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Nucleic acid motifs consist of conserved and variable nucleotide regions. For functional action, several motifs are combined to modules. The tool AIModules allows identification of such motifs including combinations of them and conservation in several nucleic acid stretches. AIModules recognizes conserved motifs and combinations of motifs (modules) allowing a number of interesting biological applications such as analysis of promoter and transcription factor binding sites (TFBS), identification of conserved modules shared between several gene families, e.g. promoter regions, but also analysis of shared and conserved other DNA motifs such as enhancers and silencers, in mRNA (motifs or regulatory elements e.g. for polyadenylation) and lncRNAs. The tool AIModules presented here is an integrated solution for motif analysis, offered as a Web service as well as downloadable software. Several nucleotide sequences are queried for TFBSs using predefined matrices from the JASPAR DB or by using one’s own matrices for diverse types of DNA or RNA motif discovery. Furthermore, AIModules can find TFBSs common to two or more sequences. Demanding high or low conservation, AIModules outperforms other solutions in speed and finds more modules (specific combinations of TFBS) than alternative available software. The application also searches RNA motifs such as polyadenylation site or RNA–protein binding motifs as well as DNA motifs such as enhancers as well as user-specified motif combinations (https://bioinfo-wuerz.de/aimodules/; alternative entry pages: https://aimodules.heinzelab.de or https://www.biozentrum.uni-wuerzburg.de/bioinfo/computing/aimodules). The application is free and open source whether used online, on-site, or locally.
Effect of inhalation exposure regimen on DNA binding potency of 1,2-dichloroethane in the rat
(1991)
1 ,2-Dichloroethane (DCE) was reported to be carcinogenic in rats in a long-tenn bioassay using gavage in com oil (24 and 48 mg/kg/day), but not by inhalation (up to 150-250 ppm, 7 h/day, 5 days/week). The daily dose metabolized was similar in the two experiments. In order to address this discrepancy, the genotoxicity of DCE was investigated in vivo under different exposure conditions. Fernale F-344 rats (183-188 g) were exposed to [1,2-14C]DCE in a closed inhalation chamber to either a low, constant concentration (0.3 mg/l = 80 ppm for 4 h) or to a peak concentration (up to 18 mg/1 = 4400 ppm) for a few minutes. After 12 h in the chamber, the dose metabolized under the two conditions was 34 mg/kg and 140 mg/k:g. DNA was isolated from liver and lung and was purified to constant specific radioactivity. DNA was enzymaticaBy hydrolyzed to the 3' -nucleotides which were separated by reverse phase HPLC. Most radioactivity eluted without detectable or with little optical density' indicating that the major part of the DNA radioactivity was due to covalent binding of the test compound. The Ievel of DNA adducts was expressed in the dose-nonnalized units ofthe Covalent Binding Index, CBI = f.Lmol adduct per mol DNA nucleotide/ mmol DCE per kg body wt. In liver DNA, the different exposure regimens resulted in markedly different CBI values of 1.8 and 69, for "constant-low" and ''peak" DCE exposure Ievels. In the Jung, the respective values were 0.9 and 31. It is concluded that the DNA darnage by DCE depends upon the concentration-time profile and that the carcinogenic potency determined in the gavage study should not be used for low-Ievel inhalation exposure.
Natural DNA storage allows cellular differentiation, evolution, the growth of our children and controls all our ecosystems. Here, we discuss the fundamental aspects of DNA storage and recent advances in this field, with special emphasis on natural processes and solutions that can be exploited. We point out new ways of efficient DNA and nucleotide storage that are inspired by nature. Within a few years DNA-based information storage may become an attractive and natural complementation to current electronic data storage systems. We discuss rapid and directed access (e.g. DNA elements such as promotors, enhancers), regulatory signals and modulation (e.g. lncRNA) as well as integrated high-density storage and processing modules (e.g. chromosomal territories). There is pragmatic DNA storage for use in biotechnology and human genetics. We examine DNA storage as an approach for synthetic biology (e.g. light-controlled nucleotide processing enzymes). The natural polymers of DNA and RNA offer much for direct storage operations (read-in, read-out, access control). The inbuilt parallelism (many molecules at many places working at the same time) is important for fast processing of information. Using biology concepts from chromosomal storage, nucleic acid processing as well as polymer material sciences such as electronical effects in enzymes, graphene, nanocellulose up to DNA macramé , DNA wires and DNA-based aptamer field effect transistors will open up new applications gradually replacing classical information storage methods in ever more areas over time (decades).
