Search results

1

Mikromacierze DNA

100%

Kisiel A., Skąpska A., Markiewicz W. T., Figlerowicz M.

Kosmos

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2004

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vol. 53

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issue 3-4

295-303

EN

Summary The breakthrough in DNA sequencing technology and completion of sequencing of first eucaryotic genomes raised the need for high-throughput methods of gene function analysis. To solve this problem the DNA microarray technology has been developed. It is based on traditional transcript profiling methods which use the hybridization ability of DNA and RNA to monitor gene expression. Due to miniaturisation and the use of fluorescent dyes DNA microarrays allow for simultaneous monitoring of the expression of tens of thousands genes. There are two main types of DNA microarrays: cDNA microarrays and oligonucleotide microarrays, also called DNA chips. In the former, several- hundred-nucleotide long cDNA probes are printed on a glass plate and hybridized to a fluorescently labeled target cDNA obtained from the tissue of interest. In the latter, type of microarrays, each gene is represented by several short oligonucleotides (about 30 nt), perfectly matching the target gene, and several oligomers with a single mismatch. Oligonucleotides are usually synthetised on a glass slide using the photolithographic technique and the slide is hybridized to fluorescently labeled target RNA. Analysis ofmicroarray data includes the comparison of gene expression in the experimental and control probes or the comparison of gene expression profiles obtained in several experiments, by different clustering methods. Medicine is one of the fields where DNA microarrays have already found practical application. At present they are especially useful in cancer research. DNA microarray analysis of tumor tissues allows to differentiate among various cancer types, to prognose the illness progress and plan the therapy. DNA microarrays are also an irreplaceable tool in a search for new drugs.

2

Computational prediction of non-enzymatic RNA degradation patterns

100%

Rybarczyk A., Jackowiak P., Figlerowicz M., Blazewicz J.

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2016

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vol. 63

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issue 4

745-751

EN

Since the beginning of the 21st century, an increasing interest in the research of ribonucleic acids has been observed in response to a surprising discovery of the role that RNA molecules play in the biological systems. It was demonstrated that they do not only take part in the protein synthesis (mRNA, rRNA, tRNA) but also are involved in the regulation of gene expression. Several classes of small regulatory RNAs have been discovered (e.g. microRNA, small interfering RNA, piwiRNA). Most of them are excised from specific double-stranded RNA precursors by enzymes that belong to the RNaseIII family (Drosha, Dicer or Dicer-like proteins). More recently, it has been shown that small regulatory RNAs are also generated as stable intermediates of RNA degradation (the so called RNA fragments originating from tRNA, snRNA, snoRNA etc.). Unfortunately, the mechanisms underlying biogenesis of the RNA fragments remain unclear. It is thought that several factors may be involved in the formation of the RNA fragments. The most important are the specific RNases, RNA-protein interactions and RNA structure. In this work, we focus on the RNA primary and secondary structures as factors influencing the RNA stability and consequently the pattern of RNA fragmentation. Earlier, we identified the major structural factors affecting non-enzymatic RNA degradation. Now, based on these data, we developed a new branch-and-cut algorithm that is able to predict the products of large RNA molecules' hydrolysis in vitro. We also present the experimental data that verify the results generated using this algorithm.

3

Two types of non-homologous RNA recombination in brome mosaic virus

100%

Alejska M., Malinowska N., Urbanowicz A., Figlerowicz M.

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2005

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vol. 52

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issue 4

833-844

EN

Non-homologous RNA recombination is a process enabling the exchange of genetic material between various (related or unrelated) RNA-based viruses. Despite extensive investigations its molecular mechanism remains unclear. Studies on genetic recombination in brome mosaic virus (BMV) have shown that local hybridization between genomic RNAs induces frequent non-homologous crossovers. A detailed analysis of recombinant structures suggested that local complementary regions might be involved in two types of non-homologous recombination in BMV: site-specific and heteroduplex-mediated. To verify the above hypothesis and better recognize the mechanism of the phenomenon studied we have tested how the putative types of recombination are affected by a specific mutation in the BMV polymerase gene or by changes in RNA structure. The experiments undertaken revealed substantial differences between site-specific and heteroduplex-mediated recombination, indicating that they occur according to different mechanisms. The former can be classified as homology-assisted, and the latter as homology-independent. In addition to local RNA/RNA hybridization, short regions of homology are required for site-specific crossovers to occur. They are most efficiently mediated if one homologous sequence is located at the beginning of and the second just before a double-stranded region. At present it is difficult to state what is the mechanism of heteroduplex-mediated recombination. Earlier it was postulated that strong RNA/RNA interaction enforces template switching by the viral replicase. There are, however, several observations questioning this model and indicating that some other factors, which are still unknown, may influence heteroduplex-mediated crossovers.

