Sequence Context Research Articles

The identification of the methylation patterns of tomato curly stunt virus in resistant and susceptible tomato lines.

Tomato curly stunt virus (ToCSV) is a monopartite begomovirus infecting tomatoes in South Africa, with sequence similarity to tomato yellow leaf curl virus (TYLCV). While there are numerous reports on the mechanism of TYLCV resistance in tomato, the underlying mechanisms in the tomato-ToCSV pathosystem is still relatively unknown. The main aim of this study was to investigate and compare the global methylation profile of ToCSV in two near-isogenic tomato lines, one with a tolerant phenotype (T, NIL396) and one with a susceptible phenotype (S, NIL395). Bisulfite conversion and PCR amplification, coupled with a next-generation sequencing approach, were used to elucidate the global pattern of methylation of ToCSV cytosine residues in T and S leave tissue at 35 days post-infection (dpi). The extent of methylation was more pronounced in tolerant plants compared to susceptible plants in all sequence (CG, CHG and CHH) contexts, however, the overall methylation levels were relatively low (<3%). Notably, a significant interaction (p < 0.05) was observed between the viral genomic region and susceptible vs. tolerant status for CG methylated regions where it was observed that the 3'IR CG methylation was significantly (p < 0.05) higher than CG methylation of other genomic regions in tolerant and susceptible plants. Additionally, statistically significant (EdgeR p < 0.05) differentially methylated cytosines were located primarily in the genomic regions V2/V1 and C4/C1 of ToCSV. The relative expression, using RT-qPCR, was also employed in order to quantify the expression of various key methylation-related genes, MET1, CMT2, KYP4/SUVH4, DML2, RDM1, AGO4 and AGO6 in T vs. S plants at 35dpi. The differential expression between T and S was significant for MET1, KYP4/SUVH4 and RDM1 at p<0.05 which further supports more pronounced methylation observed in ToCSV from T plants vs. S plants. While this study provides new insights into the differences in methylation profiles of ToCSV in S vs. T tomato plants, further research is required to link tolerance and susceptibility to ToCSV.

Coupling of conformation and CPD damage in nucleosomal DNA

UV-light can cause photodimerization and hence damages in DNA. Most frequent are cyclobutane pyrimidine dimer (CPD) damages, which predominantly form at TpT (thymine-thymine) steps. It is well known that CPD damage probability is different for single-stranded or double stranded DNA and depends on the sequence context. However, DNA deformation due to packing in nucleosomes can also influence CPD formation. Quantum mechanical calculations and Molecular Dynamics simulations indicate little CPD damage probability for DNA's equilibrium structure. We find that DNA needs to be deformed in a specific way to allow the HOMO → LUMO transition required for CPD damage formation. The simulation studies further show that the periodic CPD damage patterns measured in chromosomes and nucleosomes can be directly explained by the periodic deformation pattern of the DNA in the nucleosome complex. It supports previous findings on characteristic deformation patterns found in experimental nucleosome structures that relate to CPD damage formation. The result may have important implications for our understanding of UV-induced DNA mutations in human cancers.

Front Cover: The Wild‐Type tRNA Adenosine Deaminase Enzyme TadA Is Capable of Sequence‐Specific DNA Base Editing (ChemBioChem 16/2023)

The directed evolution of the adenosine deaminase enzyme TadA from an RNA-editing enzyme to an efficient DNA-editing enzyme was crucial for the development of the adenine base editor (ABE). Previous experiments showed no detectable DNA-editing activity by an ABE utilizing wild-type TadA (ABE0.1), but here we show ABE0.1 is able to edit DNA strictly within TACG motifs. This sequence requirement is partially governed by the C-terminal α5 helix of TadA, which accumulated many mutations during the directed evolution of TadA. The evolution of TadA to better access adenines within a diverse range of sequences is reflective of the Galapagos finches, which evolved different beak shapes in order to better access the food that was available to them. This cover art shows the protein structures of ecTadA (PDB: 1Z3A) and the evolved TadA8e (PDB: 6VCA), along with two species of Galapagos finches using the different α5 helix structures to access a target adenine within different sequence contexts. More information can be found in the Research Article by A. C. Komor et al.