Introduction:
Individuals carrying pathogenic mutations in the BRCA1 and BRCA2 genes have a high lifetime risk of breast cancer. BRCA1 and BRCA2 are involved in DNA double-strand break repair, DNA alterations that can be caused by exposure to reactive oxygen species, a main source of which are mitochondria. Mitochondrial genome variations affect electron transport chain efficiency and reactive oxygen species production. Individuals with different mitochondrial haplogroups differ in their metabolism and sensitivity to oxidative stress. Variability in mitochondrial genetic background can alter reactive oxygen species production, leading to cancer risk. In the present study, we tested the hypothesis that mitochondrial haplogroups modify breast cancer risk in BRCA1/2 mutation carriers.
Methods:
We genotyped 22,214 (11,421 affected, 10,793 unaffected) mutation carriers belonging to the Consortium of Investigators of Modifiers of BRCA1/2 for 129 mitochondrial polymorphisms using the iCOGS array. Haplogroup inference and association detection were performed using a phylogenetic approach. ALTree was applied to explore the reference mitochondrial evolutionary tree and detect subclades enriched in affected or unaffected individuals.
Results:
We discovered that subclade T1a1 was depleted in affected BRCA2 mutation carriers compared with the rest of clade T (hazard ratio (HR) = 0.55; 95% confidence interval (CI), 0.34 to 0.88; P = 0.01). Compared with the most frequent haplogroup in the general population (that is, H and T clades), the T1a1 haplogroup has a HR of 0.62 (95% CI, 0.40 to 0.95; P = 0.03). We also identified three potential susceptibility loci, including G13708A/rs28359178, which has demonstrated an inverse association with familial breast cancer risk.
Conclusions:
This study illustrates how original approaches such as the phylogeny-based method we used can empower classical molecular epidemiological studies aimed at identifying association or risk modification effects.
Background:
Chloroplast-encoded genes (matK and rbcL) have been formally proposed for use in DNA barcoding efforts targeting embryophytes. Extending such a protocol to chlorophytan green algae, though, is fraught with problems including non homology (matK) and heterogeneity that prevents the creation of a universal PCR toolkit (rbcL). Some have advocated the use of the nuclear-encoded, internal transcribed spacer two (ITS2) as an alternative to the traditional chloroplast markers. However, the ITS2 is broadly perceived to be insufficiently conserved or to be confounded by introgression or biparental inheritance patterns, precluding its broad use in phylogenetic reconstruction or as a DNA barcode. A growing body of evidence has shown that simultaneous analysis of nucleotide data with secondary structure information can overcome at least some of the limitations of ITS2. The goal of this investigation was to assess the feasibility of an automated, sequence-structure approach for analysis of IT2 data from a large sampling of phylum Chlorophyta.
Methodology/Principal Findings:
Sequences and secondary structures from 591 chlorophycean, 741 trebouxiophycean and 938 ulvophycean algae, all obtained from the ITS2 Database, were aligned using a sequence structure-specific scoring matrix. Phylogenetic relationships were reconstructed by Profile Neighbor-Joining coupled with a sequence structure-specific, general time reversible substitution model. Results from analyses of the ITS2 data were robust at multiple nodes and showed considerable congruence with results from published phylogenetic analyses.
Conclusions/Significance:
Our observations on the power of automated, sequence-structure analyses of ITS2 to reconstruct phylum-level phylogenies of the green algae validate this approach to assessing diversity for large sets of chlorophytan taxa. Moreover, our results indicate that objections to the use of ITS2 for DNA barcoding should be weighed against the utility of an automated, data analysis approach with demonstrated power to reconstruct evolutionary patterns for highly divergent lineages.
