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Now showing items 1 - 16 of 68

  • AthCNV: A Map of DNA Copy Number Variations in the Arabidopsis Genome

    Zmienko, Agnieszka   Marszalek-Zenczak, Malgorzata   Wojciechowski, Pawel   Samelak-Czajka, Anna   Luczak, Magdalena   Kozlowski, Piotr   Karlowski, Wojciech M.   Figlerowicz, Marek  

    A catalog of large copy number variations (CNVs), CNV-overlapping genes, and their genotypes in 1060 Arabidopsis accessions will stimulate studies on the genetic basis of phenotypic variation. Copy number variations (CNVs) greatly contribute to intraspecies genetic polymorphism and phenotypic diversity. Recent analyses of sequencing data for >1000 Arabidopsis (Arabidopsis thaliana) accessions focused on small variations and did not include CNVs. Here, we performed genome-wide analysis and identified large indels (50 to 499 bp) and CNVs (500 bp and larger) in these accessions. The CNVs fully overlap with 18.3% of protein-coding genes, with enrichment for evolutionarily young genes and genes involved in stress and defense. By combining analysis of both genes and transposable elements (TEs) affected by CNVs, we revealed that the variation statuses of genes and TEs are tightly linked and jointly contribute to the unequal distribution of these elements in the genome. We also determined the gene copy numbers in a set of 1060 accessions and experimentally validated the accuracy of our predictions by multiplex ligation-dependent probe amplification assays. We then successfully used the CNVs as markers to analyze population structure and migration patterns. Finally, we examined the impact of gene dosage variation triggered by a CNV spanning the SEC10 gene on SEC10 expression at both the transcript and protein levels. The catalog of CNVs, CNV-overlapping genes, and their genotypes in a top model dicot will stimulate the exploration of the genetic basis of phenotypic variation.
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  • The RNA-RNA base pairing potential of human Dicer and Ago2 proteins

    Pokornowska, Maria   Milewski, Marek C.   Ciechanowska, Kinga   Szczepanska, Agnieszka   Wojnicka, Marta   Radogostowicz, Ziemowit   Figlerowicz, Marek  

    The ribonuclease Dicer produces microRNAs (miRNAs) and small interfering RNAs that are handed over to Ago proteins to control gene expression by targeting complementary sequences within transcripts. Interestingly, a growing number of reports have demonstrated that the activity of Dicer may extend beyond the biogenesis of small regulatory RNAs. Among them, a report from our latest studies revealed that human Dicer facilitates base pairing of complementary sequences present in two nucleic acids, thus acting as a nucleic acid annealer. Accordingly, in this manuscript, we address how RNA structure influences the annealing activity of human Dicer. We show that Dicer supports hybridization between a small RNA and a complementary sequence of a longer RNA in vitro, even when both complementary sequences are trapped within secondary structures. Moreover, we show that under applied conditions, human Ago2, a core component of RNA-induced silencing complex, displays very limited annealing activity. Based on the available data from new-generation sequencing experiments regarding the RNA pool bound to Dicer in vivo, we show that multiple Dicer-binding sites within mRNAs also contain miRNA targets. Subsequently, we demonstrate in vitro that Dicer but not Ago2 can anneal miRNA to its target present within mRNA. We hypothesize that not all miRNA duplexes are handed over to Ago proteins. Instead, miRNA-Dicer complexes could target specific sequences within transcripts and either compete or cooperate for binding sites with miRNA-Ago complexes. Thus, not only Ago but also Dicer might be directly involved in the posttranscriptional control of gene expression.
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  • L1Base 2: more retrotransposition-active LINE-1s, more mammalian genomes

    Penzkofer, Tobias   Jaeger, Marten   Figlerowicz, Marek   Badge, Richard   Mundlos, Stefan   Robinson, Peter N.   Zemojtel, Tomasz  

    LINE-1 (L1) insertions comprise as much as 17% of the human genome sequence, and similar proportions have been recorded for other mammalian species. Given the established role of L1 retrotransposons in shaping mammalian genomes, it becomes an important task to track and annotate the sources of this activity: full length elements, able to encode the cis and trans acting components of the retrotransposition machinery. The L1Base database ( contains annotated full-length sequences of LINE-1 transposons including putatively active L1s. For the new version of L1Base, a LINE-1 annotation tool, L1Xplorer, has been used to mine potentially active L1 retrotransposons from the reference genome sequences of 17 mammals. The current release of the human genome, GRCh38, contains 146 putatively active L1 elements or full length intact L1 elements (FLIs). The newest versions of the mouse, GRCm38 and the rat, Rnor_6.0, genomes contain 2811 and 492 FLIs, respectively. Most likely reflecting the current level of completeness of the genome project, the latest reference sequence of the common chimpanzee genome, PT 2.19, only contains 19 FLIs. Of note, the current assemblies of the dog, CF 3.1 and the sheep, OA 3.1, genomes contain 264 and 598 FLIs, respectively. Further developments in the new version of L1Base include an updated website with implementation of modern web server technologies. including a more responsive design for an improved user experience, as well as the addition of data sharing capabilities for L1Xplorer annotation.
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  • AmiRNA Designer - new method of artificial miRNA design