Audible gestures

Abstract This study focuses on one type of audible gesture, designedly single claps (DSCs), as used by different people at an educational institution. The institution is designed to provide second language English users with opportunities to use English in various situations. Through the use of Multimodal Conversation Analysis, the analysis first focuses on the shape of DSCs and what makes them visible as not projecting further claps. Next, the analysis focuses on how DSCs are used within their sequential context. DSCs can take a variety of shapes, in that there are different ways not to project further claps; they can be used to attract attention of multiple recipients, and thus as one resource to manage interaction; and they are used as such a resource by representatives of the educational institution, who take on teacher roles within the interaction, with responsibility and deontic authority to manage shifts in activity and participation framework.

Interrogating the Function of Bicistronic Translational Control Elements to Improve Consistency of Gene Expression.

Context independent gene expression is required for genetic circuits to maintain consistent and predicable behavior. Previous efforts to develop context independent translation have leveraged the helicase activity of translating ribosomes via bicistronic design translational control elements (BCDs) located within an efficiently translated leader peptide. We have developed a series of bicistronic translational control elements with strengths that span several orders of magnitude, maintain consistent expression levels across diverse sequence contexts, and are agnostic to common ligation sequences used in modular cloning systems. We have used this series of BCDs to investigate several features of this design, including the spacing of the start and stop codons, the nucleotide identity upstream of the start codon, and factors affecting translation of the leader peptide. To demonstrate the flexibility of this architecture and their value as a generic modular expression control cassette for synthetic biology, we have developed a set of robust BCDs for use in several Rhodococcus species.

On the accuracy of the epigenetic copy machine: comprehensive specificity analysis of the DNMT1 DNA methyltransferase.

The specificity of DNMT1 for hemimethylated DNA is a central feature for the inheritance of DNA methylation. We investigated this property in competitive methylation kinetics using hemimethylated (HM), hemihydroxymethylated (OH) and unmethylated (UM) substrates with single CpG sites in a randomized sequence context. DNMT1 shows a strong flanking sequence dependent HM/UM specificity of 80-fold on average, which is slightly enhanced on long hemimethylated DNA substrates. To explain this strong effect of a single methyl group, we propose a novel model in which the presence of the 5mC methyl group changes the conformation of the DNMT1-DNA complex into an active conformation by steric repulsion. The HM/OH preference is flanking sequence dependent and on average only 13-fold, indicating that passive DNA demethylation by 5hmC generation is not efficient in many flanking contexts. The CXXC domain of DNMT1 has a moderate flanking sequence dependent contribution to HM/UM specificity during DNA association to DNMT1, but not if DNMT1 methylates long DNA molecules in processive methylation mode. Comparison of genomic methylation patterns from mouse ES cell lines with various deletions of DNMTs and TETs with our data revealed that the UM specificity profile is most related to cellular methylation patterns, indicating that de novo methylation activity of DNMT1 shapes the DNA methylome in these cells.

Systematic assessment of the flexibility of uracil damaged DNA

Uracil is a common DNA lesion which is recognized and removed by uracil DNA-glycosylase (UDG) as a part of the base excision repair pathway. Excision proceeds by base flipping, and UDG efficiency is thought to depend on the ease of deformability of the bases neighboring the lesion. We used molecular dynamics simulations to assess the flexibility of a large library of dsDNA strands, containing all tetranucleotide motifs with U:A, U:G, T:A or C:G base pairs. Our study demonstrates that uracil damaged DNA largely follows trends in flexibility of undamaged DNA. Measured bending persistence lengths, groove widths, step parameters and base flipping propensities demonstrate that uracil increases the flexibility of DNA, and that U:G base paired strands are more flexible than U:A strands. Certain sequence contexts are more deformable than others, with a key role for the 3′ base next to uracil. Flexibilities are large when this base is an A or G, and repressed for a C or T. A 5′ T adjacent to the uracil strongly promotes flexibility, but other 5′ bases are less influential. DNA bending is correlated to step deformations and base flipping, and bending aids flipping. Our study implies that the link between substrate flexibility and UDG efficiency is widely valid, helps explain why UDG prefers to bind U:G base paired strands, and suggests that the DNA bending angle of the UDG-substrate complex is optimal for base flipping. Communicated by Ramaswamy H. Sarma

