Methods of artificial evolution such as SELEX and in vitro selection have made it possible to isolate RNA and DNA motifs with a wide range of functions from large random sequence libraries. Once the primary sequence of a functional motif is known, the sequence space around it can be comprehensively explored using a combination of random mutagenesis and selection. However, methods to explore the sequence space of a secondary structure are not as well characterized. Here we address this question by describing a method to construct libraries in a single synthesis which are enriched for sequences with the potential to form a specific secondary structure, such as that of an aptamer, ribozyme, or deoxyribozyme. Although interactions such as base pairs cannot be encoded in a library using conventional DNA synthesizers, it is possible to modulate the probability that two positions will have the potential to pair by biasing the nucleotide composition at these positions. Here we show how to maximize this probability for each of the possible ways to encode a pair (in this study defined as A-U or U-A or C-G or G-C or G.U or U.G). We then use these optimized coding schemes to calculate the number of different variants of model stems and secondary structures expected to occur in a library for a series of structures in which the number of pairs and the extent of conservation of unpaired positions is systematically varied. Our calculations reveal a tradeoff between maximizing the probability of forming a pair and maximizing the number of possible variants of a desired secondary structure that can occur in the library. They also indicate that the optimal coding strategy for a library depends on the complexity of the motif being characterized. Because this approach provides a simple way to generate libraries enriched for sequences with the potential to form a specific secondary structure, we anticipate that it should be useful for the optimization and structural characterization of functional nucleic acid motifs.
- MeSH
- aptamery nukleotidové genetika MeSH
- DNA katalytická genetika MeSH
- genová knihovna * MeSH
- konformace nukleové kyseliny MeSH
- mutageneze MeSH
- nukleotidové motivy genetika MeSH
- obrácené repetice genetika MeSH
- párování bází MeSH
- pravděpodobnost MeSH
- řízená evoluce molekul metody MeSH
- RNA katalytická genetika MeSH
- syntetická biologie metody MeSH
- techniky in vitro MeSH
- Publikační typ
- časopisecké články MeSH
Mechanisms of transcriptional control in malaria parasites are still not fully understood. The positioning patterns of G-quadruplex (G4) DNA motifs in the parasite's AT-rich genome, especially within the var gene family which encodes virulence factors, and in the vicinity of recombination hotspots, points towards a possible regulatory role of G4 in gene expression and genome stability. Here, we carried out the most comprehensive genome-wide survey, to date, of G4s in the Plasmodium falciparum genome using G4Hunter, which identifies G4 forming sequences (G4FS) considering their G-richness and G-skewness. We show an enrichment of G4FS in nucleosome-depleted regions and in the first exon of var genes, a pattern that is conserved within the closely related Laverania Plasmodium parasites. Under G4-stabilizing conditions, i.e., following treatment with pyridostatin (a high affinity G4 ligand), we show that a bona fide G4 found in the non-coding strand of var promoters modulates reporter gene expression. Furthermore, transcriptional profiling of pyridostatin-treated parasites, shows large scale perturbations, with deregulation affecting for instance the ApiAP2 family of transcription factors and genes involved in ribosome biogenesis. Overall, our study highlights G4s as important DNA secondary structures with a role in Plasmodium gene expression regulation, sub-telomeric recombination and var gene biology.