4

RNA interference and its role in the regulation of eucaryotic gene expression.

100%

Szweykowska-Kulińska Z., Jarmołowski A., Figlerowicz M.

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2003

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vol. 50

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issue 1

217-229

EN

Several years ago it was discovered that plant transformation with a transcribed sense transgene could shut down the expression of a homologous endogenous gene. Moreover, it was shown that the introduction into the cell of dsRNA (double-stranded RNA) containing nucleotide sequence complementary to an mRNA sequence causes selective degradation of the latter and thus silencing of a specific gene. This phenomenon, called RNA interference (RNAi) was demonstrated to be present in almost all eukaryotic organisms. RNAi is also capable of silencing transposons in germ line cells and fighting RNA virus infection. Enzymes involved in this process exhibit high homology across species. Some of these enzymes are involved in other cellular processes, for instance developmental timing, suggesting strong interconnections between RNAi and other metabolic pathways. RNAi is probably an ancient mechanism that evolved to protect eukaryotic cells against invasive forms of nucleic acids.

5

Nonenzymatic hydrolysis of oligoribonucleotides. V. The elements affecting the process of self-hydrolysis

100%

Bibiłło A., Ziomek K., Figlerowicz M., Kierzek R.

Acta Biochimica Polonica

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1999

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vol. 46

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issue 1

145-153

6

Low recombination activity of R region located at both ends of the HIV-1 genome

88%

Urbanowicz A., Kurzyńska-Kokorniak A., Jankowska A., Alejska M., Figlerowicz M.

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2012

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vol. 59

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issue 4

619-626

EN

Although two strand transfer events are indispensable for the synthesis of double-stranded DNA and establishing HIV-1 infection, the molecular basis of these phenomena is still unclear. The first obligatory template switching event occurs just at the beginning of the virus replication cycle and involves two copies of the 97-nucleotide long R region, located one each at the both ends of the HIV-1 genome (HIV-1 R). Thus, one can expect that the molecular mechanism of this process is similar to the mechanism of homologous recombination which operates in RNA viruses. To verify the above-mentioned hypothesis, we attempted to assess the recombination activity of HIV-1 R. To this end, we tested in vitro, how effectively it induces template switching by HIV-1 RT in comparison with another well-characterized sequence supporting frequent homologous crossovers in an unrelated virus (R region derived from Brome mosaic virus - BMV R). We also examined if the RNA sequences neighboring HIV-1 R influence its recombination activity. Finally, we tested if HIV-1 R could cause BMV polymerase complex to switch between RNA templates in vivo. Overall, our results have revealed a relatively low recombination activity of HIV-1 R as compared to BMV R. This observation suggests that different factors modulate the efficiency of the first obligatory strand transfer in HIV-1 and the homology-driven recombination in RNA viruses.

7

Structural and functional characteristics of two novel members of pathogensis-related multigene family of class 10 from yellow lupine+

88%

Handschuh L., Femiak I., Kasperska A., Figlerowicz M., Sikorski M. M.

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2007

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vol. 54

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issue 4

783-796

EN

PR-10 proteins (pathogensis-related), ubiquitous within the plant kingdom, are usually encoded by multigene families. To date we have identified 10 homologous pr-10 genes in a yellow lupine cDNA library. Here, the structure and expression of two newly identified yellow lupine pr-10 genes (LlYpr10-2b and LlYpr10-2f) are presented. Many potential regulatory sites were found in both gene promoters including common ones as well as those unique for each gene. However, promoter deletion analysis in transgenic tobacco plants revealed similar patterns of reporter gene (gus) expression. Shortened fragments of both gene promoters studied caused high GUS activity in leaves (along vascular bundles), stamen stigma, anthers and pollen grains. When conjugated with longer LlYpr-10.2 promoter fragments, GUS was additionally present in petal edges. Only a long fragment of the LlYpr10-2b gene promoter caused GUS expression in the stem. In yellow lupine the pr-10.2 genes are present in all studied organs, but their level of expression depends on the stage of development and is affected by wounding, oxidative stress and salicylic acid treatment. Silencing of the Llpr-10.2b gene in 4-week-old yellow lupine plants did not lead to any visible symptoms, which suggests that the function of the silenced gene is supplemented by its close homologues, still present in the studied plants.