The covalent binding of [3H]aflatoxin B1 (AF) to liver DNA was determined, 6 h after oral administration to male rabbits. A Covalent Binding Index, CBI (flmol AF/mol DNA-P)/(mmol AF/kg b. w.) = 8,500 was found. Pretreatment of rabbits with AF coupled to bovine serum albumin in Freund's adjuvant led to the production of AF-directed antibodies. Administration of [3H]AF to such immunized rabbits resulted in a CJH of only 2,500, i.e., the iiDJ{.lUnization provided a protection by a factor of more than 3. Although this is encouraging evidence for the potential of active immunization against genotoxic carcinogens, a nurober of pointswill have to be clarified, such as the time course for the DNA binding and the question of a possible shift to other target cells.
DPF3 (BAF45c) is a member of the BAF chromatin remodeling complex. Two isoforms have been described, namely DPF3a and DPF3b. The latter binds to acetylated and methylated lysine residues of histones. Here, we elaborate on the role of DPF3a and describe a novel pathway of cardiac gene transcription leading to pathological cardiac hypertrophy. Upon hypertrophic stimuli, casein kinase 2 phosphorylates DPF3a at serine 348. This initiates the interaction of DPF3a with the transcriptional repressors HEY, followed by the release of HEY from the DNA. Moreover, BRG1 is bound by DPF3a, and is thus recruited to HEY genomic targets upon interaction of the two components. Consequently, the transcription of downstream targets such as NPPA and GATA4 is initiated and pathological cardiac hypertrophy is established. In human, DPF3a is significantly up-regulated in hypertrophic hearts of patients with hypertrophic cardiomyopathy or aortic stenosis. Taken together, we show that activation of DPF3a upon hypertrophic stimuli switches cardiac fetal gene expression from being silenced by HEY to being activated by BRG1. Thus, we present a novel pathway for pathological cardiac hypertrophy, whose inhibition is a long-term therapeutic goal for the treatment of the course of heart failure.
The self-assembly of molecules based on π-π-interactions and hydrogen bonding is of significant importance in nature. These processes enable the formation of complex supramolecular structures with diverse functions. For the transfer of the concepts from nature to artificial supramolecular structures, a basic understanding of those processes is needed. For this purpose, π-conjugated aromatic molecules with an easy synthetic access are suitable as their functionalities can be changed effortless. Perylene bisimide (PBIs) dyes are attractive candidates since they fulfill these requirements owing to their tendency to self-assemble in solution due to their large aromatic π-surfaces. Furthermore, the changes of the optical properties (for instance absorption, emission or circular dichroism) of PBI dyes, caused by their self-assembly, are easy to study experimentally. Structural variations of PBI dyes including additional non-covalent interactions, such as hydro-gen bonding, enable to direct their self-assembly process. Thus, the formation of interesting su-pramolecular structures of PBI dyes could be realized, although, often of undefined size. The aim of this thesis was to develop strategies to restrict the aggregate size of PBI dyes. Therefore, de-fined structural features of PBI molecules were combined and a variation of external influences such as solvent and concentration included. Furthermore, DNA was utilized as a template for the limitation of the aggregate size of PBI dyes.
Chapters 1 and 2 provide general information and describe examples from literature which are necessary to understand the following experimental work. The first chapter is based on the inter-actions of various molecules with DNA. Therefore, DNA is considered as a supramolecular biom-acromolecule containing specific structural and functional features to interact with small mole-cules. Afterwards, the main interaction modes of small molecules with DNA such as electrostatic interaction, intercalation and groove binding with corresponding examples are discussed. Among all techniques applied to study the interaction of ligands with DNA, UV/Vis absorption, fluores-cence and circular dichroism spectroscopy were described in detail. At the end of this chapter, examples of already pre-associated systems showing interactions with DNA are presented.
The second chapter is focused on the determination and mathematic evaluation of the self-assembly processes. The simplest models such as monomer-dimer and isodesmic model are de-scribed and supplemented by examples. Furthermore, the simplest modification of the isodesmic model, the K2-K model, is presented. Additionally, experimental problems, which may arise dur-ing the investigations of the self-assembly processes, are addressed. For the description of the entire self-assembly process, a sufficiently large concentration range and an appropriate measure-ment method that is sensitive in this concentration range is necessary. Furthermore, the full transi-tion from the monomeric to the aggregated species has to be spectroscopically ascertainable. This enables an accurate mathematic evaluation of the self-assembly process and provides meaningful binding constants. The self-assembly pathway can be controlled by the variation of solvent, con-centration or temperature. However, this pathway can also be directed by a rational design of the molecular structure of the considered system. For example, a specific interplay of π-π-interactions and hydrogen bonding may promote isodesmic as well as cooperative growth into large struc-tures.
