Biological, Mucin, Breast, Gene, CHEMICAL SCIENCES, Cell nucleus, Base Sequence, Recombinant Proteins, Mucins, Molecular Sequence Data, Restriction Mapping, and Medical and Health Sciences
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Research Interests: Molecular Biology, Biology, Biological Chemistry, Medicine, Biological Sciences, and 15 moreHumans, Biological, Mucin, CHEMICAL SCIENCES, Cell nucleus, Epithelium, Base Sequence, DNA binding proteins, Binding Site, Oligonucleotides, Gene Expression Regulation, Molecular Sequence Data, binding sites, Medical and Health Sciences, and cytidine
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The history of rDNA research started almost 90 years ago when the geneticist, Barbara McClintock observed that in interphase nuclei of maize the nucleolus was formed in association with a specific region normally located near the end of a... more
The history of rDNA research started almost 90 years ago when the geneticist, Barbara McClintock observed that in interphase nuclei of maize the nucleolus was formed in association with a specific region normally located near the end of a chromosome, which she called the nucleolar organizer region (NOR). Cytologists in the twentieth century recognized the nucleolus as a common structure in all eukaryotic cells, using both light and electron microscopy and biochemical and genetic studies identified ribosomes as the subcellular sites of protein synthesis. In the mid- to late 1960s, the synthesis of nuclear-encoded rRNA was the only system in multicellular organisms where transcripts of known function could be isolated, and their synthesis and processing could be studied. Cytogenetic observations of NOR regions with altered structure in plant interspecific hybrids and detailed knowledge of structure and function of rDNA were prerequisites for studies of nucleolar dominance, epistatic i...
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Trabajo presentado en el 7th Workshop on the Application of Next Generation Sequencing to Repetitive DNA Analysis, celebrado en České Budějovice (República Checa), del 22 al 24 de mayo de 2018Peer reviewe
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Plant genomes consist, to a considerable extent, of non-coding repetitive DNA. Several studies showed that phylogenetic signals can be extracted from such repeatome data by using among-species dissimilarities from the RepeatExplorer2... more
Plant genomes consist, to a considerable extent, of non-coding repetitive DNA. Several studies showed that phylogenetic signals can be extracted from such repeatome data by using among-species dissimilarities from the RepeatExplorer2 pipeline as distance measures. Here, we advanced this approach by adjusting the read input for comparative clustering indirectly proportional to genome size and by summarizing all clusters into a main distance matrix subjected to Neighbor Joining algorithms and Principal Coordinate Analyses. Thus, our multivariate statistical method works as a “repeatomic fingerprint,” and we proved its power and limitations by exemplarily applying it to the family Rosaceae at intrafamilial and, in the genera Fragaria and Rosa, at the intrageneric level. Since both taxa are prone to hybridization events, we wanted to show whether repeatome data are suitable to unravel the origin of natural and synthetic hybrids. In addition, we compared the results based on complete rep...
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Trabajo presentado en el XIX Jornades de Biologia Evolutiva, celebrada en Barcelona (Espana), el 28 de junio de 2019
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The essential components of splicing are the splicing factors accumulated in nuclear speckles; thus, we studied how DNA damaging agents and A-type lamin depletion affect the properties of these regions, positive on the SC-35 protein. We... more
The essential components of splicing are the splicing factors accumulated in nuclear speckles; thus, we studied how DNA damaging agents and A-type lamin depletion affect the properties of these regions, positive on the SC-35 protein. We observed that inhibitor of PARP (poly (ADP-ribose) polymerase), and more pronouncedly inhibitors of RNA polymerases, caused DNA damage and increased the SC-35 protein level. Interestingly, nuclear blebs, induced by PARP inhibitor and observed in A-type lamin-depleted or senescent cells, were positive on both the SC-35 protein and another component of the spliceosome, SRRM2. In the interphase cell nuclei, SC-35 interacted with the phosphorylated form of RNAP II, which was A-type lamin-dependent. In mitotic cells, especially in telophase, the SC-35 protein formed a well-visible ring in the cytoplasmic fraction and colocalized with β-catenin, associated with the plasma membrane. The antibody against the SRRM2 protein showed that nuclear speckles are alr...
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Background and AimsThree out of four RNA components of ribosomes are encoded by 45S rDNA loci, whose transcripts are processed into 18S, 5.8S and 26S ribosomal RNAs. The loci are organized as long head-to-tail tandem arrays of nearly... more
Background and AimsThree out of four RNA components of ribosomes are encoded by 45S rDNA loci, whose transcripts are processed into 18S, 5.8S and 26S ribosomal RNAs. The loci are organized as long head-to-tail tandem arrays of nearly identical units spanning over several megabases of sequence. Due to this peculiar structure, the number of rRNA genes, their sequence composition and expression status remain unclear, especially in complex polyploid genomes harbouring multiple loci. Here we conducted a complex study to decipher structure and activity of both major and minor rRNA loci in hexaploid bread wheat (Triticum aestivum).MethodsWe employed an original, multi-omics approach, combining chromosome flow sorting and optical mapping with transcriptome and methylome sequencing.Key ResultsThe former two techniques enabled unbiased quantification of rDNA units in particular loci of the wheat genome. Total number of rRNA genes organized in tandem arrays was 4388, with 64.1, 31.4, 3.9 and 0...