Action formation, ascription, and the talk of people with aphasia

Background: This article uncovers why people with severe expressive aphasia’s turns-at-talk are sometimes not treated as producing an action by their communication partners, and the impact this has on the person with aphasia’s (PWA’s) agency. We demonstrate resources PWAs use to pursue talk and which assist with the production of a recognizable action. Method: We examined turns produced by four PWAs and their communication partners (CPs), where present, using conversation analysis, identifying features that do not receive a response and features promoting action ascription. Analysis: The PWAs’ semantically empty or unclear turns, turns lacking sequential context, or the CPs’ focus on their own actions led to a lack of action ascription. However, CPs do attend to PWAs’ multimodal features of interaction, and PWAs’ repetition accompanied by an upgraded gesture was shown to pursue a response. Action ascription was aided by the PWAs’ preserved use of silence as a communicative device. Discussion: When PWAs’ actions are not appropriately ascribed, their agency may be diminished. Communication partners should attend to all features of the PWA’s turns, including gesture and silence, to progress the PWA’s action, rather than their own misappropriated action. This may mean accepting a delay in progressivity while the PWA pursues an appropriate response. Through this, the PWA’s agency in interaction can be maintained, and intersubjectivity achieved.

Statistical inference reveals the role of length, GC content, and local sequence in V(D)J nucleotide trimming.

To appropriately defend against a wide array of pathogens, humans somatically generate highly diverse repertoires of B cell and T cell receptors (BCRs and TCRs) through a random process called V(D)J recombination. Receptor diversity is achieved during this process through both the combinatorial assembly of V(D)J-genes and the junctional deletion and insertion of nucleotides. While the Artemis protein is often regarded as the main nuclease involved in V(D)J recombination, the exact mechanism of nucleotide trimming is not understood. Using a previously published TCRβ repertoire sequencing data set, we have designed a flexible probabilistic model of nucleotide trimming that allows us to explore various mechanistically interpretable sequence-level features. We show that local sequence context, length, and GC nucleotide content in both directions of the wider sequence, together, can most accurately predict the trimming probabilities of a given V-gene sequence. Because GC nucleotide content is predictive of sequence-breathing, this model provides quantitative statistical evidence regarding the extent to which double-stranded DNA may need to be able to breathe for trimming to occur. We also see evidence of a sequence motif that appears to get preferentially trimmed, independent of GC-content-related effects. Further, we find that the inferred coefficients from this model provide accurate prediction for V- and J-gene sequences from other adaptive immune receptor loci. These results refine our understanding of how the Artemis nuclease may function to trim nucleotides during V(D)J recombination and provide another step toward understanding how V(D)J recombination generates diverse receptors and supports a powerful, unique immune response in healthy humans.

Super Papers Private Limited: preparation of operating budgets

Learning outcomes Through this case, participants will be able to: ▪ understand the different approaches to preparing operating budgets; ▪ classify the costs based on traceability to its cost centres; ▪ understand the difference in budget preparation and its analysis under different cost centres; ▪ put together the required information, identify the format and prepare major operating budgets; and ▪ evaluate operating budgets and give suggestions to the company based on budget analysis. Case overview/synopsis This case is about a manufacturing company that is going to introduce a budgeting system. It highlights the process of information collecting from key employees for budget preparation. This case also deals with various decisions to be made during the implementation of the new system such as the context of budgets, cost units and sequence of budgets.This case will help students to enhance their understanding of the operating budgets. The students will able to visualize the difficulty faced by companies to implement a new system. Complexity academic level This case is applicable in the courses such as Master of Business Administration, Master of Commerce or other postgraduate studies. This can also be discussed in professional courses such as Chartered Accountants, Certified Management Accountants, Company Secretaries, Institute of Cost and Works Accountants of India and Chartered Financial Analysts. Supplementary materials Teaching Notes are available for educators only. Subject code CSS 1: Accounting and Finance.

