2021
|
Gumińska, Natalia; Łukomska-Kowalczyk, Maja; Chaber, Katarzyna; Zakryś, Bożena; Milanowski, Rafał Evaluation of V2 18S rDNA barcode marker and assessment of sample collection and DNA extraction methods for metabarcoding of autotrophic euglenids Journal Article Environmental Microbiology, 23 (6), pp. 2992–3008 , 2021. Abstract | Links | BibTeX @article{https://doi.org/10.1111/1462-2920.15495,
title = {Evaluation of V2 18S rDNA barcode marker and assessment of sample collection and DNA extraction methods for metabarcoding of autotrophic euglenids},
author = {Natalia Gumińska and Maja Łukomska-Kowalczyk and Katarzyna Chaber and Bożena Zakryś and Rafał Milanowski},
url = {https://sfamjournals.onlinelibrary.wiley.com/doi/abs/10.1111/1462-2920.15495},
doi = {https://doi.org/10.1111/1462-2920.15495},
year = {2021},
date = {2021-04-08},
journal = {Environmental Microbiology},
volume = {23},
number = {6},
pages = {2992–3008 },
abstract = {Summary Even though the interest in metabarcoding in environmental studies is growing, euglenids are still underrepresented in both sea and freshwater bodies researches. The reason for this situation could be the unsuitability of universal eukaryotic DNA barcodes and primers as well as the lack of a verified protocol, suitable to assess euglenid diversity. In this study, using specific primers for the V2 hypervariable region of 18S rDNA for metabarcoding resulted in obtaining a high fraction (85%) of euglenid reads and species-level identification of almost 90% of them. Fifty species were detected by the metabarcoding method, including almost all species observed using a light microscope. We investigated three biomass harvesting methods (filtering, centrifugation and scraping the side of a collection vessel) and determined that centrifugation and filtration outperformed scrapes, but the choice between them is not crucial for the reliability of the analysis. In addition, eight DNA extraction methods were evaluated. We compared five commercially available DNA isolation kits, two CTAB-based protocols and a chelating resin. For this purpose, the efficiency of extraction, quality of obtained DNA, preparation time and generated costs were taken into consideration. After examination of the aforementioned criteria, we chose the GeneMATRIX Soil DNA Purification Kit as the most suitable for DNA isolation.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Summary Even though the interest in metabarcoding in environmental studies is growing, euglenids are still underrepresented in both sea and freshwater bodies researches. The reason for this situation could be the unsuitability of universal eukaryotic DNA barcodes and primers as well as the lack of a verified protocol, suitable to assess euglenid diversity. In this study, using specific primers for the V2 hypervariable region of 18S rDNA for metabarcoding resulted in obtaining a high fraction (85%) of euglenid reads and species-level identification of almost 90% of them. Fifty species were detected by the metabarcoding method, including almost all species observed using a light microscope. We investigated three biomass harvesting methods (filtering, centrifugation and scraping the side of a collection vessel) and determined that centrifugation and filtration outperformed scrapes, but the choice between them is not crucial for the reliability of the analysis. In addition, eight DNA extraction methods were evaluated. We compared five commercially available DNA isolation kits, two CTAB-based protocols and a chelating resin. For this purpose, the efficiency of extraction, quality of obtained DNA, preparation time and generated costs were taken into consideration. After examination of the aforementioned criteria, we chose the GeneMATRIX Soil DNA Purification Kit as the most suitable for DNA isolation. |
Gumińska, Natalia; Zakryś, Bożena; Milanowski, Rafał A New Type of Circular RNA derived from Nonconventional Introns in Nuclear Genes of Euglenids Journal Article Journal of Molecular Biology, 433 (3), pp. 166758, 2021. Abstract | Links | BibTeX @article{GUMINSKA2021166758,
title = {A New Type of Circular RNA derived from Nonconventional Introns in Nuclear Genes of Euglenids},
author = {Natalia Gumińska and Bożena Zakryś and Rafał Milanowski},
url = {https://www.sciencedirect.com/science/article/pii/S0022283620306835?via%3Dihub},
doi = {https://doi.org/10.1016/j.jmb.2020.166758},
year = {2021},
date = {2021-02-05},
journal = {Journal of Molecular Biology},
volume = {433},
