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Convergent evolution, the evolution of the same or similar phenotypes in phylogenetically independent lineages, is a widespread phenomenon in nature. If the genetic basis for convergent evolution is predictable to some extent, it may be possible to infer organismic phenotypes and the capability of organisms to utilize new ecological resources based on genome sequence data. While repeated amino acid changes have been studied in association with convergent evolution, relatively little is known about the potential contribution of repeated gene copy number changes. In this study, we explore whether gene copy number changes of particular gene families are linked to diet shifts in mammals and assess whether trophic ecology can be inferred from the copy numbers of a specific set of gene families. Using 86 mammalian genome sequences, we identified 24 gene families with a trend toward higher copy numbers in herbivores, carnivores, and omnivores, even after phylogenetic corrections. We were able to confirm previous findings on genes such as amylase, olfactory receptors, and xenobiotic metabolism genes, and identify novel gene families whose copy numbers correlate with dietary patterns. For example, omnivores exhibited higher copy numbers of genes encoding regulators of translation. We also established a discriminant function based on the copy numbers of 13 gene families that can help predict trophic ecology to some extent. These findings highlight a possible association between convergent evolution and repeated copy number changes in specific gene families, suggesting the potential to develop a method for predicting animal ecology from genome sequence data., (© The Author(s) 2025. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Enhancers are remotely located noncoding DNA sequences that regulate gene expression in response to developmental, homeostatic, and environmental cues. Canonical short-range enhancers located <50 kb from their cognate promoters function by binding transcription factors, coactivators, and chromatin modifiers. In this review, we discuss recent evidence that medium-range (50-400 kb) and long-range (>400 kb) enhancers rely on additional mechanisms, including cohesin, CCCTC-binding factor, and high-affinity protein-protein interactions. These mechanisms are crucial for establishing the physical proximity and interaction between enhancers and their target promoters over extended genomic distances and ensuring robust gene activation during mammalian development. Future studies will be critical to unravel their prevalence and evolutionary significance across various genomic loci, cell types, and species., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Studying the functional consequences of structural variants (SVs) in mammalian genomes is challenging because (i) SVs arise much less commonly than single-nucleotide variants or small indels and (ii) methods to generate, map, and characterize SVs in model systems are underdeveloped. To address these challenges, we developed Genome-Shuffle-seq, a method that enables the multiplex generation and mapping of thousands of SVs (deletions, inversions, translocations, and extrachromosomal circles) throughout mammalian genomes. We also demonstrate the co-capture of SV identity with single-cell transcriptomes, facilitating the measurement of SV impact on gene expression. We anticipate that Genome-Shuffle-seq will be broadly useful for the systematic exploration of the functional consequences of SVs on gene expression, the chromatin landscape, and three-dimensional nuclear architecture, while also initiating a path toward a minimal mammalian genome.

Gene expression or gene regulation studies often assume one gene expresses one mRNA. However, contrary to the conventional idea, a single gene in mammalian cells can express multiple transcript variants translated into several different proteins. The transcript variants are generated through transcription from alternative start sites and alternative post-transcriptional processing of the precursor mRNA (pre-mRNA). In addition, gene mutations and RNA editing further enhance the diversity of the transcript variants. The transcript variants can encode proteins with various domains, expanding the functional repertoire of a single gene. Some transcript variants may not encode proteins but function as non-coding RNAs and regulate gene expression. The expression level of the transcript variants may vary between cell types or within the same cells under different biological conditions. Transcript variants are characteristic of cell differentiation in a particular tissue, and the variants may play a key role in normal development and aging. Studies also reported that some transcript variants may have roles in disease pathogenesis. The biological significances urge studying the complexity of gene expression at the transcript level. This article updates the molecular basis of transcript variants in mammalian cells, including the formation mechanisms and potential roles in host biology. Gaining insight into the transcript variants will not only identify novel mechanisms of gene regulation but also unravel the role of the variants in health and disease.

