Biology subjects

Gelfand, M. S.

Publications and source records attributed to Gelfand, M. S..

At least 19 recordsLinked to original sources

bioRxiv2026

Hairpin Architecture and Extended Sequence Context Shape APOBEC3 Mutagenesis in Monkeypox Virus at Different Genomic Scales

The 2022 global mpox outbreak was characterized by an unusual accumulation of more than fifty APOBEC-signature substitutions in MPXV genomes. A previous study suggested that these substitutions preferentially occur within and near hairpins or cruciforms - stem-loop structures formed by inverted repeats. Here, we show that this conclusion resulted from an inappropriate statistical analysis and, using two substantially larger datasets, demonstrate that APOBEC-signature mutations are specifically enriched at TC motifs located at the 3' ends of hairpin loops, but not in other regions of hairpins/cruciforms or in their vicinity. We further identified a preference for stem-loop structures with longer stems and shorter loops, consistent with patterns previously reported for the APOBEC mutagenesis in cancer genomes. Our analysis also revealed a positive association between APOBEC-signature mutation propensity and hairpin stability in single-stranded DNA. Nevertheless, stem-loop structures with long stems and short loops are relatively rare in the MPXV genome, limiting their contribution to the genome-wide distribution of APOBEC-signature mutations to a predominantly local effect. In contrast, extended nucleotide context downstream of the TC motif showed strong genome-wide predictive value, with a marked preference for guanine-rich trinucleotide immediately downstream. Analysis of the upstream nucleotide context was consistent with the mutational specificity of APOBEC3A. Together, these results identify distinct structural and sequence determinants of the APOBEC-associated mutagenesis in MPXV and show that local hairpin/cruciform architecture and extended nucleotide context contribute at different genomic scales.

microbiology↗

bioRxiv2026

Azospirillum: effective plasmid management

Azospirillum is a genus of nitrogen-fixing alpha-proteobacteria whose genomes consist of a chromosome and several large secondary replicons. The organization and evolution of these replicons remain poorly understood. We analyzed 22 complete Azospirillum genomes, containing six to ten replicons, and characterized their gene composition, replication, segregation, and dimer resolution systems. The largest replicon contains most of the essential genes and is the chromosome. Other replicons can be classified based on genecomposition, similarity of the plasmid replication protein RepA, and similarity of the ParAB segregation proteins. Overall, groups obtained using different approaches agree with each other. Analysis of gene composition also identified fusions or splits of secondary replicons. None of the large secondary replicons of Azospirillum strains contain the repABC replication system, and all of them are iteron plasmids. For all groups of replicons, the RepA box motifs and the structure of the plasmid replication origin oriV were predicted. Most replicons use the ParABS system for segregation. In rare cases, the replicon contains repA from one group and parAB from another. The position of the parAB operon relative to the replication origin is determined by the parAB group, and not by repA. Most secondary replicons apparently use an FtsK-dependent pathway for dimer resolution, relying instead on the chromosomally encoded XerCD proteins. In most cases, the identified dif motifs do not distinguish groups of replicons.

genomics↗

bioRxiv2026

Novel "Candidatus Endobugula" hosts and distinct microbiomes in the bryozoans from the White Sea

Marine bryozoans harbor diverse microbial communities, but not much is known about specificity of bacterial symbionts towards different hosts. We identified potential symbionts and described overall microbial communities of three marine bryozoans, Aquiloniella scabra, Rhamphostomella bilaminata (Gymnolaemata: Cheilostomatida), and Patinella verrucaria (Stenolaemata: Cyclostomatida), revealing that each species maintains a distinct microbial assemblage that differs both from the surrounding environmental microbiome and from other hosts. Previously, the bryostatin-producing gammaproteobacterial genus "Candidatus Endobugula" was known from only two bryozoan species in the family Bugulidae. Here, we report its first detection in two more cheilostome bryozoans, A. scabra and R. bilaminata, expanding its known host range to two more families.