8

AmiRNA Designer - new method of artificial miRNA design

88%

Mickiewicz A., Rybarczyk A., Sarzynska J., Figlerowicz M., Blazewicz J.

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2016

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vol. 63

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issue 1

71-77

EN

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 http://www.cs.put.poznan.pl/arybarczyk/AmiRNA/.

9

How RNA viruses exchange their genetic material.

88%

Alejska M., Kurzyńska-Kokorniak A., Broda M., Kierzek R., Figlerowicz M.

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2001

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vol. 48

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issue 2

391-407

EN

One of the most unusual features of RNA viruses is their enormous genetic variability. Among the different processes contributing to the continuous generation of new viral variants RNA recombination is of special importance. This process has been observed for human, animal, plant and bacterial viruses. The collected data reveal a great susceptibility of RNA viruses to recombination. They also indicate that genetic RNA recombination (especially the nonhomologous one) is a major factor responsible for the emergence of new viral strains or species. Although the formation and accumulation of viral recombinants was observed in numerous RNA viruses, the molecular basis of this phenomenon was studied in only a few viral species. Among them, brome mosaic virus (BMV), a model (+)RNA virus offers the best opportunities to investigate various aspects of genetic RNA recombination in vivo. Unlike any other, the BMV-based system enables homologous and nonhomologous recombination studies at both the protein and RNA levels. As a consequence, BMV is the virus for which the structural requirements for genetic RNA recombination have been most precisely established. Nevertheless, the previously proposed model of genetic recombination in BMV still had one weakness: it could not really explain the role of RNA structure in nonhomologous recombination. Recent discoveries concerning the latter problem give us a chance to fill this gap. That is why in this review we present and thoroughly discuss all results concerning nonhomologous recombination in BMV that have been obtained until now.

10

RNA recombination in brome mosaic virus, a model plus strand RNA virus

86%

Figlerowicz M., Bujarski J. J.

Acta Biochimica Polonica

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1998

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vol. 45

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issue 4

847-868

11

Impact of DNA microarray data transformation on gene expression analysis - comparison of two normalization methods

76%

Schmidt M. T., Handschuh L., Zyprych J., Szabelska A., Olejnik-Schmidt A. K., Siatkowski I., Figlerowicz M.

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2011

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vol. 58

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issue 4

573-580

EN

Two-color DNA microarrays are commonly used for the analysis of global gene expression. They provide information on relative abundance of thousands of mRNAs. However, the generated data need to be normalized to minimize systematic variations so that biologically significant differences can be more easily identified. A large number of normalization procedures have been proposed and many softwares for microarray data analysis are available. Here, we have applied two normalization methods (median and loess) from two packages of microarray data analysis softwares. They were examined using a sample data set. We found that the number of genes identified as differentially expressed varied significantly depending on the method applied. The obtained results, i.e. lists of differentially expressed genes, were consistent only when we used median normalization methods. Loess normalization implemented in the two software packages provided less coherent and for some probes even contradictory results. In general, our results provide an additional piece of evidence that the normalization method can profoundly influence final results of DNA microarray-based analysis. The impact of the normalization method depends greatly on the algorithm employed. Consequently, the normalization procedure must be carefully considered and optimized for each individual data set.

12

Assembling the SARS-CoV genome - new method based on graph theoretical approach.

64%

Błażewicz J., Figlerowicz M., Formanowicz P., Kasprzak M., Nowierski B., Styszyński R., Szajkowski Ł., Widera P., Wiktorczyk M.

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vol. 51

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issue 4

983-993

EN

Nowadays, scientists may learn a lot about the organisms studied just by analyzing their genetic material. This requires the development of methods of reading genomes with high accuracy. It has become clear that the knowledge of the changes occuring within a viral genome is indispensable for effective fighting of the pathogen. A good example is SARS-CoV, which was a cause of death of many people and frightened the entire world with its fast and hard to prevent propagation. Rapid development of sequencing methods, like shotgun sequencing or sequencing by hybridization (SBH), gives scientists a good tool for reading genomes. However, since sequencing methods can read fragments of up to 1000 bp only, methods for sequence assembling are required in order to read whole genomes. In this paper a new assembling method, based on graph theoretical approach, is presented. The method was tested on SARS-CoV and the results were compared to the outcome of other widely known methods.

13

RNA-Seq-based analysis of differential gene expression associated with hepatitis C virus infection in a cell culture

64%

Hojka-Osinska A., Budzko L., Zmienko A., Rybarczyk A., Maillard P., Budkowska A., Figlerowicz M., Jackowiak P.