- MeSH
- aminochinoliny farmakologie MeSH
- G-kvadruplexy * MeSH
- genom účinky léků MeSH
- kyseliny pikolinové farmakologie MeSH
- lidé MeSH
- malárie farmakoterapie genetika parazitologie MeSH
- nukleotidové motivy genetika MeSH
- Plasmodium falciparum genetika patogenita MeSH
- promotorové oblasti (genetika) genetika MeSH
- regulace genové exprese účinky léků MeSH
- ribozomy účinky léků genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: The i-motif is a tetrameric DNA structure based on the formation of hemiprotonated cytosine-cytosine (C+.C) base pairs. i-motifs are widely used in nanotechnology. In biological systems, i-motifs are involved in gene regulation and in control of genome integrity. In vivo, the i-motif forming sequences are subjects of epigenetic modifications, particularly 5-cytosine methylation. In plants, natively occurring methylation patterns lead to a complex network of C+.C, 5mC+.C and 5mC+.5mC base-pairs in the i-motif stem. The impact of complex methylation patterns (CMPs) on i-motif formation propensity is currently unknown. METHODS: We employed CD and UV-absorption spectroscopies, native PAGE, thermal denaturation and quantum-chemical calculations to analyse the effects of native, native-like, and non-native CMPs in the i-motif stem on the i-motif stability and pKa. RESULTS: CMPs have strong influence on i-motif stability and pKa and influence these parameters in sequence-specific manner. In contrast to a general belief, i) CMPs do not invariably stabilize the i-motif, and ii) when the CMPs do stabilize the i-motif, the extent of the stabilization depends (in a complex manner) on the number and pattern of symmetric 5mC+.5mC or asymmetric 5mC+.C base pairs in the i-motif stem. CONCLUSIONS: CMPs can be effectively used to fine-tune i-motif properties. Our data support the notion of epigenetic modifications as a plausible control mechanism of i-motif formation in vivo. GENERAL SIGNIFICANCE: Our results have implications in epigenetic regulation of telomeric DNA in plants and highlight the potential and limitations of engineered patterning of cytosine methylations on the i-motif scaffold in nanotechnological applications.
- MeSH
- cytosin metabolismus MeSH
- DNA rostlinná chemie genetika MeSH
- epigeneze genetická * MeSH
- metylace DNA * MeSH
- molekulární modely MeSH
- nanotechnologie * MeSH
- nukleotidové motivy genetika MeSH
- sekvence nukleotidů MeSH
- telomery genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Single nucleotide polymorphisms located in 5' untranslated regions (5'UTRs) can regulate gene expression and have clinical impact. Recognition of functionally significant sequences within 5'UTRs is crucial in next-generation sequencing applications. Furthermore, information about the behavior of 5'UTRs during gene evolution is scarce. Using the example of the ATP-binding cassette transporter A1 (
- MeSH
- 5' nepřekládaná oblast * genetika MeSH
- ABCA1 protein chemie genetika MeSH
- anotace sekvence MeSH
- fylogeneze MeSH
- introny MeSH
- konformace nukleové kyseliny MeSH
- konzervovaná sekvence genetika MeSH
- lidé MeSH
- messenger RNA genetika metabolismus MeSH
- nukleotidové motivy genetika MeSH
- otevřené čtecí rámce genetika MeSH
- savci genetika MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- sestřih RNA genetika MeSH
- zastoupení bazí genetika MeSH
- zesilovače transkripce genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
Triplex DNA is implicated in a wide range of biological activities, including regulation of gene expression and genomic instability leading to cancer. The tumor suppressor p53 is a central regulator of cell fate in response to different type of insults. Sequence and structure specific modes of DNA recognition are core attributes of the p53 protein. The focus of this work is the structure-specific binding of p53 to DNA containing triplex-forming sequences in vitro and in cells and the effect on p53-driven transcription. This is the first DNA binding study of full-length p53 and its deletion variants to both intermolecular and intramolecular T.A.T triplexes. We demonstrate that the interaction of p53 with intermolecular T.A.T triplex is comparable to the recognition of CTG-hairpin non-B DNA structure. Using deletion mutants we determined the C-terminal DNA binding domain of p53 to be crucial for triplex recognition. Furthermore, strong p53 recognition of intramolecular T.A.T triplexes (H-DNA), stabilized by negative superhelicity in plasmid DNA, was detected by competition and immunoprecipitation experiments, and visualized by AFM. Moreover, chromatin immunoprecipitation revealed p53 binding T.A.T forming sequence in vivo. Enhanced reporter transactivation by p53 on insertion of triplex forming sequence into plasmid with p53 consensus sequence was observed by luciferase reporter assays. In-silico scan of human regulatory regions for the simultaneous presence of both consensus sequence and T.A.T motifs identified a set of candidate p53 target genes and p53-dependent activation of several of them (ABCG5, ENOX1, INSR, MCC, NFAT5) was confirmed by RT-qPCR. Our results show that T.A.T triplex comprises a new class of p53 binding sites targeted by p53 in a DNA structure-dependent mode in vitro and in cells. The contribution of p53 DNA structure-dependent binding to the regulation of transcription is discussed.
