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1
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Cloning and characterization of Arabidopsis thaliana AtNAP57 - a homologue of yeast pseudouridine synthase Cbf5p.

100%
Maceluch J., Kmieciak M., Szweykowska-Kulińska Z., Jarmołowski A.
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2002
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vol. 49
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issue 3
699-709
EN
Rat Nap57 and its yeast homologue Cbf5p are pseudouridine synthases involved in rRNA biogenesis, localized in the nucleolus. These proteins, together with H/ACA class of snoRNAs compose snoRNP particles, in which snoRNA guides the synthase to direct site-specific pseudouridylation of rRNA. In this paper we present an Arabidopsis thaliana protein that is highly homologous to Cbf5p (72% identity and 85% homology) and NAP57 (67% identity and 81% homology). Moreover, the plant protein has conserved structural motifs that are characteristic features of pseudouridine synthases of the TruB class. We have named the cloned and characterized protein AtNAP57 (A rabidopsis t haliana homologue of NAP57 ). AtNAP57 is a 565 amino-acid protein and its calculated molecular mass is 63 kDa. The protein is encoded by a single copy gene located on chromosome 3 of the A. thaliana genome. Interestingly, the AtNAP57 gene does not contain any introns. Mutations in the human DKC1 gene encoding dyskerin (human homologue of yeast Cbf5p and rat NAP57) cause dyskeratosis congenita a rare inherited bone marrow failure syndrome characterized by abnormal skin pigmentation, nail dystrophy and mucosal leukoplakia.
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Arabidopsis thaliana microRNA162 level is posttranscriptionally regulated via splicing and polyadenylation site selection

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Barciszewska-Pacak M., Knop K., Jarmołowski A., Szweykowska-Kulińska Z.
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2016
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vol. 63
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issue 4
811-816
EN
Arabidopsis microRNA162 (miRNA162) level regulation was studied under abiotic stresses, such as drought and salinity. The TaqMan® microRNA assay proved that A. thaliana miRNA162 level was elevated under these stresses, confirming its salt and drought responsiveness. The promoter region analyses of A. thaliana miRNA162a and b genes (MIR162a and MIR162b) identified numerous salinity and drought responsive elements. However, our results indicated that Arabidopsis MIR162a was presumably the main locus responsible for the mature ath-miRNA162 accumulation under the stresses tested, and the MIR162b was generally rather weakly expressed, both in control and under the stress conditions. The MIR162a structure was confirmed to be complex and the pri-miRNA162a hairpin structure was shown to span an alternative exon and an intron. The MIR162a transcription generated a few pri-miRNA162a splicing isoforms that could be functional and non-functional. Upon drought and salinity stresses, the regulation of the pri-miRNA162a alternative splicing pattern revealed an increase of a functional pri-miR162a isoform and a preferential distal polyA site selection under the stress conditions. Apart from the potential transcriptional regulation of the miRNA genes (MIRs) expression, the data obtained point to an essential role of posttranscriptional regulation of Arabidopsis microRNA162 level.
3
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The nuclear cap-binding protein complex is not essential for nonsense-mediated mRNA decay (NMD) in plants

100%
Dzikiewicz-Krawczyk A., Piontek P., Szweykowska-Kulińska Z., Jarmołowski A.
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2008
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vol. 55
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issue 4
825-828
EN
In this study we investigated whether in plants, like in mammals, components of the nuclear cap-binding protein complex (CBC) are involved in nonsense-mediated mRNA decay (NMD). We selected several genes producing at least two alternatively spliced mRNA variants: one with a premature termination codon (PTC+) and another without it (PTC-). For each gene the PTC+/PTC- ratio was calculated using RT-PCR and direct sequencing in four Arabidopsis thaliana lines: wild type, the NMD mutant atupf3-1 and two CBC mutants: cbp20 and abh1. Whereas in the NMD mutant the ratios of PTC+/PTC- splice variants were higher than in wild-type plants, the two CBC mutants investigated showed no change in the PTC+/PTC- ratios. Our results suggest that neither CBP20 nor CBP80 is involved in NMD in A. thaliana.
4
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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.
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Effect of nuclear matrix attachment regions on transgene expression in tobacco plants.

