Hałakuc, Paweł; Karnkowska, Anna; Milanowski, Rafał Typical structure of rRNA coding genes in diplonemids points to two independent origins of the bizarre rDNA structures of euglenozoans Journal Article BMC Ecology and Evolution, 22 (1), pp. 59, 2022, ISSN: 2730-7182. Abstract | Links | BibTeX @article{Haakuc2022,
title = {Typical structure of rRNA coding genes in diplonemids points to two independent origins of the bizarre rDNA structures of euglenozoans},
author = {Paweł Hałakuc and Anna Karnkowska and Rafał Milanowski},
url = {https://doi.org/10.1186/s12862-022-02014-9
https://bmcevolbiol.biomedcentral.com/articles/10.1186/s12862-022-02014-9},
doi = {10.1186/s12862-022-02014-9},
issn = {2730-7182},
year = {2022},
date = {2022-05-09},
journal = {BMC Ecology and Evolution},
volume = {22},
number = {1},
pages = {59},
abstract = {Members of Euglenozoa (Discoba) are known for unorthodox rDNA organization. In Euglenida rDNA is located on extrachromosomal circular DNA. In Kinetoplastea and Euglenida the core of the large ribosomal subunit, typically formed by the 28S rRNA, consists of several smaller rRNAs. They are the result of the presence of additional internal transcribed spacers (ITSs) in the rDNA. Diplonemea is the third of the main groups of Euglenozoa and its members are known to be among the most abundant and diverse protists in the oceans. Despite that, the rRNA of only one diplonemid species, Diplonema papillatum, has been examined so far and found to exhibit continuous 28S rRNA. Currently, the rDNA organization has not been researched for any diplonemid. Herein we investigate the structure of rRNA genes in classical (Diplonemidae) and deep-sea diplonemids (Eupelagonemidae), representing the majority of known diplonemid diversity. The results fill the gap in knowledge about diplonemid rDNA and allow better understanding of the evolution of the fragmented structure of the rDNA in Euglenozoa.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Members of Euglenozoa (Discoba) are known for unorthodox rDNA organization. In Euglenida rDNA is located on extrachromosomal circular DNA. In Kinetoplastea and Euglenida the core of the large ribosomal subunit, typically formed by the 28S rRNA, consists of several smaller rRNAs. They are the result of the presence of additional internal transcribed spacers (ITSs) in the rDNA. Diplonemea is the third of the main groups of Euglenozoa and its members are known to be among the most abundant and diverse protists in the oceans. Despite that, the rRNA of only one diplonemid species, Diplonema papillatum, has been examined so far and found to exhibit continuous 28S rRNA. Currently, the rDNA organization has not been researched for any diplonemid. Herein we investigate the structure of rRNA genes in classical (Diplonemidae) and deep-sea diplonemids (Eupelagonemidae), representing the majority of known diplonemid diversity. The results fill the gap in knowledge about diplonemid rDNA and allow better understanding of the evolution of the fragmented structure of the rDNA in Euglenozoa. |
Treitli, Sebastian Cristian; Peña-Diaz, Priscila; Hałakuc, Paweł; Karnkowska, Anna; Hampl, Vladimír High quality genome assembly of the amitochondriate eukaryote Monocercomonoides exilis Journal Article Microbial Genomics, 7 (12), pp. 000745, 2021, ISSN: 2057-5858. Abstract | Links | BibTeX @article{mbs:/content/journal/mgen/10.1099/mgen.0.000745,
title = {High quality genome assembly of the amitochondriate eukaryote Monocercomonoides exilis},
author = {Sebastian Cristian Treitli and Priscila Peña-Diaz and Paweł Hałakuc and Anna Karnkowska and Vladimír Hampl},
url = {https://www.microbiologyresearch.org/content/journal/mgen/10.1099/mgen.0.000745},
doi = {https://doi.org/10.1099/mgen.0.000745},
issn = {2057-5858},
year = {2021},
date = {2021-12-24},
journal = {Microbial Genomics},
volume = {7},
number = {12},
pages = {000745},
publisher = {Microbiology Society},
abstract = {Monocercomonoides exilis is considered the first known eukaryote to completely lack mitochondria. This conclusion is based primarily on a genomic and transcriptomic study which failed to identify any mitochondrial hallmark proteins. However, the available genome assembly has limited contiguity and around 1.5 % of the genome sequence is represented by unknown bases. To improve the contiguity, we re-sequenced the genome and transcriptome of M. exilis using Oxford Nanopore Technology (ONT). The resulting draft genome is assembled in 101 contigs with an N50 value of 1.38 Mbp, almost 20 times higher than the previously published assembly. Using a newly generated ONT transcriptome, we further improve the gene prediction and add high quality untranslated region (UTR) annotations, in which we identify two putative polyadenylation signals present in the 3′UTR regions and characterise the Kozak sequence in the 5′UTR regions. All these improvements are reflected by higher BUSCO genome completeness values. Regardless of an overall more complete genome assembly without missing bases and a better gene prediction, we still failed to identify any mitochondrial hallmark genes, thus further supporting the hypothesis on the absence of mitochondrion.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Monocercomonoides exilis is considered the first known eukaryote to completely lack mitochondria. This conclusion is based primarily on a genomic and transcriptomic study which failed to identify any mitochondrial hallmark proteins. However, the available genome assembly has limited contiguity and around 1.5 % of the genome sequence is represented by unknown bases. To improve the contiguity, we re-sequenced the genome and transcriptome of M. exilis using Oxford Nanopore Technology (ONT). The resulting draft genome is assembled in 101 contigs with an N50 value of 1.38 Mbp, almost 20 times higher than the previously published assembly. Using a newly generated ONT transcriptome, we further improve the gene prediction and add high quality untranslated region (UTR) annotations, in which we identify two putative polyadenylation signals present in the 3′UTR regions and characterise the Kozak sequence in the 5′UTR regions. All these improvements are reflected by higher BUSCO genome completeness values. Regardless of an overall more complete genome assembly without missing bases and a better gene prediction, we still failed to identify any mitochondrial hallmark genes, thus further supporting the hypothesis on the absence of mitochondrion. |
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. |