Single-molecule tracking (SMT) has emerged as the dominant technology to investigate the dynamics of chromatin-transcription factor (TF) interactions. How long a TF needs to bind to a regulatory site to elicit a transcriptional response is a fundamentally important question. However, highly divergent estimates of TF binding have been presented in the literature, stemming from differences in photobleaching correction and data analysis. TF movement is often interpreted as specific or non-specific association with chromatin, yet the dynamic nature of the chromatin polymer is often overlooked. In this perspective, we highlight how recent SMT studies have reshaped our understanding of TF dynamics, chromatin mobility, and genome organization in the mammalian nucleus, focusing on the technical details and biological implications of these approaches. In a remarkable convergence of fixed and live-cell imaging, we show how super-resolution and SMT studies of chromatin have dovetailed to provide a convincing nanoscale view of genome organization., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Enhancer clusters, pivotal in mammalian development and diseases, can organize as enhancer networks to control cell identity and disease genes; however, the underlying mechanism remains largely unexplored. Here, we introduce eNet 2.0, a comprehensive tool for enhancer networks analysis during development and diseases based on single-cell chromatin accessibility data. eNet 2.0 extends our previous work eNet 1.0 by adding network topology, comparison and dynamics analyses to its network construction function. We reveal modularly organized enhancer networks, where inter-module interactions synergistically affect gene expression. Moreover, network alterations correlate with abnormal and dynamic gene expression in disease and development. eNet 2.0 is robust across diverse datasets. To facilitate application, we introduce eNetDB (https://enetdb.huanglabxmu.com), an enhancer network database leveraging extensive scATAC-seq (single-cell assay for transposase-accessible chromatin sequencing) datasets from human and mouse tissues. Together, our work provides a powerful computational tool and reveals that modularly organized enhancer networks contribute to gene expression robustness in mammalian development and diseases., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Semi-aquatic mammals represent a transitional phase in the evolutionary spectrum between terrestrial and aquatic mammals. The sense of balance is crucial for mammalian locomotion, and in semi-aquatic mammals, the structural foundation of this sense (the vestibular system) shows distinct morphological adaptations to both aquatic and terrestrial environments compared to their terrestrial counterparts. Despite this, the precise molecular mechanisms driving these adaptations remain elusive. Our study endeavors to unravel the genetic components associated with the sense of balance in semi-aquatic mammals and to examine the evolutionary trajectories of these genes, shed light on the molecular mechanisms underlying the adaptive evolution of balance perception in semi-aquatic mammals., Results: We selected 42 mammal species across 20 orders, 38 families, and 42 genera for analysis. We analyzed a comprehensive set of 116 genes related to the vestibular system's development or function. Our findings indicate that 27 of these genes likely experienced adaptive evolution in semi-aquatic mammals. Particularly, genes such as SLC26A2, SOX10, MYCN, and OTX1 are implicated in collectively orchestrating morphological adaptations in the semicircular canals to suit semi-aquatic environments. Additionally, genes associated with otolith development, including SLC26A2, OC90, and OTOP1, likely regulate otolith sensitivity across various locomotor modes. Moreover, genes linked to vestibular disorders, such as GJB2, GJB6, and USH1C, may provide a molecular foundation for averting vertigo amidst intricate locomotor scenarios in semi-aquatic mammals., Conclusions: Our research offers insights into the molecular mechanisms underlying the evolution of the sense of balance in semi-aquatic mammals, while also providing a new research direction for the adaptive evolution of mammals undergoing a secondary transition to an aquatic lifestyle., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Long unique genomic regions have been reported to be highly enriched for developmental genes in mice and humans. In this paper, we identify unique genomic regions using an efficient method based on fast string matching. We quantify the resource consumption and accuracy of this method before applying it to the genomes of 18 mammals. We annotate their unique regions (URs) of at least 10 kb and find that they are strongly enriched for developmental genes across the board. We then investigated the subset of URs that lack annotations, which we call "anonymous." The longest anonymous UR in the Tasmanian devil spanned 83 kb and contained the gene encoding inositol polyphosphate-5-phosphatase A, which is an essential part of intracellular signaling. This discovery of an essential gene in a UR implies that URs might be given priority when annotating mammalian genomes. Our documented pipeline for annotating URs in any mammalian genome is available from the repository github.com/evolbioinf/auger; the additional data for this study are available from the dataverse at doi.org/10.17617/3.4IKQAG., Competing Interests: Conflicts of interest: The author(s) declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Genome-wide analyses of various taxa have repeatedly shown that immune genes are important targets of positive selection. However, little is known about what factors determine which immune genes are under positive selection. To address this question, we here focus on the mammalian immune system and investigate the importance of gene function and other factors such as gene expression, protein-protein interactions, and overall selective constraint as determinants of positive selection. We compiled a list of >1,100 immune genes that were divided into six functional categories and analyzed using data from rodents. Genes encoding proteins that are in direct interactions with pathogens, such as pattern recognition receptors (PRRs), are often expected to be key targets of positive selection. We found that categories containing cytokines, cytokine receptors, and other cell surface proteins involved in, for example, cell-cell interactions were at least as important targets as PRRs, with three times higher rate of positive selection than nonimmune genes. The higher rate of positive selection on cytokines and cell surface proteins was partly an effect of these categories having lower selective constraint. Nonetheless, cytokines had a higher rate of positive selection than nonimmune genes even at a given level of selective constraint, indicating that gene function per se can also be a determinant of positive selection. These results have broad implications for understanding the causes of positive selection on immune genes, specifically the relative importance of host-pathogen coevolution versus other processes., (© The Author(s) 2025. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