    Mickiewicz, Agnieszka   Rybarczyk, Agnieszka   Sarzynska, Joanna   Figlerowicz, Marek   Blazewicz, Jacek  

    MicroRNAs (miRNAs) are small non-coding RNAs that have been found in most of the eukaryotic organisms. They are involved in the regulation of gene expression at the post-transcriptional level in a sequence specific manner. MiRNAs are produced from their precursors by Dicer-dependent small RNA biogenesis pathway. Involvement of miRNAs in a wide range of biological processes makes them excellent candidates for studying gene function or for therapeutic applications. For this purpose, different RNA-based gene silencing techniques have been developed. Artificially transformed miRNAs (amiRNAs) targeting one or several genes of interest represent one of such techniques being a potential tool in functional genomics. Here, we present a new approach to amiRNA design, implemented as AmiRNA Designer software. Our method is based on the thermodynamic analysis of the native miRNA/miRNA* and miRNA/target duplexes. In contrast to the available automated tools, our program allows the user to perform analysis of natural miRNAs for the organism of interest and to create customized constraints for the design stage. It also provides filtering of the amiRNA candidates for the potential off-targets. AmiRNA Designer is freely available at
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    The present invention relates to a Lyme disease vaccine, a genetic construct, recombinant protein, method for genetic construct design, method for vaccine delivery, method for recombinant proteins delivery, use of recombinant proteins in the production of Lyme disease vaccine. In particular, the method concerns the use of TROSPA and TROSPA- Salpl 5 recombinant proteins derived from castor bean tick {Ixodes riccinus) as a component of Lyme disease vaccine for animals. The antibodies present in blood of an immunized vertebrate directed against the TROSPA proteins considerably reduce the chance of infecting new ticks by blocking or hindering the interaction of TROSPA protein with OspA protein of Borrelia burgdorferi sensu lato. The interaction is crucial in the process of the spirochete entering a tick. The antibodies directed against the TROSPA- Salpl5 protein protect vertebrates from infection on the stage of Borellia diffusion by destroying their protective coating formed at the surface as a result of the interaction between the Salpl5 tick protein and OspC spirochete protein. The vaccine based on TROSPA tick proteins and TROSPA-Salpl5 proteins may be used independently or together with the OspA recombinant proteins and OspC protein of Borrelia burgdorferi sensu lato.
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  • Mutations in proteasome-related genes are associated with thyroid hemiagenesis

    Budny, Bartlomiej   Szczepanek-Parulska, Ewelina   Zemojtel, Tomasz   Szaflarski, Witold   Rydzanicz, Malgorzata   Wesoly, Joanna   Handschuh, Luiza   Wolinski, Kosma   Piatek, Katarzyna   Niedziela, Marek   Ziemnicka, Katarzyna   Figlerowicz, Marek   Zabel, Maciej   Ruchala, Marek  

    Purpose Human thyroid development is a complex and still unexplained process. Thyroid hemiagenesis is a congenital anomaly, where one of the thyroid lobes fails to develop. In the majority of patients with thyroid hemiagenesis, the genetic background remains unknown. The aim of the study was to search for novel genetic contributors to the etiology of thyroid hemiagenesis. Methods A cohort of 34 sporadic patients diagnosed with thyroid hemiagenesis and one three-generation family were subjected to comprehensive genomic examination. Initially, targeted screening of associated transcription factors, known to be linked to thyroid development, was performed. As a next step, genomic examinations were applied using high-resolution microarrays, whereas for the thyroid hemiagenesis family, additionally the whole exome sequencing was performed. Results Screening of transcription factors revealed no causative mutations in the studied cohort. Genomic examinations revealed the presence of four recurrent defects (three deletions and one duplication) affecting highly conservative proteasome genes PSMA1, PSMA3, and PSMD3. In a thyroid hemiagenesis family a splice site mutation in a proteasome gene PSMD2 (c.612T > C cDNA.1170T > C, g.3271T > C) was found in both affected mother and daughter. Conclusions Our results shed a new light on etiology of thyroid hemiagenesis, so far suspected to be linked only to mutations in the genes directly involved in the thyroid development. We demonstrated, for the first time, that genomic alterations in proteasome-associated genes co-occur in patients presenting this developmental anomaly.
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  • Phylogeny and molecular evolution of the hepatitis C virus.