ecology↗

bioRxiv2026

Loop statistics and fountain geometry reveal effective two-sided cohesin extrusion and crowding-induced arm desynchronization

Cohesin-driven loop extrusion shapes chromosome organization, yet what determines loop lengths and coordination between the extruding arms in vivo remains unclear. Broad extrusion "fountains" in Hi-C maps point to arm desynchronization, whose physical origin is unknown. Here we develop a kinetic theory of extrusion through transient chromatin roadblocks and neighbouring cohesins. Roadblock abundance, lifetime and partial permeability, together with cohesin crowding, combine into an effective obstacle density that renormalizes processivity and sets the mean loop length. Full loop-length distributions reveal extrusion symmetry: one-sided extrusion remains exponential, whereas effectively two-sided extrusion can generate a finite-length peak. ChIA-PET and MNase HiChIP data match the two-sided predictions, disfavouring purely one-sided extrusion. Cohesin crowding further desynchronizes the arms while leaving a residual correlation approaching {rho}=1/4. Fountain anisotropy across three vertebrates yields correlations near this value, consistent with local cohesin crowding as a sufficient mechanism for fountain formation. Fits further reveal 30-100-kb cohesin-loading regions and, in Danio rerio, a loading width comparable to the extent of enhancer enrichment, motivating a model in which an enhancer-rich loading platform acts as a collective barrier sustaining outward extrusion toward surrounding promoters. Our framework shows how cohesin and roadblock kinetics jointly determine loop statistics, shape chromosome-contact patterns, and may facilitate enhancer-promoter search.

biophysics↗

bioRxiv2025

Stochastic motility-adhesion switch within the E. coli K-12 strains revealed by gene expression clustering

Biofilm formation in Escherichia coli arises from regulatory programs that can diverge even among genetically identical cultures. Exploiting spontaneous replicate-to-replicate variation in E. coli K-12 MG1655, we mapped expression modules whose behavior tracks biofilm abundance across four genetic backgrounds: wild type; strain with deleted gene for hexuronate regulator UxuR {Delta}uxuR; an uxuR translation-disrupted retaining locus-derived sRNAs ({Delta}uxuR-tr), and strain with deleted gene for global carbon regulator cAMP-CRP ({Delta}crp). In the wild type, regulator EcpR/MatA demonstrated the highest correlation with the biofilm intensity, and its role was, in particular, seemingly reinforced by stress/pH-homeostasis and c-di-GMP-linked factors. {Delta}uxuR exhibited the strongest stochasticity in biofilm formation, while in {Delta}uxuR-tr, replicate dispersion was mostly muted, indicating that uxuR-derived sRNAs buffer motility-adhesion switching. In {Delta}crp, pgaA was de-repressed due to the absence of CsrA, with expression being highly consistent with intensity of biofilm formation, alongside stress/ion-homeostasis genes. This is in line with the suggestion that CRP regulates biofilms and motility indirectly via regulation of more local transcription factors. Overall, replicate-resolved clustering converts apparent "noise" into structured regulatory patterns, which include genes whose role in biofilm formation was not yet evident, and helps identify switch nodes between planktonic and biofilm bacterial states.

microbiology↗

bioRxiv2025

Comment on "Inverted repeats in the monkeypox virus genome are hot spots for mutation"

Recent work proposed that APOBEC-signature mutations in monkeypox virus (MPXV) are enriched in cruci-form structures formed by inverted repeats. We independently reanalyzed the same dataset with the published settings and several complementary approaches and found no statistically supported enrichment of mutations in these structures. While a modest trend at TC motif at 3 end of hairpin loop contexts cannot be excluded, current evidence is insufficient. Additional data and careful handling of overlapping structures are needed to clarify any association.

microbiology↗

bioRxiv2025

Analysis of MPXV RNA-seq Data Reveals Lack of Evidence of APOBEC3-mediated RNA Editing