number = {3},
pages = {166758},
abstract = {Nuclear protein-coding genes of euglenids (Discoba, Euglenozoa, Euglenida) contain conventional (spliceosomal) and nonconventional introns. The latter have been found only in euglenozoans. A unique feature of nonconventional introns is the ability to form a stable and slightly conserved RNA secondary structure bringing together intron ends and placing adjacent exons in proximity. To date, little is known about the mechanism of their excision (e.g. whether it involves the spliceosome or not). The tubA gene of Euglena gracilis harbors three conventional and three nonconventional introns. While the conventional introns are excised as lariats, nonconventional introns are present in the cell solely as circular RNAs with full-length ends. Based on this discovery as well as on previous observations indicating that nonconventional introns are observed frequently at unique positions of genes, we suggest that this new type of intronic circRNA might play a role in intron mobility.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Nuclear protein-coding genes of euglenids (Discoba, Euglenozoa, Euglenida) contain conventional (spliceosomal) and nonconventional introns. The latter have been found only in euglenozoans. A unique feature of nonconventional introns is the ability to form a stable and slightly conserved RNA secondary structure bringing together intron ends and placing adjacent exons in proximity. To date, little is known about the mechanism of their excision (e.g. whether it involves the spliceosome or not). The tubA gene of Euglena gracilis harbors three conventional and three nonconventional introns. While the conventional introns are excised as lariats, nonconventional introns are present in the cell solely as circular RNAs with full-length ends. Based on this discovery as well as on previous observations indicating that nonconventional introns are observed frequently at unique positions of genes, we suggest that this new type of intronic circRNA might play a role in intron mobility. |
2019
|
Ignatenko, Antonina; Gumińska, Natalia; Milanowski, Rafał Mechanizmy utraty i nabywania intronów spliceosomalnych Journal Article Postepy biochemii, 65 (4), pp. 289–298, 2019, ISSN: 00325422. Abstract | Links | BibTeX @article{Ignatenko2019,
title = {Mechanizmy utraty i nabywania intronów spliceosomalnych},
author = {Antonina Ignatenko and Natalia Gumińska and Rafał Milanowski},
url = {https://postepybiochemii.ptbioch.edu.pl/index.php/PB/article/view/292},
doi = {10.18388/pb.2019_292},
issn = {00325422},
year = {2019},
date = {2019-01-01},
journal = {Postepy biochemii},
volume = {65},
number = {4},
pages = {289--298},
abstract = {Introny to wewnątrzgenowe sekwencje niekodujące. Dawniej były uznawane za „śmieciowy” DNA, jednak obecnie uważa się, że są ważnymi elementami wpływającymi na funkcjonowanie genomu. Udowodniono, że introny zwiększają różnorodność transkryptomu i proteomu, spełniają w komórce role regulatorowe, wpływają na ekspresję genów oraz obróbkę, translację i degradację mRNA. Ze względu na sposób powstawania dzielą się na trzy główne kategorie: spliceosomalne, samowycinające się oraz introny tRNA. Introny spliceosomalne są charakterystyczne dla organizmów eukariotycznych. Analizy sekwencji genów ortologicznych w rożnych grupach eukariontów pozwoliły zidentyfikować wiele przypadków nabywania oraz utraty intronów. Niektóre z tych zdarzeń miały miejsce w dalekiej przeszłości, do innych doszło stosunkowo niedawno. Uważa się, że procesy te mogą działać jako jedna z sił napędowych w ewolucji genów eukariotycznych.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Introny to wewnątrzgenowe sekwencje niekodujące. Dawniej były uznawane za „śmieciowy” DNA, jednak obecnie uważa się, że są ważnymi elementami wpływającymi na funkcjonowanie genomu. Udowodniono, że introny zwiększają różnorodność transkryptomu i proteomu, spełniają w komórce role regulatorowe, wpływają na ekspresję genów oraz obróbkę, translację i degradację mRNA. Ze względu na sposób powstawania dzielą się na trzy główne kategorie: spliceosomalne, samowycinające się oraz introny tRNA. Introny spliceosomalne są charakterystyczne dla organizmów eukariotycznych. Analizy sekwencji genów ortologicznych w rożnych grupach eukariontów pozwoliły zidentyfikować wiele przypadków nabywania oraz utraty intronów. Niektóre z tych zdarzeń miały miejsce w dalekiej przeszłości, do innych doszło stosunkowo niedawno. Uważa się, że procesy te mogą działać jako jedna z sił napędowych w ewolucji genów eukariotycznych. |