    Jackowiak, Paulina   Kuls, Karolina   Budzko, Lucyna   Mania, Anna   Figlerowicz, Magdalena   Figlerowicz, Marek  

    The hepatitis C virus (HCV) is a globally prevalent human pathogen that causes persistent liver infections in most infected individuals. HCV is classified into seven phylogenetically distinct genotypes, which have different geographical distributions and levels of genetic diversity. Some of these genotypes are endemic and highly divergent, whereas others disseminate rapidly on an epidemic scale but display lower variability. HCV phylogeny has an important impact on disease epidemiology and clinical practice because the viral genotype may determine the pathogenesis and severity of the resultant chronic liver disease. In addition, there is a clear association between the HCV genotype and its susceptibility to antiviral treatment. Similarly to other RNA viruses, in a single host, HCV exists as a combination of related but genetically different variants. The whole formation is the actual target of selection exerted by a host organism and antiviral therapeutics. The genetic structure of the viral population is largely shaped by mutations that are constantly introduced during an error-prone replication. However, it appears that genetic recombination may also contribute to this process. This heterogeneous collection of variants has a significant ability to evolve towards the fitness optimum. Interestingly, negative selection, which restricts diversity, emerges as an essential force that drives HCV evolution. It is becoming clear that HCV evolves to become stably adapted to the host environment. In this article we review the HCV phylogeny and molecular evolution in the context of host-virus interactions. Copyright =C2=A9 2013 Elsevier B.V. All rights reserved.
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  • Copy number polymorphism in plant genomes.

    Zmienko, Agnieszka   Samelak, Anna   Kozlowski, Piotr   Figlerowicz, Marek  

    Copy number variants (CNVs) are genomic rearrangements resulting from gains or losses of DNA segments. Typically, the term refers to rearrangements of sequences larger than 1kb. This type of polymorphism has recently been shown to be a key contributor to intra-species genetic variation, along with single-nucleotide polymorphisms and short insertion-deletion polymorphisms. Over the last decade, a growing number of studies have highlighted the importance of copy number variation (CNV) as a factor affecting human phenotype and individual CNVs have been linked to risks for severe diseases. In plants, the exploration of the extent and role of CNV is still just beginning. Initial genomic analyses indicate that CNVs are prevalent in plants and have greatly affected plant genome evolution. Many CNV events have been observed in outcrossing and autogamous species. CNVs are usually found on all chromosomes, with CNV hotspots interspersed with regions of very low genetic variation. Although CNV is mainly associated with intergenic regions, many CNVs encompass protein-coding genes. The collected data suggest that CNV mainly affects the members of large families of functionally redundant genes. Thus, the effects of individual CNV events on phenotype are usually modest. Nevertheless, there are many cases in which CNVs for specific genes have been linked to important traits such as flowering time, plant height and resistance to biotic and abiotic stress. Recent reports suggest that CNVs may form rapidly in response to stress. =20
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  • Application of click chemistry to the production of DNA microarrays

    Uszczynska, Barbara   Ratajczak, Tomasz   Frydrych, Emilia   Figlerowicz, Marek   Markiewicz, Wojciech T.  

    The copper-catalyzed alkyne-azide cycloaddition (CuAAC) reaction was applied as the novel method of DNA immobilization on a modified solid support. The CuAAC click reaction enables the covalent binding of DNA modified with pentynyl groups at its 5'-end to azide-loaded slides. Click microarrays were produced using this approach and successfully employed in biological/model experiments.
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  • Functional characterization of RNA fragments using high-throughput interactome screening

    Jackowiak, Paulina   Lis, Angelika   Luczak, Magdalena   Stolarek, Ireneusz   Figlerowicz, Marek  