The main focus of this thesis is to develop strategies to control the aggregate size of PBI dyes (Chapter 3). For this purpose, a PBI scaffold was designed which contains hydrogen bonding amide functions at the imide positions derived from the amino acid L-alanine and solubilizing side groups in the periphery (Figure 81). The variations of the residues R/R’ range from didodecylox-yphenyl, didodecylphenyl, dioligo(ethylene glycol)phenyl to branched and linear alkyl chains.
The most extensive study of the aggregation behavior was performed for the PBI dye 5. Concen-tration-dependent 1H NMR and UV/Vis absorption measurements clearly revealed the formation of dimers in chloroform. Further investigations by means of 2D NMR, VPO and ITC confirmed the exclusive presence of dimer aggregates of PBI 5 in the investigated concentration range. Mo-lecular modelling studies, supported by NMR and FT-IR experiments, provided structural reasons for the absence of further growth into larger aggregates. The specific combination of π-π interac-tions and hydrogen bonds between the NH groups of the amide groups and the carbonyl oxygen atoms of the PBI core are decisive for the formation of the discrete dimer stack (see Figure 82). The investigations of the aggregation behavior of PBIs 6-9 were less extensive but consistent with the results obtained for PBI 5. However, the determined binding constants vary over a considera-ble range of 1.1 x 102 M-1 (PBI 8) to 1.4 x 104 M-1 (PBI 5). These differences could be attributed to structural variations of the dyes. The electron-rich phenyl substituent promoted the aggregation tendency of PBIs 5-7 compared with 8 and 9 that carry only alkyl side chains. Thus, the π-π in-teractions of bay-unsubstituted PBI cores in combination with hydrogen bonding of the amide functions control the formation of discrete dimers of these PBI dyes.
The variation of conditions, such as solvent, change the aggregation behavior of PBI dyes. In the solvents toluene and/or methylcyclohexane, anti-cooperative growth into larger aggregates of PBI 5 was observed (Chapter 4). The important feature of this self-assembly process is the absence of isosbestic points over the whole concentration range in the UV/Vis absorption measurements. The preference for the dimeric species of PBI 5 remained in both solvents as well as in mixtures of them, but upon increasing the concentration these dimers self-assemble into larger aggregates.
An important feature of the self-assembly process is the preferred formation of even-numbered aggregates compared to the odd-numbered ones (see Figure 83). Although, the conventional K2-K model provides plausible binding constants, it is not capable to describe the aggregation behavior adequately, since it considers a continuous size distribution. The gradual aggregation process over dimers, tetramers, hexamers, etc. was therefore analyzed with a newly developed K2-K model for anti-cooperative supramolecular polymerization. By the global analysis of the UV/Vis absorption spectra a very good agreement between the experimental and simulated spectra, which were based on the new K2-K model, was obtained. Furthermore, the calculated UV/Vis absorption spectra of a dimer and an aggregate highlighted the most important structural differences. The absorption spectrum of the dimer still has a pronounced vibronic structure which gets lost in the spectrum of the aggregate.
In another part of this work, a series of water soluble PBI dyes were described which contain similar PBI scaffolds as PBIs 5-8 (Chapter 5). These PBI dyes self-assemble into similar dimer aggregates in water due to their positively charged side chains causing electrostatic repulsion be-tween the molecules (see Figure 84). Here, however, the self-assembly behavior has not been studied thoroughly in water due to the similarities of already reported PBI dyes.