2018
|
Gumińska, Natalia; Płecha, Magdalena; Walkiewicz, Halszka; Hałakuc, Paweł; Zakryś, Bożena; Milanowski, Rafał Culture purification and DNA extraction procedures suitable for next-generation sequencing of euglenids Journal Article Journal of Applied Phycology, 30 (6), pp. 3541–3549, 2018, ISSN: 1573-5176. Abstract | Links | BibTeX @article{Guminska2018,
title = {Culture purification and DNA extraction procedures suitable for next-generation sequencing of euglenids},
author = {Natalia Gumińska and Magdalena Płecha and Halszka Walkiewicz and Paweł Hałakuc and Bożena Zakryś and Rafał Milanowski},
url = {https://doi.org/10.1007/s10811-018-1496-0},
doi = {10.1007/s10811-018-1496-0},
issn = {1573-5176},
year = {2018},
date = {2018-01-01},
journal = {Journal of Applied Phycology},
volume = {30},
number = {6},
pages = {3541--3549},
abstract = {In the present study, five different DNA extraction procedures were examined to determine their effectiveness for extracting DNA suitable for NGS applications. This included two silica-membrane spin column kits, phenol:chloroform, and two CTAB-based methods. Spectrophotometric and fluorimetric measurements as well as standard gel electrophoresis were used as criteria for evaluating the quantity and quality of the isolated DNA prior to the sequencing. Herein, the method of establishing and maintaining axenic Euglena cultures is also presented. The modified CTAB-based method proved to be highly efficient. In terms of DNA quantity and purity (according to the absorbance ratios), the chosen method resulted in DNA of high molecular weight and quality, which fulfills the library construction requirements. Genomic DNA of Euglena hiemalis (CCAP 1224/35) and E. longa (CCAP 1204-17a) isolated using the suggested protocol had been successfully sequenced on the Illumina HiSeq platform. A modified, rapid CTAB-based method of total DNA isolation from Euglena has been described. In terms of the DNA quantity and quality, the protocol devised involving the washing step with DMSO:acetonitrile proved superior to the commonly used, commercially manufactured kits and isolation with phenol:chloroform. The method is also less labor-intensive and time-consuming than the traditional CTAB-based protocol.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
In the present study, five different DNA extraction procedures were examined to determine their effectiveness for extracting DNA suitable for NGS applications. This included two silica-membrane spin column kits, phenol:chloroform, and two CTAB-based methods. Spectrophotometric and fluorimetric measurements as well as standard gel electrophoresis were used as criteria for evaluating the quantity and quality of the isolated DNA prior to the sequencing. Herein, the method of establishing and maintaining axenic Euglena cultures is also presented. The modified CTAB-based method proved to be highly efficient. In terms of DNA quantity and purity (according to the absorbance ratios), the chosen method resulted in DNA of high molecular weight and quality, which fulfills the library construction requirements. Genomic DNA of Euglena hiemalis (CCAP 1224/35) and E. longa (CCAP 1204-17a) isolated using the suggested protocol had been successfully sequenced on the Illumina HiSeq platform. A modified, rapid CTAB-based method of total DNA isolation from Euglena has been described. In terms of the DNA quantity and quality, the protocol devised involving the washing step with DMSO:acetonitrile proved superior to the commonly used, commercially manufactured kits and isolation with phenol:chloroform. The method is also less labor-intensive and time-consuming than the traditional CTAB-based protocol. |
Gumińska, Natalia; Płecha, Magdalena; Zakryś, Bożena; Milanowski, Rafał Order of removal of conventional and nonconventional introns from nuclear transcripts of Euglena gracilis Journal Article PLoS Genetics, 14 (10), pp. e1007761, 2018, ISSN: 15537404. Abstract | Links | BibTeX @article{Guminska2018b,
title = {Order of removal of conventional and nonconventional introns from nuclear transcripts of Euglena gracilis},
author = {Natalia Gumińska and Magdalena Płecha and Bożena Zakryś and Rafał Milanowski},
doi = {10.1371/journal.pgen.1007761},
issn = {15537404},
year = {2018},
date = {2018-01-01},
journal = {PLoS Genetics},
volume = {14},
number = {10},
pages = {e1007761},