- MeSH
- aktivace transkripce genetika MeSH
- DNA vazebné proteiny chemie genetika MeSH
- DNA chemie genetika MeSH
- konformace nukleové kyseliny MeSH
- lidé MeSH
- nádorový supresorový protein p53 chemie genetika MeSH
- nukleotidové motivy genetika MeSH
- plazmidy genetika MeSH
- promotorové oblasti (genetika) MeSH
- regulační oblasti nukleových kyselin genetika MeSH
- sekvenční delece genetika MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Telomeres are nucleoprotein complexes protecting the physical ends of linear eukaryotic chromosomes and therefore helping to ensure their stability and integrity. Additionally, telomeric sequences can be localized in non-terminal regions of chromosomes, forming so-called interstitial telomeric sequences (ITSs). ITSs are traditionally considered to be relics of chromosomal rearrangements and thus very informative in the reconstruction of the evolutionary history of karyotype formation. We examined the distribution of the telomeric motifs (TTAGGG)n using fluorescence in situ hybridization (FISH) in 30 species, representing 17 families of squamate reptiles, and compared them with the collected data from another 38 species from literature. Out of the 68 squamate species analyzed, 35 possess ITSs in pericentromeric regions, centromeric regions and/or within chromosome arms. We conclude that the occurrence of ITSs is rather common in squamates, despite their generally conserved karyotypes, suggesting frequent and independent cryptic chromosomal rearrangements in this vertebrate group.
Cyphellophora and Phialophora (Chaetothyriales, Pezizomycota) comprise species known from skin infections of humans and animals and from a variety of environmental sources. These fungi were studied based on the comparison of cultural and morphological features and phylogenetic analyses of five nuclear loci, i.e., internal transcribed spacer rDNA operon (ITS), large and small subunit nuclear ribosomal DNA (nuc28S rDNA, nuc18S rDNA), β-tubulin, DNA replication licensing factor (mcm7) and second largest subunit of RNA polymerase II (rpb2). Phylogenetic results were supported by comparative analysis of ITS1 and ITS2 secondary structure of representatives of the Chaetothyriales and the identification of substitutions among the taxa analyzed. Base pairs with non-conserved, co-evolving nucleotides that maintain base pairing in the RNA transcript and unique evolutionary motifs in the ITS2 that characterize whole clades or individual taxa were mapped on predicted secondary structure models. Morphological characteristics, structural data and phylogenetic analyses of three datasets, i.e., ITS, ITS-β-tubulin and 28S-18S-rpb2-mcm7, define a robust clade containing eight species of Cyphellophora (including the type) and six species of Phialophora. These taxa are now accommodated in the Cyphellophoraceae, a novel evolutionary lineage within the Chaetothyriales. Cyphellophora is emended and expanded to encompass species with both septate and nonseptate conidia formed on discrete, intercalary, terminal or lateral phialides. Six new combinations in Cyphellophora are proposed and a dichotomous key to species accepted in the genus is provided. Cyphellophora eugeniae and C. hylomeconis, which grouped in the Chaetothyriaceae, represent another novel lineage and are introduced as the type species of separate genera.
- MeSH
- Ascomycota genetika MeSH
- fylogeneze * MeSH
- genetické lokusy genetika MeSH
- geny hub genetika MeSH
- konformace nukleové kyseliny * MeSH
- konsenzuální sekvence MeSH
- mezerníky ribozomální DNA chemie genetika MeSH
- molekulární evoluce * MeSH
- molekulární sekvence - údaje MeSH
- nukleotidové motivy genetika MeSH
- RNA ribozomální chemie genetika MeSH
- sekvence nukleotidů MeSH
- spory hub cytologie MeSH
- tubulin genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