The 2022 outbreak of monkeypox virus (MPXV), a double-stranded DNA virus, is remarkable for an unusually high number of single-nucleotide substitutions compared to earlier strains, with a strong bias toward C[->]T and G[->]A transitions consistent with the APOBEC3 cytidine deaminase activity. While APOBEC3-induced mutagenesis is well documented at the DNA level, its potential impact on MPXV RNA transcripts remains unclear. To assess whether APOBEC3 enzymes act on MPXV RNA, we analyzed RNA-seq data from infected samples. The enrichment of APOBEC-signature substitutions among high-frequency mismatched positions led us to consider two possibilities: RNA editing at hotspots or fixed DNA mutations. Multiple lines of evidence support the conclusion that these substitutions arise from DNA-level mutagenesis rather than RNA editing. These include a substantial number of G[->]A substitutions remaining after normalization by gene strand direction, a largely neutral impact of substitutions on protein-coding sequences, the lack of positional correlation with transcriptional features or RNA secondary structure typically associated with APOBEC action hotspots, and an overlap with known genomic mutations in MPXV strains. Analysis of the nucleotide context of observed substitutions indicated that APOBEC3A or APOBEC3B were likely drivers of DNA-level mutagenesis. ImportanceThe 2022 monkeypox virus (MPXV) outbreak showed an unusually high number of mutations thought to result from human antiviral enzymes of the APOBEC3 family. While such mutations have been clearly documented in the viral DNA, whether APOBEC3 also edits viral messenger RNA molecules remained unclear. In this study, we analyzed multiple publicly available MPXV RNA sequencing datasets to address this question. We found that the apparent APOBEC-like changes in RNA are best explained by fixed DNA mutations rather than active RNA editing. This finding helps clarify how MPXV evolves and adapts, suggesting that APOBEC3s role in shaping the virus likely operates at the DNA level. Understanding where and how these mutations occur provides insight into the viruss interaction with the human immune system and informs future studies on viral evolution and antiviral defenses.

microbiology↗

bioRxiv2024

Temporal dynamics of gene expression during metamorphosis in two distant Drosophila species

Complete metamorphosis of holometabolous insects is a complex biological process characterized by profound morphological, physiological, and transcriptional changes. To reveal the temporal dynamics of gene expression during this critical developmental transition, we conducted a detailed analysis of the developmental transcriptomes of two Drosophila species, Drosophila melanogaster and Drosophila virilis. We confirmed partial recapitulation of the embryonic transcriptional program in pupae, but unlike the traditional hourglass model, suggesting maximal conservation at mid-embryonic stages, at different stages of pupae we observed a more complicated pattern of alternating low and high diversity, resembling an inverted hourglass, or "spindle". This underscores the complexity of developmental processes during complete metamorphosis. Notably, recently formed genes (specific to insects) exhibit pronounced expression peaks during mid-pupal development, indicating their potential role in developmental transitions. Significance StatementThis study describes the transcriptomic dynamics of complete metamorphosis in holometabolous insects. By analyzing developmental transcriptomes of Drosophila melanogaster and Drosophila virilis, we characterize the spindle pattern of gene expression during pupal stages, contrasting with the classical hourglass model of embryogenesis. This alternating pattern of low and high transcriptional diversity highlights the unique complexity of the metamorphose processes.

developmental biology↗

bioRxiv2024

Convergent gene pairs restrict chromatin looping in Dictyostelium discoideum, acting as directional barriers for extrusion