Instead, the focus here is on the characterization of the interactions of these dyes with DNA/RNA. The comprehensive studies using thermal denaturation experiments showed the high stability of these PBI/polynucleotide complexes. The spermine-functionalized PBI dyes having six positive charges showed strong interactions with DNA/RNA which was expressed in a signif-icant increase of the melting temperatures of DNA/RNA (ΔTm values between 7 and > 35 ° C). The dioxa analogues containing only two positive charges had lower enhancement of the melting temperature of DNA/RNA (ΔTm values between 3 and 30 ° C). A similar trend has been observed in the fluorimetric titrations. The spermine-functionalized PBI dyes showed high binding con-stants (log Ks = 9.2 - 9.8), independently of the used polynucleotides. In contrast, the dioxa ana-logues displayed smaller binding constants (log Ks = 6.5 - 7.9) without any correlation between binding affinity and binding strength of the PBI dyes and the applied polynucleotides. The CD-spectroscopic measurements revealed significant differences in the binding properties of the dyes with DNA/RNA. They were dependent on the steric hindrance of the amino acid residues at the imide position and their configuration on one side and the grooves properties of ds-DNA/RNA on the other side. The spectroscopic results confirmed the formation of excitonically coupled PBI dimers in the minor groove of ds-DNA and the major groove of ds-RNA. Depending on the se-quence, the grooves of the polynucleotides provide different amount of space for embedding molecules. The guanine amino groups protrude into the minor groove of the polynucleotide poly(dG-dC)2 increasing the steric hindrance, which is not the case for poly(dA-dT)2. Molecular modeling studies showed that the PBI dimers penetrate deeper into the groove of poly(dA-dT)2 due to the absence of the steric hindrance, in comparison to the groove of poly(dG-dC)2 (see Figure 85).
Male rats were treated for 2 months with 1000 ppm nafenopin in the diet or for 4 or 7 days with a choline-devoid low-methionine diet. DNA was isolated from the livers and analyzed for the presence of cis-thymidine glycol-3'-phosphate (cis-dTGp) by 32P-postlabeling and for the Ievel of 8-hydroxy-deoxyguanosine (8-0H-dG) by electrochemical detection (ECD). In no DNA sample was the Ievel of cis-dTGp above the Iimit of detection of 1 modified thymidine per 106 nucleotides. With 8-0H-dG, a background Ievel of this modification of 20 8-0H-dG per 106 nucleosides was found in liver DNA of control rats, which was not affected by either treatment. It is postulated for thymidine glycol that a potential increase was below the Iimit of detection or was rapidly repaired in vivo and that the steady-state Ievel of endogenous 8-hydroxydeoxyguanosine appears not tobe influenced by the treatments chosen.
Pigment cells and neuronal cells both are derived from the neural crest. Here, we describe the Pit-Oct-Unc (POU) domain transcription factor Brn3a, normally involved in neuronal development, to be frequently expressed in melanoma, but not in melanocytes and nevi. RNAi-mediated silencing of Brn3a strongly reduced the viability of melanoma cell lines and decreased tumour growth in vivo. In melanoma cell lines, inhibition of Brn3a caused DNA double-strand breaks as evidenced by Mre11/Rad50-containing nuclear foci. Activated DNA damage signalling caused stabilization of the tumour suppressor p53, which resulted in cell cycle arrest and apoptosis. When Brn3a was ectopically expressed in primary melanocytes and fibroblasts, anchorage-independent growth was increased. In tumourigenic melanocytes and fibroblasts, Brn3a accelerated tumour growth in vivo. Furthermore, Brn3a cooperated with proliferation pathways such as oncogenic BRAF, by reducing oncogene-induced senescence in non-malignant melanocytes. Together, these results identify Brn3a as a new factor in melanoma that is essential for melanoma cell survival and that promotes melanocytic transformation and tumourigenesis.