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We report on a major update to the animal rDNA loci database, which now contains cytogenetic information for 45S and 5S rDNA loci in more than 2600 and 1000 species, respectively. The data analyses show the following: (i) A high... more
We report on a major update to the animal rDNA loci database, which now contains cytogenetic information for 45S and 5S rDNA loci in more than 2600 and 1000 species, respectively. The data analyses show the following: (i) A high variability in 5S and 45S loci numbers, with both showing 50-fold or higher variability. However, karyotypes with an extremely high number of loci were rare, and medians generally converged to two 5S sites and two 45S rDNA sites per diploid genome. No relationship was observed between the number of 5S and 45S loci. (ii) The position of 45S rDNA on sex chromosomes was relatively frequent in some groups, particularly in arthropods (14% of karyotypes). Furthermore, 45S rDNA was almost exclusively located in microchromosomes when these were present (in birds and reptiles). (iii) The proportion of active NORs (positively stained with silver staining methods) progressively decreased with an increasing number of 45S rDNA loci, and karyotypes with more than 12 loci ...
Research Interests: Genetics, Biology, Medicine, Animals, Genome, and 7 moreMolecular sciences, Karyotype, rDNA, Ribosomal DNA, Database, Sex Chromosome, and Ploidy
Template-free nonenzymatic polymerization of 3′,5′ cyclic nucleotides is an emerging topic of the origin of life research. In the last ten years, a number of papers have been published addressing various aspects of this process. These... more
Template-free nonenzymatic polymerization of 3′,5′ cyclic nucleotides is an emerging topic of the origin of life research. In the last ten years, a number of papers have been published addressing various aspects of this process. These works evoked a vivid discussion among scientists working in the field of prebiotic chemistry. The aim of the current review is to answer the most frequently raised questions related to the detection and characterization of oligomeric products as well as to the geological context of this chemistry.
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Genome sizes of eukaryotic organisms vary substantially, with whole-genome duplications (WGD) and transposable element expansion acting as main drivers for rapid genome size increase. The two North American mudminnows, Umbra limi and... more
Genome sizes of eukaryotic organisms vary substantially, with whole-genome duplications (WGD) and transposable element expansion acting as main drivers for rapid genome size increase. The two North American mudminnows, Umbra limi and Umbra pygmaea, feature genomes about twice the size of their sister lineage Esocidae (e.g., pikes and pickerels). However, it is unknown whether all Umbra species share this genome expansion and which causal mechanisms drive this expansion. Using flow cytometry, we find that the genome of the European mudminnow is expanded similarly to both North American species, ranging between 4.5 and 5.4 pg per diploid nucleus. Observed blocks of interstitially located telomeric repeats in U. limi suggest frequent Robertsonian rearrangements in its history. Comparative analyses of transcriptome and genome assemblies show that the genome expansion in Umbra is driven by the expansion of DNA transposon and unclassified repeat sequences without WGD. Furthermore, we find...
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The genus Rosa comprises more than 100 woody species characterized by intensive hybridization, introgression, and an overall complex evolutionary history. Besides many diploid species (2n = 2x = 14) polyploids ranging from 3x to 10x are... more
The genus Rosa comprises more than 100 woody species characterized by intensive hybridization, introgression, and an overall complex evolutionary history. Besides many diploid species (2n = 2x = 14) polyploids ranging from 3x to 10x are frequently found. Here we analyzed 5S ribosomal DNA in 19 species covering two subgenera and the major sections within subg. Rosa. In addition to diploids and polyploids with regular meiosis, we focused on 5x dogroses (Rosa sect. Caninae), which exhibit an asymmetric meiosis differentiating between bivalent- and univalent-forming chromosomes. Using genomic resources, we reconstructed 5S rDNA units to reveal their phylogenetic relationships. Additionally, we designed locus-specific probes derived from intergenic spacers (IGSs) and determined the position and number of 5S rDNA families on chromosomes. Two major 5S rDNA families (termed 5S_A and 5S_B, respectively) were found at variable ratios in both diploid and polyploid species including members of ...
Research Interests: Genetics, Biology, Medicine, Meiosis, Concerted Evolution, and 3 morePhylogenetic Tree, Ploidy, and Subgenus
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This article comments on: Nicola Schmidt, Kathrin M. Seibt, Beatrice Weber, Trude Schwarzacher, Thomas Schmidt, and Tony Heitkam, Broken, silent, and in hiding: tamed endogenous pararetroviruses escape elimination from the genome of sugar... more
This article comments on: Nicola Schmidt, Kathrin M. Seibt, Beatrice Weber, Trude Schwarzacher, Thomas Schmidt, and Tony Heitkam, Broken, silent, and in hiding: tamed endogenous pararetroviruses escape elimination from the genome of sugar beet (Beta vulgaris), Annals of Botany Volume 128, Issue 3, 26 August 2021, Pages 281–291, https://doi.org/10.1093/aob/mcab042
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The genomic diversity of SARS-CoV-2 has been a focus during the ongoing COVID-19 pandemic. Here, we analyzed the distribution and character of emerging mutations in a data set comprising more than 95,000 virus genomes covering eight major... more
The genomic diversity of SARS-CoV-2 has been a focus during the ongoing COVID-19 pandemic. Here, we analyzed the distribution and character of emerging mutations in a data set comprising more than 95,000 virus genomes covering eight major SARS-CoV-2 lineages in the GISAID database, including genotypes arising during COVID-19 therapy. Globally, the C>U transitions and G>U transversions were the most represented mutations, accounting for the majority of single-nucleotide variations. Mutational spectra were not influenced by the time the virus had been circulating in its host or medical treatment. At the amino acid level, we observed about a 2-fold excess of substitutions in favor of hydrophobic amino acids over the reverse. However, most mutations constituting variants of interests of the S-protein (spike) lead to hydrophilic amino acids, counteracting the global trend. The C>U and G>U substitutions altered codons towards increased amino acid hydrophobicity values in more ...