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2016

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vol. 63

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issue 4

789-798

EN

Hepatitis C virus (HCV) infection is one of the major causes of chronic liver diseases. Unfortunately, the mechanisms of HCV infection-induced liver injury and host-virus interactions are still not well recognized. To better understand these processes we determined the changes in the host gene expression that occur during HCV infection of Huh-7.5 cells. As a result, we identified genes that may contribute to the immune and metabolic cellular responses to infection. Pathway enrichment analysis indicated that HCV induced an increased expression of genes involved in mitogen-activated protein kinases signaling, adipocytokine signaling, cell cycle and nitrogen metabolism. In addition, the enrichment analyses of processes and molecular functions revealed that the up-regulated genes were mainly implicated in the negative regulation of phosphorylation. Construction of the pathway-gene-process network enabled exploration of a much more complex landscape of molecular interactions. Consequently, several essential processes altered by HCV infection were identified: negative regulation of cell cycle, response to endoplasmic reticulum stress, response to reactive oxygen species, toll-like receptor signaling and pattern recognition receptor signaling. The analyses of genes whose expression was decreased upon HCV infection showed that the latter were engaged in the metabolism of lipids and amino acids. Moreover, we observed disturbance in the cellular antiviral defense. Altogether, our results demonstrated that HCV infection elicits host response that includes a very wide range of cellular mechanisms. Our findings significantly broaden the understanding of complex processes that accompany HCV infection. Consequently, they may be used for developing new host-oriented therapeutic strategies.

14

How short RNAs impact the human ribonuclease Dicer activity: putative regulatory feedback-loops and other RNA-mediated mechanisms controlling microRNA processing

64%

Koralewska N., Hoffmann W., Pokornowska M., Milewski M., Lipinska A., Bienkowska-Szewczyk K., Figlerowicz M., Kurzynska-Kokorniak A.

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2016

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vol. 63

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issue 4

773-783

EN

Ribonuclease Dicer plays a pivotal role in RNA interference pathways by processing long double-stranded RNAs and single-stranded hairpin RNA precursors into small interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. While details of Dicer regulation by a variety of proteins are being elucidated, less is known about non-protein factors, e.g. RNA molecules, that may influence this enzyme's activity. Therefore, we decided to investigate the question of whether the RNA molecules can function not only as Dicer substrates but also as its regulators. Our previous in vitro studies indicated that the activity of human Dicer can be influenced by short RNA molecules that either bind to Dicer or interact with its substrates, or both. Those studies were carried out with commercial Dicer preparations. Nevertheless, such preparations are usually not homogeneous enough to carry out more detailed RNA-binding studies. Therefore, we have established our own system for the production of human Dicer in insect cells. In this manuscript, we characterize the RNA-binding and RNA-cleavage properties of the obtained preparation. We demonstrate that Dicer can efficiently bind single-stranded RNAs that are longer than ~20-nucleotides. Consequently, we revisit possible scenarios of Dicer regulation by single-stranded RNA species ranging from ~10- to ~60-nucleotides, in the context of their binding to this enzyme. Finally, we show that siRNA/miRNA-sized RNAs may affect miRNA production either by binding to Dicer or by participating in regulatory feedback-loops. Altogether, our studies suggest a broad regulatory role of short RNAs in Dicer functioning.

Refine search results

Mikromacierze DNA

Computational prediction of non-enzymatic RNA degradation patterns

Two types of non-homologous RNA recombination in brome mosaic virus

RNA interference and its role in the regulation of eucaryotic gene expression.

Nonenzymatic hydrolysis of oligoribonucleotides. V. The elements affecting the process of self-hydrolysis

Low recombination activity of R region located at both ends of the HIV-1 genome

Structural and functional characteristics of two novel members of pathogensis-related multigene family of class 10 from yellow lupine+

AmiRNA Designer - new method of artificial miRNA design

How RNA viruses exchange their genetic material.

RNA recombination in brome mosaic virus, a model plus strand RNA virus

Impact of DNA microarray data transformation on gene expression analysis - comparison of two normalization methods

Assembling the SARS-CoV genome - new method based on graph theoretical approach.

RNA-Seq-based analysis of differential gene expression associated with hepatitis C virus infection in a cell culture

How short RNAs impact the human ribonuclease Dicer activity: putative regulatory feedback-loops and other RNA-mediated mechanisms controlling microRNA processing