In human cells, the intergenic spacers (IGS), which separate ribosomal genes, are complex approximately 30 kb-long loci. Recent studies indicate that all, or almost all, parts of IGS may be transcribed, and that at least some of them are involved in the regulation of the ribosomal DNA (rDNA) transcription, maintenance of the nucleolar architecture, and response of the cell nucleus to stress. However, since each cell contains hundreds not quite identical copies of IGS, the structure and functions of this locus remain poorly understood, and the dynamics of its products has not been specially studied. In this work, we used quantitative PCR to measure the expression levels of various rDNA regions at different times after inhibition of the transcription by Actinomycin D applied in high doses. This approach allowed us to measure real or extrapolated half-life times of some IGS loci. Our study reveals characteristic dynamic patterns suggestive of various pathways of RNA utilization and decay.
- Klíčová slova
- Intergenic spacer, Processing, RNA decay, lncRNAs, rDNA,
- MeSH
- HeLa buňky MeSH
- lidé MeSH
- mezerníky ribozomální DNA chemie genetika metabolismus MeSH
- RNA analýza biosyntéza genetika izolace a purifikace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- mezerníky ribozomální DNA MeSH
- RNA MeSH
The intergenic spacer (IGS) of rDNA is frequently built of long blocks of tandem repeats. To estimate the intragenomic variability of such knotty regions, we employed PacBio sequencing of the Cucurbita moschata genome, in which thousands of rDNA copies are distributed across a number of loci. The rRNA coding regions are highly conserved, indicating intensive interlocus homogenization and/or high selection pressure. However, the IGS exhibits high intragenomic structural diversity. Two repeated blocks, R1 (300-1250 bp) and R2 (290-643 bp), account for most of the IGS variation. They exhibit minisatellite-like features built of multiple periodically spaced short GC-rich sequence motifs with the potential to adopt non-canonical DNA conformations, G-quadruplex-folded and left-handed Z-DNA. The mutual arrangement of these motifs can be used to classify IGS variants into five structural families. Subtle polymorphisms exist within each family due to a variable number of repeats, suggesting the coexistence of an enormous number of IGS variants. The substantial length and structural heterogeneity of IGS minisatellites suggests that the tempo of their divergence exceeds the tempo of the homogenization of rDNA arrays. As frequently occurring among plants, we hypothesize that their instability may influence transcription regulation and/or destabilize rDNA units, possibly spreading them across the genome.
- Klíčová slova
- Cucurbita moschata, DNA-minisatellite, intragenomic structural heterogeneity, non-canonical DNA conformations, ribosomal DNA intergenic spacer,
- MeSH
- Cucurbita genetika MeSH
- genetická variace * MeSH
- konformace nukleové kyseliny * MeSH
- mezerníky ribozomální DNA chemie genetika metabolismus MeSH
- minisatelitní repetice * MeSH
- sekvenční analýza DNA MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- mezerníky ribozomální DNA MeSH
Our understanding of fungal diversity is far from complete. Species descriptions generally focus on morphological features, but this approach may underestimate true diversity. Using the morphological species concept, Hesperomyces virescens (Ascomycota, Laboulbeniales) is a single species with global distribution and wide host range. Since its description 120 years ago, this fungal parasite has been reported from 30 species of ladybird hosts on all continents except Antarctica. These host usage patterns suggest that H. virescens could be made up of many different species, each adapted to individual host species. Using sequence data from three gene regions, we found evidence for distinct clades within Hesperomyces virescens, each clade corresponding to isolates from a single host species. We propose that these lineages represent separate species, driven by adaptation to different ladybird hosts. Our combined morphometric, molecular phylogenetic and ecological data provide support for a unified species concept and an integrative taxonomy approach.
- MeSH
- analýza hlavních komponent MeSH
- Ascomycota klasifikace genetika izolace a purifikace fyziologie MeSH
- brouci parazitologie MeSH
- DNA fungální chemie genetika metabolismus MeSH
- fylogeneze MeSH
- mezerníky ribozomální DNA chemie genetika metabolismus MeSH
- sekvenční analýza DNA MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA fungální MeSH
- mezerníky ribozomální DNA MeSH
New specific primers AR1 and AR2 were successfully used for the amplification of a specific part of internal transcribed spacer (ITS) of rDNA of Armillaria isolated from soil samples. DNA was isolated from 0.5 g of forest soil and ITS region was amplified by nested PCR reaction with external primers ITS1 and ITS4 and internal primers AR1 and AR2. The individual species were distinguished by restriction fragment length polymorphisms (RFLPs) analysis with restriction endonuclease HinfI. The fragments were analysed by ion-exchange HPLC that is more sensible and more rapid than electrophoresis. The amplicons were sequenced to improve the discrimination between the species. The method enables the identification of Armillaria species within one day directly from soil samples without the need for previous isolation and cultivation of mycelium of Armillaria.
- MeSH
- Agaricales klasifikace genetika izolace a purifikace MeSH
- DNA fungální analýza izolace a purifikace metabolismus MeSH
- mezerníky ribozomální DNA analýza izolace a purifikace metabolismus MeSH
- polymerázová řetězová reakce * MeSH
- polymorfismus délky restrikčních fragmentů * MeSH
- půdní mikrobiologie * MeSH
- restrikční endonukleasy typu II metabolismus MeSH
- sekvenční analýza DNA MeSH
- senzitivita a specificita MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA fungální MeSH
- GANTC-specific type II deoxyribonucleases MeSH Prohlížeč
- mezerníky ribozomální DNA MeSH
- restrikční endonukleasy typu II MeSH