Dictyostelium discoideum is a unicellular slime mold, developing into a multicellular fruiting body upon starvation. Development is accompanied by large-scale shifts in gene expression program, but underlying features of chromatin spatial organization remain unknown. Here, we report that the Dictyostelium 3D genome is organized into positionally conserved, largely consecutive, non-hierarchical and weakly insulated loops at the onset of multicellular development. The transcription level within the loop interior tends to be higher than in adjacent regions. Loop interiors frequently contain functionally linked genes and genes which coherently change expression level during development. Loop anchors are predominantly positioned by the genes in convergent orientation. Our data suggest that the loop profile may arise from the interplay between transcription and extrusion-driven chromatin folding. In particular, a convergent gene pair serves as a bidirectional extrusion barrier or a "diode" that controls passage of the cohesin extruder by relative transcription level of paired genes.

molecular biology↗

bioRxiv2023

Genome rearrangements drive evolution of ANK genes in Wolbachia

IntroductionGenus Wolbachia comprises endosymbionts infecting many arthropods and nematodes; it is a model for studying symbiosis as its members feature numerous, diverse mutualistic and parasitic adaptations to different hosts. In contrast to nematode-infecting Wolbachia, genomes of arthropod-infecting strains contain a high fraction of repetitive elements creating possibilities for multiple recombination events and causing genome rearrangements. The mechanisms and role of these features are still not fully understood. ResultsTransposons cover up to 18% of an arthropod-infecting Wolbachia genome and drive numerous genome rearrangements including inversions and segmental amplifications. ANK (ankyrin-repeat domain family) genes are also often found at the breakpoints of rearrangements, while less than 7% of them were found within locally collinear blocks (LCBs). We observed a strong correlation between the number of ANK genes and the genome size as well as significant overrepresentation of transposons adjacent to these genes. We also revealed numerous cases of integration of transposases to the ANK genes affecting the sequences and putative products of the latter. Our results uncover the role of mobile elements in the amplification and diversification of ANK genes. ConclusionsEvolution of arthropod-infecting Wolbachia was accompanied by diverse genome rearrangements driving the evolution of ANK genes important for bacteria-host interactions. This study demonstrates the effectiveness of our LCB-based approach to the Wolbachia genomics and provides a framework for understanding the impact of genome rearrangements on their rapid host adaptation.

evolutionary biology↗

bioRxiv2023

Extrusion fountains are hallmarks of chromosome organization emerging upon zygotic genome activation

The initiation of gene expression during development, known as zygotic genome activation (ZGA), is accompanied by massive changes in chromosome organization. However, the earliest events of chromosome folding and their functional roles remain unclear. Using Hi-C on zebrafish embryos, we discovered that chromosome folding begins early in development with the formation of "fountains", a novel element of chromosome organization. Emerging preferentially at enhancers, fountains exhibit an initial accumulation of cohesin, which later redistributes to CTCF sites at TAD borders. Knockouts of pioneer transcription factors driving ZGA enhancers result in the specific loss of fountains, establishing a causal link between enhancer activation and fountain formation. Polymer simulations demonstrate that fountains may arise as sites of facilitated cohesin loading, requiring two-sided but desynchronized loop extrusion, potentially caused by cohesin collisions with obstacles or internal switching. Moreover, we detected similar fountain patterns at enhancers in mouse cells. Fountains disappear upon acute cohesin depletion, as well as during mitosis, and reappear with cohesin loading in early G1. Altogether, fountains represent the first known enhancer-specific elements of chromosome organization and constitute starting points for chromosome folding during development, likely through facilitated cohesin loading.