A Pragmatic Study of Turn Taking and Adjacency Pairs in Online Conversations

This study tries to investigate one type of synchronous online communication, which is Internet Relay Chat, in terms of conversational analysis, particularly turn-taking and adjacency pairs. Synchronous online communication can be viewed as a novel medium that combines spoken, written and electronic properties. There may be a direct causal link between the lack of coherence in synchronous online communication and its propensity for language play that leads to the conversational analysis of synchronous online communication, which is associated with a reduction of coherence, disruption of turn adjacency and phantom turn adjacency. In synchronous online communication, there is a difficulty in interpreting messages in their sequential context that arises from the fact that turn sequencing is partly user-controlled and partly system-controlled. This leads to disrupted turn-taking and adjacency pairs as other stands of conversation get inserted between their parts.

T Residues Preceded by Runs of G Are Hotspots of T→G Mutation in Bacteria.

The rate of mutation varies among positions in a genome. Local sequence context can affect the rate and has different effects on different types of mutation. Here, I report an effect of local context that operates to some extent in all bacteria examined: the rate of T→G mutation is greatly increased by preceding runs of three or more G residues. The strength of the effect increases with the length of the run. In Salmonella, in which the effect is strongest, a G run of length three 3 increases the rate by a factor of ∼26, a run of length 4 increases it by almost a factor of 100, and runs of length 5 or more increase it by a factor of more than 400 on average. The effect is much stronger when the T is on the leading rather than the lagging strand of DNA replication. Several observations eliminate the possibility that this effect is an artifact of sequencing error.

PiCRISPR: Physically informed deep learning models for CRISPR/Cas9 off-target cleavage prediction

CRISPR/Cas programmable nuclease systems have become ubiquitous in the field of gene editing. With progressing development, applications in in vivo therapeutic gene editing are increasingly within reach, yet limited by possible adverse side effects from unwanted edits. Recent years have thus seen continuous development of off-target prediction algorithms trained on in vitro cleavage assay data gained from immortalised cell lines. It has been shown that in contrast to experimental epigenetic features, computed physically informed features are so far underutilised despite bearing considerably larger correlation with cleavage activity. Here, we implement state-of-the-art deep learning algorithms and feature encodings for off-target prediction with emphasis on physically informed features that capture the biological environment of the cleavage site, hence terming our approach piCRISPR. Features were gained from the large, diverse crisprSQL off-target cleavage dataset. We find that our best-performing models highlight the importance of sequence context and chromatin accessibility for cleavage prediction and compare favourably with literature standard prediction performance. We further show that our novel, environmentally sensitive features are crucial to accurate prediction on sequence-identical locus pairs, making them highly relevant for clinical guide design. The source code and trained models can be found ready to use at github.com/florianst/picrispr.

Prior Movement of One Arm Facilitates Motor Adaptation in the Other.

Many movements in daily life are embedded in motion sequences that involve more than one limb, demanding the motor system to monitor and control different body parts in quick succession. During such movements, systematic changes in the environment or the body might require motor adaptation of specific segments. However, previous motor adaptation research has focused primarily on motion sequences produced by a single limb, or on simultaneous movements of several limbs. For example, adaptation to opposing force fields is possible in unimanual reaching tasks when the direction of a prior or subsequent movement is predictive of force field direction. It is unclear, however, whether multilimb sequences can support motor adaptation processes in a similar way. In the present study (38 females, 38 males), we investigated whether reaches can be adapted to different force fields in a bimanual motor sequence when the information about the perturbation is associated with the prior movement direction of the other arm. In addition, we examined whether prior perceptual (visual or proprioceptive) feedback of the opposite arm contributes to force field-specific motor adaptation. Our key finding is that only active participation in the bimanual sequential task supports pronounced adaptation. This result suggests that active segments in bimanual motion sequences are linked across limbs. If there is a consistent association between movement kinematics of the linked and goal movement, the learning process of the goal movement can be facilitated. More generally, if motion sequences are repeated often, prior segments can evoke specific adjustments of subsequent movements.SIGNIFICANCE STATEMENT Movements in a limb's motion sequence can be adjusted based on linked movements. A prerequisite is that kinematics of the linked movements correctly predict which adjustments are needed. We show that use of kinematic information to improve performance is even possible when a prior linked movement is performed with a different limb. For example, a skilled juggler might have learned how to correctly adjust his catching movement of the left hand when the right hand performed a throwing action in a specific way. Linkage is possibly a key mechanism of the human motor system for learning complex bimanual skills. Our study emphasizes that learning of specific movements should not be studied in isolation but within their motor sequence context.