5-Methylation (m5C) on mRNA molecules is a prevalent internal posttranscriptional modification in eukaryotes. Although m5C modification has been reported to regulate some biological processes, whether most mRNA m5C modifications are functional is unknown. To address this question, we analyzed the genome-wide evolutionary characteristics of m5C modifications in protein-coding genes of humans and mice. Our analysis of RNA sequencing data from 13 tissues of both species revealed that (i) the occurrence of m5C modification is exceedingly low, (ii) the fraction of m5Cs decreases with the amount of Cs across genes or tissues, (iii) m5C modifications are mostly unshared between species, and (iv) m5C sites and motifs do not exhibit greater evolutionary conservation. Additionally, we estimate that a large fraction of the observed mRNA m5C modifications may be deleterious. Together, these observations suggest that most m5C modifications in mammalian mRNAs are nonadaptive, which has important implications for understanding the biological significance of m5C and other posttranscriptional modifications., Competing Interests: Conflict of Interest: The authors declare no conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2025. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Combinations of transcription factors govern the identity of cell types, which is reflected by genomic enhancer codes. We used deep learning to characterize these enhancer codes and devised three metrics to compare cell types in the telencephalon across amniotes. To this end, we generated single-cell multiome and spatially resolved transcriptomics data of the chicken telencephalon. Enhancer codes of orthologous nonneuronal and γ-aminobutyric acid-mediated (GABAergic) cell types show a high degree of similarity across amniotes, whereas excitatory neurons of the mammalian neocortex and avian pallium exhibit varying degrees of similarity. Enhancer codes of avian mesopallial neurons are most similar to those of mammalian deep-layer neurons. With this study, we present generally applicable deep learning approaches to characterize and compare cell types on the basis of genomic regulatory sequences.

In recent decades, the field of synthetic biology has witnessed remarkable progress, driving advances in both research and practical applications. One pivotal area of development involves the design of transgene switches capable of precisely regulating specified outputs and controlling cell behaviors in response to physical cues, which encompass light, magnetic fields, temperature, mechanical forces, ultrasound, and electricity. In this review, we delve into the cutting-edge progress made in the field of physically controlled protein expression in engineered mammalian cells, exploring the diverse genetic tools and synthetic strategies available for engineering targeting cells to sense these physical cues and generate the desired outputs accordingly. We discuss the precision and efficiency limitations inherent in these tools, while also highlighting their immense potential for therapeutic applications., Competing Interests: Declaration of interest The authors declare no competing interest., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The DNA methylation field has matured from a phase of discovery and genomic characterization to one seeking deeper functional understanding of how this modification contributes to development, ageing and disease. In particular, the past decade has seen many exciting mechanistic discoveries that have substantially expanded our appreciation for how this generic, evolutionarily ancient modification can be incorporated into robust epigenetic codes. Here, we summarize the current understanding of the distinct DNA methylation landscapes that emerge over the mammalian lifespan and discuss how they interact with other regulatory layers to support diverse genomic functions. We then review the rising interest in alternative patterns found during senescence and the somatic transition to cancer. Alongside advancements in single-cell and long-read sequencing technologies, the collective insights made across these fields offer new opportunities to connect the biochemical and genetic features of DNA methylation to cell physiology, developmental potential and phenotype., Competing Interests: Competing interests: A.M. and Z.D.S. are inventors on a patent related to hypermethylated CGI targets in cancer. Z.D.S. and A.M. are co-founders and scientific advisers of Harbinger Health. S.H. declares no competing interests., (© 2024. Springer Nature Limited.)