molecular biology↗

bioRxiv2023

Evolutionary trajectories of secondary replicons in multipartite genomes

Most bacterial genomes have a single chromosome that may be supplemented by smaller, dispensable plasmids. However, approximately 10% of bacteria with completely sequenced genomes, mostly pathogens and plant symbionts, have more than one stable large replicon. Some secondary replicons are species-specific, carrying pathogenicity or symbiotic factors. Other replicons are common on at least the genus level, carry house-keeping genes, and may have a size of several million base pairs. We analyzed the abundance and sizes of large secondary replicons in different groups of bacteria and identified two patterns in the evolution of multipartite genomes. In nine genera of four families, Pseudoalteromonadaceae, Burkholderiaceae, Vibrionaceae, and Brucellaceae, we observed a positive correlation between the sizes of the chromosome and the secondary replicon with the slope in the range of 0.6-1.2. This indicates that in these genera the replicons evolve in a coordinated manner, with comparable rates of gene gain/loss, hence supporting classification of such secondary replicons as chromids. The second, more common pattern, features gene gains and losses mainly occurring in the primary replicon, yielding a stable size of the secondary replicon. Such secondary replicons are usually present in only a low fraction of the genus species. Hence, such replicons behave as megaplasmids. A mixed situation was observed in symbiotic genera from the Rhizobiaceae family where the large secondary replicons are of stable size, but present in all species. These results may provide a general framework for understanding the evolution of genome complexity in prokaryotes. SignificanceLarge secondary replicons are observed in representatives of many taxonomic groups of bacteria. Traditionally, they are referred to as second chromosomes, chromids, or megaplasmids, with little consistency, in particular because their evolution remains understudied. Here we demonstrate that the sizes of secondary replicons follow two main evolutionary trends: replicons whose size scales linearly with the size of the main chromosome (the suggested term chromids) typically contain numerous essential genes (rRNA, tRNA, ribosomal proteins), while large secondary replicons of stable size (termed megaplasmids) contain fewer or none such genes.

genomics↗

bioRxiv2023

Mutational signatures in wild type Escherichia coli strains reveal dominance of DNA polymerase errors

While mutational processes operating in the Escherichia coli genome have been revealed by multiple laboratory experiments, the contribution of these processes to accumulation of bacterial polymorphism and evolution in natural environments is unknown. To address this question, we reconstruct signatures of distinct mutational processes from experimental data on E. coli hypermutators, and ask how these processes contribute to differences between naturally occurring E. coli strains. We show that both mutations accumulated in the course of evolution of wild type strains in nature and in the lab-grown non-mutator laboratory strains are explained predominantly by the low fidelity of DNA polymerases II and III. By contrast, contributions specific to disruption of DNA repair systems cannot be detected, suggesting that temporary accelerations of mutagenesis associated with such disruptions are unimportant for within-species evolution. These observations demonstrate that accumulation of diversity in bacterial strains in nature is predominantly associated with errors of DNA polymerases.

evolutionary biology↗

bioRxiv2022

Non-triplet genetic code in ciliate Euplotes ciliates is a result of neutral evolution

Although several variants of the standard genetic code are known, its triplet character is universal with an exception in ciliates Euplotes, where stop codons at internal mRNA positions specify ribosomal frameshifting. How did Euplotes spp. evolved and maintained such an unusual genetic code remains a mystery. To investigate these questions, we explored the evolution of frameshifting occurrence in Euplotes genes. We sequenced and analyzed several transcriptomes from different Euplotes spp to characterize the gain-and-loss dynamics of frameshift sites. Surprisingly, we found a sharp asymmetry between frameshift gain and frameshift loss events with the former exceeding the latter by about 10 folds. Further analysis of mutation rates in protein-coding and non-coding regions revealed that this asymmetry is expected based on single nucleotide mutation rates and does not require positive selection for frameshifting. We found that the number of frameshift sites in Euplotes spp is increasing and is far from the steady state. The steady equilibrium state is expected in about 0.1 to 1 billion years leading to about a 10 fold increase in the number of frameshift sites in Euplotes genes.

evolutionary biology↗

bioRxiv2022

Recapitulation of the embryonic transcriptional program in insect pupae

Metamorphosis of holometabolous insects is predominantly a motionless stage, when no active feeding is observed and the body is enclosed into the pupa. These physiological properties as well as undergoing processes resemble embryogenesis since at the pupal stage organs and systems of the imago are formed. Therefore recapitulation of the embryonic expression program during pupa development could be hypothesized. To assess this hypothesis at the transcriptome level, we have performed a comprehensive analysis of the developmental datasets available in the public domain. Indeed, for most datasets, the pupal gene expression resembles the embryonic rather than the larval pattern, interrupting gradual changes of the transcriptome. Moreover, changes in the transcriptome profile during the pupa-to-imago transition are positively correlated with those at the embryo-to-larvae transition, suggesting that similar expression programs are activated. Gene sets that change their expression level during the larval stage and revert it to the embryonic-like state during the metamorphosis are enriched with genes associated with metabolism and development.