100%
Nowak W., Gawłowska0 M., Jarmołowski A., Augustyniak J.
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2001
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vol. 48
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issue 3
637-646
EN
Matrix attachment regions (MARs) are thought to participate in the organization and segregation of independent chromosomal loop domains. Although there are several reports on the action of natural MARs in the context of heterologous genes in transgenic plants, in our study we tested a synthetic MAR (sMAR) with the special property of unpairing when under superhelical strain, for its effect on reporter gene expression in tobacco plants. The synthetic MAR was a multimer of a short sequence from the MAR 3' end of the immunoglobulin heavy chain (IgH) enhancer. This sMAR sequence was used to flank the β-glucuronidase (GUS) reporter gene within the T-DNA of the binary vector pBI121. Vectors with or without the sMARs were then used to transform tobacco plants by Agrobacterium tumefaciens. Transgenic plants containing the sMAR sequences flanking the GUS gene exhibited higher levels of transgene expression compared with transgenic plants which lacked the sMARs. This effect was observed independently of the position of the sMAR at the 5' side of the reporter gene. However, variation of the detected transgene expression was significant in all transformed plant populations, irrespective of the construct used. Most genes whose expression has been studied in transgenic plants are generally expressed in appropriate patterns. However, transgene expression can vary within an extremely wide range, often showing only a very low level [1, 2]. Variation in transgene expression is frequently attributed to corresponding variation in the transcription potential of different chromosomal insertion sites. DNA sequences called scaffold/matrix attachment regions (S/MARs) are involved in the structural and functional organization of all eukaryotic genomes. Evolutionarily, the structures of these sequences seem to be conserved. Typically, S/MARs are located every 5 to 200 kb of sequence and are known to bind specifically to components of the nuclear scaffold, therefore suggesting that they are responsible for loop domain base formation [3, 4].Of the MAR elements reported, many do not display extensive sequence homology. It is therefore reasonable to assume that the scaffold probably recognizes some structural features of the MAR DNA rather than a specific sequence [5].MARs appear to be functionally conserved, since animal MARs can bind to plant nuclear scaffolds and vice versa [6, 7].Most MARs have been generally characterized as AT-rich sequences. However, AT-richness per se is not a sufficient criterion for specific sequence recognition of MARs by specific binding proteins [7]. Their capacity to bind to the nuclear matrix is determined by the specific structure of DNA. A prominent structural characteristic of different MARs is their strong potential for extensive unpairing when subjected to superhelical strain [8, 9]. The ability to assume a stably unpaired conformation has been described for several MARs. For example, within the MAR 3' end of immunoglobulin heavy chain (IgH) enhancer there is an AATATATTT motif that is a nucleation site for DNA unwinding [10]. Concatamerized oligonucleotides containing seven repeats of this sequence exhibited a strong affinity for the nuclear scaffold and increased SV40 promoter activity in stably transformed mouse cells [11].In this paper we present results of our studies that concern the effect of a synthetic MAR on transgene expression in tobacco plants. The synthetic MAR sequences were used to flank the β-glucuronidase (GUS) gene whose transcription was under control of the 35S CaMV promoter in the binary vector pBI121. This construct was introduced into tobacco plants and the GUS reporter gene expression was monitored in stably transformed plants.
6
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Analysis of potyvirus terminal protein VPg-transgenic Arabidopsis thaliana plants

76%
Wojtal I., Piontek P., Grzela R., Jarmołowski A., Zagórski W., Chroboczek J.
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2011
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vol. 58
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issue 3
349-353
EN
Virus-coded VPg protein of Potato virus Y (PVY) does not have homologs apart from other VPgs. Since VPg is indispensable for the potyvirus life cycle, it appeared a good candidate for eliciting pathogen-derived resistance to PVY. Following agroinfection used to obtain PVY VPg-transgenic Arabidopsis thaliana plants, only few transgenic seeds were recovered giving rise to six transgenic plants that contained the VPg gene with the correct sequence. They generated VPg mRNA, but VPg protein was not detected. Some plants were immune to PVY infection suggesting post-transcriptional gene silencing. However, the likely PVY VPg toxicity exerted at an early stage of transformed seeds development precludes its use for engineering pathogen-derived resistance.
7
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Abscisic acid does not influence the subcellular distribution of the HYL1 protein from Arabidopsis thaliana

76%
Lesicka-Górecka J., Szarzyńska B., Sawczak M., Bagdiul I., Górski P., Jarmołowski A., Szweykowska-Kulińska Z.
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2008
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vol. 55
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issue 3
517-524
EN
HYL1 is a nuclear protein involved in the processing of miRNAs but its exact function remains unknown. Arabidopsis thaliana hyl1 mutants exhibit hypersensitivity to ABA. We decided to answer the question whether ABA affects the HYL1 protein localization within the cell and show that it does not. We also studied the expression of HYL1 in different tissues and organs. In this paper we show for the first time the expression profile of the HYL1 protein using anti-HYL1 antibodies. The protein is present in seedlings and mature plants in all organs studied, with the highest amount in inflorescences. A. thaliana HYL1 protein has several repetitions of a 28-amino-acid sequence at the C-terminus that confer protein instability. Our bioinformatic analysis of HYL1 homologs in different Brassica species shows that this repetition is typical only for Arabidopsis. This may suggest a relatively late evolutionary acquisition of the C-terminal domain.
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