Evolution of coronavirus frameshifting elements: Competing stem networks explain conservation and variability

The frameshifting RNA element (FSE) in coronaviruses (CoVs) regulates the programmed -1 ribosomal frameshift (-1 PRF) mechanism common to many viruses. The FSE is of particular interest as a promising drug candidate. Its associated pseudoknot or stem loop structure is thought to play a large role in frameshifting and thus viral protein production. To investigate the FSE structural evolution, we use our graph theory-based methods for representing RNA secondary structures in the RNA-As-Graphs (RAG) framework to calculate conformational landscapes of viral FSEs with increasing sequence lengths for representative 10 Alpha and 13 Beta-CoVs. By following length-dependent conformational changes, we show that FSE sequences encode many possible competing stems which in turn favor certain FSE topologies, including a variety of pseudoknots, stem loops, and junctions. We explain alternative competing stems and topological FSE changes by recurring patterns of mutations. At the same time, FSE topology robustness can be understood by shifted stems within different sequence contexts and base pair coevolution. We further propose that the topology changes reflected by length-dependent conformations contribute to tuning the frameshifting efficiency. Our work provides tools to analyze virus sequence/structure correlations, explains how sequence and FSE structure have evolved for CoVs, and provides insights into potential mutations for therapeutic applications against a broad spectrum of CoV FSEs by targeting key sequence/structural transitions.

A DddA ortholog-based and transactivator-assisted nuclear and mitochondrial cytosine base editors with expanded target compatibility

Bacterial double-stranded DNA (dsDNA) cytosine deaminase DddAtox-derived cytosine base editor (DdCBE) and its evolved variant, DddA11, guided by transcription-activator-like effector (TALE) proteins, enable mitochondrial DNA (mtDNA) editing at TC or HC (H= A, C, or T) sequence contexts, while it remains relatively unattainable for GC targets. Here, we identified a dsDNA deaminase originated from a Roseburia intestinalis interbacterial toxin (riDddAtox) and generated CRISPR-mediated nuclear DdCBEs (crDdCBEs) and mitochondrial CBEs (mitoCBEs) using split riDddAtox, which catalyzed C-to-T editing at both HC and GC targets in nuclear and mitochondrial genes. Moreover, transactivator (VP64, P65, or Rta) fusion to the tail of DddAtox- or riDddAtox-mediated crDdCBEs and mitoCBEs substantially improved nuclear and mtDNA editing efficiencies by up to 3.5- and 1.7-fold, respectively. We also used riDddAtox-based and Rta-assisted mitoCBE to efficiently stimulate disease-associated mtDNA mutations in cultured cells and in mouse embryos with conversion frequencies of up to 58% at non-TC targets.

8-Oxo-2'-deoxyguanosine Replication in Mutational Hot Spot Sequences of the p53 Gene in Human Cells Is Less Mutagenic than That of the Corresponding Formamidopyrimidine.

7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-OxodGuo) is a ubiquitous DNA damage formed by oxidation of 2'-deoxyguanosine. In this study, plasmid DNA containing 8-OxodGuo located in three mutational hot spots of human cancers, codons 248, 249, and 273 of the Tp53 tumor suppressor gene, was replicated in HEK 293T cells. 8-OxodGuo was only a weak block of replication, and the bypass was largely error-free. The mutations (1-5%) were primarily G → T transversions, and the mutation frequency was generally lower than that of the chemically related Fapy·dG. A unique 8-OxodGuo mutation spectrum was observed at each site, as reflected by replication in translesion synthesis (TLS) polymerase- or hPol λ-deficient cells. In codon 248 (CG*G) and 249 (AG*G), where G* denotes 8-OxodGuo, hPol η and hPol ζ carried out largely error-free bypass of the lesion, whereas hPol κ and hPol ι were involved mostly in error-prone TLS, resulting in G → T mutations. 8-OxodGuo bypass in codon 273 (CG*T) was unlike the other two sites, as hPol κ participated in the mostly error-free bypass of the lesion. Yet, in all three sites, including codon 273, simultaneous deficiency of hpol κ and hPol ι resulted in reduction of G → T transversions. This indicates a convincing role of these two TLS polymerases in error-prone bypass of 8-OxodGuo. Although the dominant mutation was G → T in each site, in codon 249, and to a lesser extent in codon 248, significant semi-targeted single-base deletions also occurred, which suggests that 8-OxodGuo can initiate slippage of a base near the lesion site. This study underscores the importance of sequence context in 8-OxodGuo mutagenesis in human cells. It also provides a more comprehensive comparison between 8-OxodGuo and the sister lesion, Fapy·dG. The greater mutagenicity of the latter in the same sequence contexts indicates that Fapy·dG is a biologically significant lesion and biomarker on par with 8-OxodGuo.