abstract = {Nuclear genes of euglenids and marine diplonemids harbor atypical, nonconventional introns which are not observed in the genomes of other eukaryotes. Nonconventional introns do not have the conserved borders characteristic for spliceosomal introns or the sequence complementary to U1 snRNA at the 5' end. They form a stable secondary structure bringing together both exon/intron junctions, nevertheless, this conformation does not resemble the form of self-splicing or tRNA introns. In the genes studied so far, frequent nonconventional introns insertions at new positions have been observed, whereas conventional introns have been either found at the conserved positions, or simply lost. In this work, we examined the order of intron removal from Euglena gracilis transcripts of the tubA and gapC genes, which contain two types of introns: nonconventional and spliceosomal. The relative order of intron excision was compared for pairs of introns belonging to different types. Furthermore, intermediate products of splicing were analyzed using the PacBio Next Generation Sequencing system. The analysis led to the main conclusion that nonconventional introns are removed in a rapid way but later than spliceosomal introns. Moreover, the observed accumulation of transcripts with conventional introns removed and nonconventional present may suggest the existence of a time gap between the two types of splicing.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Nuclear genes of euglenids and marine diplonemids harbor atypical, nonconventional introns which are not observed in the genomes of other eukaryotes. Nonconventional introns do not have the conserved borders characteristic for spliceosomal introns or the sequence complementary to U1 snRNA at the 5' end. They form a stable secondary structure bringing together both exon/intron junctions, nevertheless, this conformation does not resemble the form of self-splicing or tRNA introns. In the genes studied so far, frequent nonconventional introns insertions at new positions have been observed, whereas conventional introns have been either found at the conserved positions, or simply lost. In this work, we examined the order of intron removal from Euglena gracilis transcripts of the tubA and gapC genes, which contain two types of introns: nonconventional and spliceosomal. The relative order of intron excision was compared for pairs of introns belonging to different types. Furthermore, intermediate products of splicing were analyzed using the PacBio Next Generation Sequencing system. The analysis led to the main conclusion that nonconventional introns are removed in a rapid way but later than spliceosomal introns. Moreover, the observed accumulation of transcripts with conventional introns removed and nonconventional present may suggest the existence of a time gap between the two types of splicing. |
2016
|
Milanowski, Rafał; Gumińska, Natalia; Karnkowska, Anna; Ishikawa, Takao; Zakryś, Bożena Intermediate introns in nuclear genes of euglenids – are they a distinct type? Journal Article BMC Evolutionary Biology, 16 (1), pp. 49, 2016, ISSN: 1471-2148. Abstract | Links | BibTeX @article{Milanowski2016,
title = {Intermediate introns in nuclear genes of euglenids – are they a distinct type?},
author = {Rafał Milanowski and Natalia Gumińska and Anna Karnkowska and Takao Ishikawa and Bożena Zakryś},
url = {https://bmcecolevol.biomedcentral.com/articles/10.1186/s12862-016-0620-5},
doi = {10.1186/s12862-016-0620-5},
issn = {1471-2148},
year = {2016},
date = {2016-12-01},
journal = {BMC Evolutionary Biology},
volume = {16},
number = {1},
pages = {49},
abstract = {textcopyright 2016 Milanowski et al. Background: Nuclear genes of euglenids contain two major types of introns: Conventional spliceosomal and nonconventional introns. The latter are characterized by variable non-canonical borders, RNA secondary structure that brings intron ends together, and an unknown mechanism of removal. Some researchers also distinguish intermediate introns, which combine features of both types. They form a stable RNA secondary structure and are classified into two subtypes depending on whether they contain one (intermediate/nonconventional subtype) or both (conventional/intermediate subtype) canonical spliceosomal borders. However, it has been also postulated that most introns classified as intermediate could simply be special cases of conventional or nonconventional introns. Results: Sequences of tubB, hsp90 and gapC genes from six strains of Euglena agilis were obtained. They contain four, six, and two or three