Mechanistic possibilitles responsible for nonlinear shapes of the dose-response relationship in chemical carcinogenesis are discussed. (i) Induction and saturation of enzymatic activation and detoxification processes and of DNA repair affect the relationship between dose and steady-state DNA adduct Ievel; (ii) The fixation of DNA adducts in the form of mutations is accelerated by stimulation of the cell division, for Jnstance due to regenerative hyperplasia at cytotoxic dose Ievels; (iii) The rate of tumor formation results from a superposition of the rates of the individual steps. It can become exponential with dose if more than one step is accelerated by the DNA damage exerted by the genotoxic carcinogen. The strongly sigmoidal shapes often observed for dose-tumor incidence relationships in animal bioassays supports this analysis. A power of four for the dose in the su~linear part of the curve is the maximum observed (formaldehyde). In contrast to animal experiments, epidemiological data ln humans rarely show a slgnificant deviation from linearity. The discrepancy might be explained by the fact that a I arge nu mber of genes contribute to the overall sensitivity of an individual and to the respective heterogeneity within the human population. Mechanistic nonlinearities are flattened out in the presence of genetic and life-style factors which affect the sensitivity for the development of cancer. For a risk assessment, linear extrapolation from the high-dose lncidence to the spontaneaus rate can therefore be approprlate in a heterogeneous population even if the mechanism of action would result in a nonlinear shape of the dose-response curve in a homogeneaus population.
Male Fischer F-344 rats were given ethanol in the drinking water and/or by single oral administration. Following this, the animals received p.o. 100 ng/kg of the hepatocarcinogen eHJaflatoxin BI (AFBI)' 24 h later, the level of DNA-bound AFBI was determined in the liver and was found not to be affected by any type of ethanol pretreatment. A cocarcinogenic effect of ethanol in the liver is therefore unlikely to be due to an effect on the metabolic activation and inactivation processes governing the formation of DNA-binding AFBI metabolites.
In aqueous environment, hydrophobic interactions play an important role for DNA. The introduction of modifications based on hydrophobic aromatic moieties offers additional ways for controlling recognition and reactivity of functional groups in DNA. Modifications are introduced through an artificial backbone or in the form of an extension of the nucleobases, resulting in additional properties of the DNA.
This dissertation focuses on the use of hydrophobic units for the functionalization of DNA.
In the first part of the work, the tolane (i. e. diphenylacetylene) motif was used in combination with the acyclic backbone of GNA and BuNA to generate recognition units in the DNA context. Fluorination of the aromatic rings in the tolane moiety provided the basis for a supramolecular language based on arene-fluoroarene interactions. The specific recognition was investigated by thermodynamic, kinetic and NMR spectroscopic methods.
In the second part of the work, deoxyuridine derivatives with a hydrophobic aromatic modification were prepared and incorporated into DNA duplexes. The irradiation with UV light led to a [2+2] cycloaddition reaction between two modified nucleosides in the DNA. This reaction product was structurally characterized and the reaction was used in various biochemical and nanotechnological DNA applications.
Arene‐fluoroarene interactions offer outstanding possibilities for engineering of supramolecular systems, including nucleic acids. Here, we implement the tolane‐perfluorotolane interaction as base pair replacement in DNA. Tolane (THH) and perfluorotolane (TFF) moieties were connected to acyclic backbone units, comprising glycol nucleic acid (GNA) or butyl nucleic acid (BuNA) building blocks, that were incorporated via phosphoramidite chemistry at opposite positions in a DNA duplex. Thermodynamic analyses by UV thermal melting revealed a compelling stabilization by THH/TFF heteropairs only when connected to the BuNA backbone, but not with the shorter GNA linker. Detailed NMR studies confirmed the preference of the BuNA backbone for enhanced polar π‐stacking. This work defines how orthogonal supramolecular interactions can be tailored by small constitutional changes in the DNA backbone, and it inspires future studies of arene‐fluoroarene‐programmed assembly of DNA.
The genomes of kinetoplastids are organized into polycistronic gene clusters that are flanked by the modified DNA base J. Previous work has established a role of base J in promoting RNA polymerase II termination in Leishmania spp. where the loss of J leads to termination defects and transcription into adjacent gene clusters. It remains unclear whether these termination defects affect gene expression and whether read through transcription is detrimental to cell growth, thus explaining the essential nature of J. We now demonstrate that reduction of base J at specific sites within polycistronic gene clusters in L. major leads to read through transcription and increased expression of downstream genes in the cluster. Interestingly, subsequent transcription into the opposing polycistronic gene cluster does not lead to downregulation of sense mRNAs. These findings indicate a conserved role for J regulating transcription termination and expression of genes within polycistronic gene clusters in trypanosomatids. In contrast to the expectations often attributed to opposing transcription, the essential nature of J in Leishmania spp. is related to its role in gene repression rather than preventing transcriptional interference resulting from read through and dual strand transcription.