    Populations of small eukaryotic RNAs, in addition to relatively well recognized molecules such as miRNAs or siRNAs, also contain fragments derived from all classes of constitutively expressed non-coding RNAs. It has been recently demonstrated that the formation and accumulation of RNA fragments (RFs) is cell-/tissue-specific and depends on internal and external stimuli. Unfortunately, the mechanisms underlying RF biogenesis and function remain unclear. To better understand them, we employed RNA pull-down and mass spectrometry methods to characterize the interactions of seven RFs originating from tRNA, snoRNA and snRNA. By integrating our results with publicly available data on physical protein-protein interactions, we constructed an RF interactome network. We determined that the RF interactome comprises proteins generally different from those that interact with their parental full length RNAs. Proteins captured by the RFs were involved in mRNA splicing, tRNA processing, DNA recombination/replication, protein biosynthesis and carboxylic acid metabolism. Our data suggest that RFs can be endogenous aptamer-like molecules and potential players in recently revealed RNA-protein regulatory networks. Significance: In the recent decade it has become evident that RNAs with well-known functions (for example tRNA, snoRNA or rRNA) can be cleaved to yield short fragments, whose role in cells remains only partially characterized. At the same time, unconventional interactions between mRNA and proteins without RNA-binding domains have been demonstrated, revealing novel layers of possible RNA-mediated regulation. Considering the above, we hypothesized that RNA fragments (RFs) can be endogenous aptamer-like molecules that unconventionally interact with proteins. In this study we identified protein partners of seven selected RFs. We found that RFs bind different set of proteins than their parental full length RNAs and identified proteins differentially bound by the particular RFs. These observations suggest biological relevance of the discovered interactions. Our data provide a novel perspective on the significance of RFs and point to this pool of molecules as to a rich collection of potential components of the recently discovered RNA-protein regulatory networks.
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  • RNA-RNA Recombination in Plant Virus Replication and Evolution

    Sztuba-Solinska, Joanna   Urbanowicz, Anna   Figlerowicz, Marek   Bujarski, Jozef J.  

    RNA-RNA recombination is one of the strongest forces shaping the genomes of plant RNA viruses. The detection of recombination is a challenging task that prompted the development of both in vitro and in vivo experimental systems. In the divided genome of Brome mosaic virus system, both inter- and intrasegmental crossovers are described. Other systems utilize satellite or defective interfering RNAs (DI-RNAs) of Turnip crinkle virus, Tomato bushy stunt virus, Cucumber necrosis virus, and Potato virus X. These assays identified the mechanistic details of the recombination process, revealing the role of RNA structure and proteins in the replicase-mediated copy-choice mechanism. In copy choice, the polymerase and the nascent RNA chain from which it is synthesized switch from one RNA template to another. RNA recombination was found to mediate the rearrangement of viral genes, the repair of deleterious mutations, and the acquisition of nonself sequences influencing the phylogenetics of viral taxa. The evidence for recombination, not only between related viruses but also among distantly related viruses, and even with host RNAs, suggests that plant viruses unabashedly test recombination with any genetic material at hand.
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  • An MLPA-Based Strategy for Discrete CNV Genotyping: CNV-miRNAs as an Example

    Marcinkowska-Swojak, Malgorzata   Uszczynska, Barbara   Figlerowicz, Marek  

    Copy number variation (CNV) has become well recognized in recent years. It has been estimated that common CNVs account for approximately 10% of the human genome and that they overlap hundreds of genes and other functional genetic elements. Although substantial progress in genome-wide CNV analysis has been made recently, there is still a need for a method that allows precise genotyping of selected CNVs. Here, we describe a novel strategy for CNV genotyping, taking advantage of the general principles of the multiplex ligation-dependent probe amplification (MLPA) method and short oligonucleotide probes, allowing easy custom design and generation of assays for almost any genomic region of interest. As a proof-of-concept, we developed two assays covering 17 candidate CNV regions that overlap human miRNA genes. Extensive quality control analysis demonstrated high reproducibility and reliability of the genotypes determined using our method. Detailed analysis of identified CNVs revealed that they are highly differentiated among the HapMap populations. The main advantages of the developed strategy include the simplicity of the assay design, its flexibility in terms of the selection of genomic regions, and its low cost (<$1$10/genotype, depending on scale of experiment). These advantages make the presented strategy attractive for large-scale genetic analyses.
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  • Two types of defective RNAs arising from the tomato black ring virus genome

    Hasiow-Jaroszewska, Beata   Borodynko, Natasza   Figlerowicz, Marek   Pospieszny, Henryk  

    Short defective RNAs (D-RNAs) associated with tomato black ring virus (TBRV) were isolated, cloned and sequenced. As a result, two types of D-RNAs associated with different TBRV isolates were identified. Both types were derived from RNA1. The first one contained sequences from the 5' and 3' untranslated regions (UTR) and from the 5' region of a single large open reading frame. The second one included a portion of the coding region for the RNA-dependent RNA polymerase flanked by a short fragment of the 5' UTR and the entire 3' UTR. The possible nature and origin of these RNA species is discussed.
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  • Quasispecies nature of Pepino mosaic virus and its evolutionary dynamics