introns, respectively (the third intron in the gapC gene is unique for just one strain). Conventional introns were present at three positions: Two in the tubB gene (at one position conventional/intermediate introns were also found) and one in the gapC gene. Nonconventional introns are present at ten positions: T wo in the tubB gene (at one position intermediate/nonconventional introns were also found), six in hsp90 (at four positions intermediate/nonconventional introns were also found), and two in the gapC gene. Conclusions: Sequence and RNA secondary structure analyses of nonconventional introns confirmed that their most strongly conserved elements are base pairing nucleotides at positions +4, +5 and +6/-8,-7 and-6 (in most introns CAG/CTG nucleotides were observed). It was also confirmed that the presence of the 5' GT/C end in intermediate/nonconventional introns is not the result of kinship with conventional introns, but is due to evolutionary pressure to preserve the purine at the 5' end. However, an example of a nonconventional intron with GC-AG ends was shown, suggesting the possibility of intron type conversion between nonconventional and conventional. Furthermore, an analysis of conventional introns revealed that the ability to form a stable RNA secondary structure by some introns is probably not a result of their relationship with nonconventional introns. It was also shown that acquisition of new nonconventional introns is an ongoing process and can be observed at the level of a single species. In the recently acquired intron in the gapC gene an extended direct repeats at the intron-exon junctions are present, suggesting that double-strand break repair process could be the source of new nonconventional introns.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
textcopyright 2016 Milanowski et al. Background: Nuclear genes of euglenids contain two major types of introns: Conventional spliceosomal and nonconventional introns. The latter are characterized by variable non-canonical borders, RNA secondary structure that brings intron ends together, and an unknown mechanism of removal. Some researchers also distinguish intermediate introns, which combine features of both types. They form a stable RNA secondary structure and are classified into two subtypes depending on whether they contain one (intermediate/nonconventional subtype) or both (conventional/intermediate subtype) canonical spliceosomal borders. However, it has been also postulated that most introns classified as intermediate could simply be special cases of conventional or nonconventional introns. Results: Sequences of tubB, hsp90 and gapC genes from six strains of Euglena agilis were obtained. They contain four, six, and two or three introns, respectively (the third intron in the gapC gene is unique for just one strain). Conventional introns were present at three positions: Two in the tubB gene (at one position conventional/intermediate introns were also found) and one in the gapC gene. Nonconventional introns are present at ten positions: T wo in the tubB gene (at one position intermediate/nonconventional introns were also found), six in hsp90 (at four positions intermediate/nonconventional introns were also found), and two in the gapC gene. Conclusions: Sequence and RNA secondary structure analyses of nonconventional introns confirmed that their most strongly conserved elements are base pairing nucleotides at positions +4, +5 and +6/-8,-7 and-6 (in most introns CAG/CTG nucleotides were observed). It was also confirmed that the presence of the 5' GT/C end in intermediate/nonconventional introns is not the result of kinship with conventional introns, but is due to evolutionary pressure to preserve the purine at the 5' end. However, an example of a nonconventional intron with GC-AG ends was shown, suggesting the possibility of intron type conversion between nonconventional and conventional. Furthermore, an analysis of conventional introns revealed that the ability to form a stable RNA secondary structure by some introns is probably not a result of their relationship with nonconventional introns. It was also shown that acquisition of new nonconventional introns is an ongoing process and can be observed at the level of a single species. In the recently acquired intron in the gapC gene an extended direct repeats at the intron-exon junctions are present, suggesting that double-strand break repair process could be the source of new nonconventional introns. |
Zainteresowania badawcze