Human herpesvirus 6A (HHV-6A) replicates in peripheral blood mononuclear cells (PBMCs) and various T-cell lines in vitro. Intriguingly, the virus can also establish latency in these cells, but it remains unknown what influences the decision between lytic replication and the latency of the virus. Incoming virus genomes are confronted with the nuclear domain 10 (ND10) complex as part of an intrinsic antiviral response. Most herpesviruses can efficiently subvert ND10, but its role in HHV-6A infection remains poorly understood. In this study, we investigated if the ND10 complex affects HHV-6A replication and contributes to the silencing of the virus genome during latency. We could demonstrate that ND10 complex was not dissociated upon infection, while the number of ND10 bodies was reduced in lytically infected cells. Virus replication was significantly enhanced upon knock down of the ND10 complex using shRNAs against its major constituents promyelocytic leukemia protein (PML), hDaxx, and Sp100. In addition, we could demonstrate that viral genes are more efficiently silenced in the presence of a functional ND10 complex. Our data thereby provides the first evidence that the cellular ND10 complex plays an important role in suppressing HHV-6A lytic replication and the silencing of the virus genome in latently infected cells.
Prenatal stress-induced programming of genome-wide promoter DNA methylation in 5-HTT-deficient mice
(2014)
The serotonin transporter gene (5-HTT/SLC6A4)-linked polymorphic region has been suggested to have a modulatory role in mediating effects of early-life stress exposure on psychopathology rendering carriers of the low-expression short (s)-variant more vulnerable to environmental adversity in later life. The underlying molecular mechanisms of this gene-by-environment interaction are not well understood, but epigenetic regulation including differential DNA methylation has been postulated to have a critical role. Recently, we used a maternal restraint stress paradigm of prenatal stress (PS) in 5-HTT-deficient mice and showed that the effects on behavior and gene expression were particularly marked in the hippocampus of female 5-Htt+/- offspring. Here, we examined to which extent these effects are mediated by differential methylation of DNA. For this purpose, we performed a genome-wide hippocampal DNA methylation screening using methylated-DNA immunoprecipitation (MeDIP) on Affymetrix GeneChip Mouse Promoter 1.0 R arrays. Using hippocampal DNA from the same mice as assessed before enabled us to correlate gene-specific DNA methylation, mRNA expression and behavior. We found that 5-Htt genotype, PS and their interaction differentially affected the DNA methylation signature of numerous genes, a subset of which showed overlap with the expression profiles of the corresponding transcripts. For example, a differentially methylated region in the gene encoding myelin basic protein (Mbp) was associated with its expression in a 5-Htt-, PS- and 5-Htt × PS-dependent manner. Subsequent fine-mapping of this Mbp locus linked the methylation status of two specific CpG sites to Mbp expression and anxiety-related behavior. In conclusion, hippocampal DNA methylation patterns and expression profiles of female prenatally stressed 5-Htt+/- mice suggest that distinct molecular mechanisms, some of which are promoter methylation-dependent, contribute to the behavioral effects of the 5-Htt genotype, PS exposure and their interaction.
Nucleic acids are one of the important classes of biomolecules together with carbohydrates, proteins and lipids. Both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are most well known for their respective roles in the storage and expression of genetic information.
Over the course of the last decades, nucleic acids with a variety of other functions have been discovered in biological organisms or created artificially. Examples of these functional nucleic acids are riboswitches, aptamers and ribozymes. In order to gain information regarding their function, several analytical methods can be used.
Electron paramagnetic resonance (EPR) spectroscopy is one of several techniques which can be used to study nucleic acid structure and dynamics. However, EPR spectroscopy requires unpaired electrons and because nucleic acids themselves are not paramagnetic, the incorporation of spin labels which carry a radical is necessary.
Here, three new spin labels for the analysis of nucleic acids by EPR spectroscopy are presented. All of them share two important design features. First, the paramagnetic center is located at a nitroxide, flanked by ethyl groups to prevent nitroxide degradation, for example during solid phase synthesis. Furthermore, they were designed with rigidity as an important quality, in order to be useful for applications like pulsed electron double resonance (PELDOR) spectroscopy, where independent motion of the spin labels relative to the macromolecule has a noticeable negative effect on the precision of the measurements.