Modulation of DNA-protein Interactions by Proximal Genetic Elements as Uncovered by Interpretable Deep Learning

Transcription factors (TF) recognize specific motifs in the genome that are typically 6–12 bp long to regulate various aspects of the cellular machinery. Presence of binding motifs and favorable genome accessibility are key drivers for a consistent TF-DNA interaction. Although these pre-requisites may occur thousands of times in the genome, there seems to be a high degree of selectivity for the sites that are actually bound. Here, we present a deep-learning framework that identifies and characterizes the upstream and downstream genetic elements to the binding motif, for their role in enforcing the mentioned selectivity. The proposed framework is based on an interpretable recurrent neural network architecture that enables for the relative analysis of sequence context features. We apply the framework to model twenty-six transcription factors and score the TF-DNA binding at a base-pair resolution. We find significant differences in activations of DNA context features for bound and unbound sequences. In addition to standardized evaluation protocols, we offer outstanding interpretability that enables us to identify and annotate DNA sequence with possible elements that modulate TF-DNA binding. Also, differences in data processing have a huge influence on the overall model performance. Overall, the proposed framework allows for novel insights on the non-coding genetic elements and their role in facilitating a stable TF-DNA interaction.

Embryonic Stem Cell-Derived Neurons as a Model System for Epigenome Maturation during Development.

DNA methylation in neurons is directly linked to neuronal genome regulation and maturation. Unlike other tissues, vertebrate neurons accumulate high levels of atypical DNA methylation in the CH sequence context (mCH) during early postnatal brain development. Here, we investigate to what extent neurons derived in vitro from both mouse and human pluripotent stem cells recapitulate in vivo DNA methylation patterns. While human ESC-derived neurons did not accumulate mCH in either 2D culture or 3D organoid models even after prolonged culture, cortical neurons derived from mouse ESCs acquired in vivo levels of mCH over a similar time period in both primary neuron cultures and in vivo development. mESC-derived neuron mCH deposition was coincident with a transient increase in Dnmt3a, preceded by the postmitotic marker Rbfox3 (NeuN), was enriched at the nuclear lamina, and negatively correlated with gene expression. We further found that methylation patterning subtly differed between in vitro mES-derived and in vivo neurons, suggesting the involvement of additional noncell autonomous processes. Our findings show that mouse ESC-derived neurons, in contrast to those of humans, can recapitulate the unique DNA methylation landscape of adult neurons in vitro over experimentally tractable timeframes, which allows their use as a model system to study epigenome maturation over development.

Sex differences in autistic adults: A preliminary study showing differences in mentalizing, but not in narrative coherence

Studying autism might be a complex endeavor due to its clinical heterogeneity. Little is currently known about potential sex differences in autistic adults, especially regarding mentalizing and narrative coherence. In this study, male and female participants told a personal story about one of their most positive and most negative life events and performed two mentalizing tasks. One of these mentalizing tasks was a recently developed Picture and Verbal Sequencing task that has shown cerebellar recruitment, and which requires mentalizing in a sequential context (i.e., participants chronologically ordered scenarios that required true and false belief mentalizing). Our preliminary comparison shows that males were faster and more accurate on the Picture Sequencing task compared to female participants when ordering sequences involving false beliefs, but not true beliefs. No sex differences were found for the other mentalizing and narrative tasks. These results highlight the importance of looking at sex differences in autistic adults and provide a possible explanation for sex-related differences in daily life mentalizing functions, which suggest a need for more sensitive diagnosis and tailored support.