developmental biology↗

bioRxiv2021

Absence of enterotypes in the human gut microbiomes reanalyzed with non-linear dimensionality reduction methods

Enterotypes of the human gut microbiome have been proposed to be a powerful prognostic tool to evaluate the correlation between lifestyle, nutrition, and disease. However, the number of enterotypes suggested in the literature ranged from two to four. The growth of available metagenome data and the use of exact, non-linear methods of data analysis challenges the very concept of clusters in the multidimensional space of bacterial microbiomes. Using several published human gut microbiome datasets, we demonstrate the presence of a lower-dimensional structure in the microbiome space, with high-dimensional data concentrated near a low-dimensional non-linear submanifold, but the absence of distinct and stable clusters that could represent enterotypes. This observation is robust with regard to diverse combinations of dimensionality reduction techniques and clustering algorithms.

bioinformatics↗

bioRxiv2021

Chromosome-encoded IpaH ubiquitin ligases indicate non-human pathogenic Escherichia

Until recently, Shigella and enteroinvasive Escherichia coli were thought to be primate-restricted pathogens. The base of their pathogenicity is the type 3 secretion system (T3SS) encoded by the pINV virulence plasmid, which facilitates host cell invasion and subsequent proliferation. A large family of T3SS effectors, E3 ubiquitin-ligases encoded by the ipaH genes, have a key role in the Shigella pathogenicity through the modulation of cellular ubiquitination that degrades host proteins. However, recent genomic studies identified ipaH genes in the genomes of Escherichia marmotae, a potential marmot pathogen, and an E. coli extracted from fecal samples of bovine calves, suggesting that non-human hosts may also be infected by these strains, potentially pathogenic to humans. We performed a comparative genomic study of the functional repertoires in the ipaH gene family in Shigella and enteroinvasive Escherichia from human and predicted non-human hosts. We found that fewer than half of Shigella genomes had a complete set of ipaH genes, with frequent gene losses and duplications that were not consistent with the species tree and nomenclature. Non-human host IpaH proteins had a diverse set of substrate-binding domains and, in contrast to the Shigella proteins, two different types of the NEL C-terminal domain. Only the ipaH9.8 gene was found in Escherichia derived from both human and non-human hosts. These results provide a framework for understanding of ipaH-mediated host-pathogens interactions and suggest a need for a genomic study of fecal samples from diseased animals.

microbiology↗

bioRxiv2021

APOBEC mutagenesis is low in most types of non-B DNA structures, unlike other types of cancer mutagenesis

While somatic mutations are known to be enriched in genome regions with non-canonical DNA secondary structure, the impact of particular mutagens still needs to be elucidated. Here, we demonstrate that in human cancers, the APOBEC mutagenesis is not enriched in direct repeats, mirror repeats, short tandem repeats, and G-quadruplexes, and even decreased below its level in B-DNA for cancer samples with very high APOBEC activity. In contrast, we observe that the APOBEC-induced mutational density is positively associated with APOBEC activity in inverted repeats (cruciform structures), where the impact of cytosine at the 3-end of the hairpin loop is substantial. Surprisingly, the APOBEC-signature mutation density per TC motif in the single-stranded DNA of a G-quadruplex (G4) is lower than in the four-stranded part of G4 and in B-DNA. The APOBEC mutagenesis, as well as the UV-mutagenesis in melanoma samples are absent in Z-DNA regions, due to depletion of their mutational signature motifs.

cancer biology↗