Benzi-spin is a spin label which differs from most previous examples of rigid spin labels in that rather than being based on a canonical nucleoside, with a specific base pairing partner, it is supposed to be a universal nucleoside which is sufficiently rigid for EPR measurements when placed opposite to a number of different nucleosides. Benzi-spin was successfully incorporated into a 20 nt oligonucleotide and its base pairing behavior with seven different nucleosides was examined by UV/VIS thermal denaturation and continuous wave (CW) EPR experiments. The results show only minor differences between the different nucleosides, thus confirming the ability of benzi-spin to act as a universally applicable spin label.
Lumi-spin is derived from lumichrome. It features a rigid scaffold, as well as a free 2'-hydroxy group, which should make it well suited for PELDOR experiments once it is incorporated into RNA oligonucleotides.
EÇr is based on the Ç family of spin labels, which contains the most well known rigid spin labels for nucleic acids to this day. It is essentially a version of EÇm with a free 2'-hydroxy group. It was converted to triphosphate EÇrTP and used for primer extension experiments to test the viability of enzymatic incorporation of rigid spin labels into oligonucleotides as an alternative to solid-phase synthesis. Incorporation into DNA by Therminator III DNA polymerase in both single-nucleotide and full-length primer extensions was achieved.
All three of these spin labels represent further additions to the expanding toolbox of EPR spectroscopy on nucleic acids and might prove valuable for future research.
In order to investigate the DNA localization within Ehrlich tumor cell nucleoli during mitosis, two recent immunocytochemical methods using either an anti-DNA or an anti-bromodeoxyuridine (BrdU) monoclonal antibody have been applied. In both cases, the immunogold labeling has been performed on ultrathin sections of cells embedded either in Lowicryl K4M or in Epon, respectively. Identical results are observed with both immunocytochemical approaches. In the interphase nucleolus, besides the labeling of the perinucleolar chromatin shell and of its intranucleolar invaginations which penetrate into the nucleolar body and often terminate at the fibrillar centers, a few gold particles are also preferentially found towards the peripheral region of the fibrillar centers. In contrast, the dense fibrillar component and the granular component are never labeled. During mitosis, the fibrillar centers persist at the chromosomal nucleolus organizing regions (NOR's) and can be selectively stained by the silver method. However, these metaphase fibrillar centers are no longer decorated by the DNA- or BrdU antibodies. These results indicate that until the end of prophase, rRNA genes are present inside the fibrillar center material, disappear during metaphase and reappear in reconstituting nucleoli during telophase. Thus, fibrillar centers appear to represent structures sui generis, which are populated by rRNA genes only when the nucleolus is functionally active. In segregated nucleoli after actinomycin D treatment, the DNA labeling is exclusively restricted to the perinucleolar chromatin blocks. These findings also suggest that the DNA content of the fibrillar center material varies according to the rRNA transcription level of the cells. The results are discussed in the light of the present knowledge of the functional organization of the nucleolus.
The distribution of DNA in Ehrlich tumour cell nucleoli was investigated by means of an immunocytochemical approach , involving a monoclonal antibody directed against double- and single-stranded DNA. Immunolabelling was performed . either before or after the embedding process. The postembedding labelling method allows better ultrastructural preservation than the preembedding labelling method. In particular, the various nucleolar components are well preserved and identifiable. In the nucleolus, labelling is particularly concentrated over the perinucleolar chromatin and over its intranucleolar invaginations, which penetrate the nucleolar body and often terminate at the fibrillar centres. In addition, aggregates of gold particles are found in the fibrillar centres, preferentially towards the peripheral regions. By contrast, the dense fibrillar component is completely devoid of labelling. The results seem to indicate that DNA containing the rDNA genes is located in the fibrillar centres, with a preference for the peripheral regions. This finding suggests that transcription of the rDNA genes should occur within the confines of the fibrillar centre, probably close to the boundary region of the surrounding dense fibrillar component. The results are discussed in the light of present knowledge of the functional organization of the nucleolus.