    Hasiow-Jaroszewska, Beata   Jackowiak, Paulina   Borodynko, Natasza   Figlerowicz, Marek   Pospieszny, Henryk  

    Genetic variability is an essential feature of RNA viruses. It allows them to adapt to the ever-changing environmental conditions. Important biological properties of the viruses, their infectivity, adaptability, and host range, may also depend on the level of quasispecies diversity. Here, we present the analysis of the genetic polymorphism of Pepino mosaic virus (PepMV). The examined populations were isolated from the naturally infected tomato plants (Solanum lycopersicum). In order to determine the complexity of the PepMV populations, the number of different viral variants and their genetic diversity was established. Moreover, phylogenetic trees were created to depict relations between the identified variants. For the first time we have shown that the PepMV exists as a quasispecies. The observed level of genetic variability allows PepMV for a quick and flexible adaptation to different hosts. Our results suggest that the level of PepMV variability possibly influences the course of infection.
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  • Chronic kidney disease-related atherosclerosis - proteomic studies of blood plasma

    Luczak, Magdalena   Formanowicz, Dorota   Pawliczak, Elzbieta   Wanic-Kossowska, Maria   Wykretowicz, Andrzej   Figlerowicz, Marek  

    Background: Atherosclerosis is considered the major cause of the dramatic increase in cardiovascular mortality among patients suffering from chronic kidney disease (CKD). Although the close connection between atherosclerosis and kidney dysfunction is undeniable, factors enhancing CKD-mediated plaque formation are still not well recognized. Results: To increase our knowledge of this process we carried out a comparative proteomic analysis of blood plasma proteins isolated from 75 patients in various stages of renal dysfunction (CKD group), 25 patients with advanced cardiovascular disease (CVD group) and 25 healthy volunteers (HV group). The collected samples were subjected to 2D electrophoresis. Then, individual proteins were identified by mass spectrometry. The comparative analysis involving CKD and HV groups showed a differential accumulation of alpha-1-microglobulin, apolipoprotein A-IV, gamma-fibrinogen and haptoglobin in patients with kidney disease. Exactly the same proteins were identified as differentially expressed when proteomes of CVD patients and HV were compared. However, a direct comparison of CKD and CVD groups revealed significant differences in the accumulation of two proteins: alpha-1-microglobulin and apolipoprotein A-IV. Conclusions: The obtained results indicate that at least two processes differentially contribute to the plaque formation in CKD- and CVD-mediated atherosclerosis. It seems that the inflammatory process is more intense in CKD patients. On the other hand, the down-and up-regulation of apolipoprotein A-IV in CVD and CKD groups, respectively, suggests that substantial differences exist in the efficacy of cholesterol transport in both groups of patients.
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  • Selection of RNA Oligonucleotides That Can Modulate Human Dicer Activity In Vitro

    Tyczewska, Agata   Kurzynska-Kokorniak, Anna   Koralewska, Natalia   Szopa, Aleksandra   Kietrys, Anna M.   Wrzesinski, Jan   Twardowski, Tomasz   Figlerowicz, Marek  

    Human ribonuclease Dicer is an enzyme that excises small regulatory RNAs from perfectly or partially double-stranded RNA precursors. Although Dicer substrates and products have already been quite well characterized, our knowledge about cellular factors regulating the activity of this enzyme is still limited. To learn more about this problem, we attempted to determine whether RNA could function not only as a Dicer substrate but also as its regulator. To this end, we applied an in vitro selection method. We identified 120 RNA oligomers binding human Dicer. Sixteen of them were subjected to more detailed in vitro studies. We found that 6 out of 16 oligomers affected Dicer ability to digest pre-microRNAs (miRNAs), although most of them were cleaved by this enzyme. For the 6 most active oligomers the putative mechanism of Dicer inhibition was determined. Three oligomers were classified as typical competitive inhibitors and one as an allosteric inhibitor. The remaining 2 oligomers acted as selective inhibitors. They affected the production of 1 miRNA, whereas the formation of other miRNAs was hardly influenced. In general, the data obtained suggest that one can modulate the generation of specific miRNAs by using RNA oligomers. Moreover, we found that sequences similar to those of the selected oligomers can be found within the molecules composing human transcriptome.
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