Mutations can be beneficial by bringing innovation to their bearer, allowing them to adapt to environmental change. These mutations are typically unpredictable since they respond to an unforeseen change in the environment. However, mutations can also be beneficial because they are simply restoring a state of higher fitness that was lost due to genetic drift in a stable environment. In contrast to adaptive mutations, these beneficial non-adaptive mutations can be predicted if the underlying fitness landscape is stable and known. The contribution of such non-adaptive mutations to molecular evolution has been widely neglected mainly because their detection is very challenging. We have here reconstructed protein-coding gene fitness landscapes shared between mammals, using mutation-selection models and a multi-species alignments across 87 mammals. These fitness landscapes have allowed us to predict the fitness effect of polymorphisms found in 28 mammalian populations. Using methods that quantify selection at the population level, we have confirmed that beneficial non-adaptive mutations are indeed positively selected in extant populations. Our work confirms that deleterious substitutions are accumulating in mammals and are being reverted, generating a balance in which genomes are damaged and restored simultaneously at different loci. We observe that beneficial non-adaptive mutations represent between 15% and 45% of all beneficial mutations in 24 of 28 populations analyzed, suggesting that a substantial part of ongoing positive selection is not driven solely by adaptation to environmental change in mammals., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Latrille et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Mammalia comprises a great diversity of diet types and associated adaptations. An understanding of the genomic mechanisms underlying these adaptations may offer insights for improving human health. Comparative genomic studies of diet that employ taxonomically restricted analyses or simplified diet classifications may suffer reduced power to detect molecular convergence associated with diet evolution. Here, we use a quantitative carnivory score-indicative of the amount of animal protein in the diet-for 80 mammalian species to detect significant correlations between the relative evolutionary rates of genes and changes in diet. We have identified six genes- ACADSB , CLDN16 , CPB1 , PNLIP , SLC13A2 , and SLC14A2 -that experienced significant changes in evolutionary constraint alongside changes in carnivory score, becoming less constrained in lineages evolving more herbivorous diets. We further consider the biological functions associated with diet evolution and observe that pathways related to amino acid and lipid metabolism, biological oxidation, and small molecule transport experienced reduced purifying selection as lineages became more herbivorous. Liver and kidney functions show similar patterns of constraint with dietary change. Our results indicate that these functions are important for the consumption of animal matter and become less important with the evolution of increasing herbivory. So, genes expressed in these tissues experience a relaxation of evolutionary constraint in more herbivorous lineages., (© 2024 Pollard et al.; Published by Cold Spring Harbor Laboratory Press.)

Growing evidence suggests that somatic mutations may be a major cause of the aging process. However, it remains to be tested whether the predictions of the theory also apply to species with longer life spans than humans. Hydra is a genus of freshwater polyps with remarkable regeneration abilities and a potentially unlimited life span under laboratory conditions. By genome sequencing of single cells and whole animals, we found that the mutation rates in Hydra 's stem cells are even slightly higher than in humans or mice. A potential explanation for this deviation from the prediction of the theory may lie in the adaptability offered by a higher mutation rate, as we were able to show that the genome of the widely studied Hydra magnipapillata s train 105 has undergone a process of strong positive selection since the strain's cultivation 50 years ago. This most likely represents a rapid adaptation to the drastically altered environmental conditions associated with the transition from the wild to laboratory conditions. Processes under positive selection in captive animals include pathways associated with Hydra 's simple nervous system, its nucleic acid metabolic process, cell migration, and hydrolase activity., (© 2024 Sahm et al.; Published by Cold Spring Harbor Laboratory Press.)

Taking into consideration recent reports on the successful creation of cloned rhesus monkeys [...].

N 6 -methyladenosine (m6A) is the most prevalent chemical modification in eukaryotic mRNAs and plays key roles in diverse cellular processes. Precise localization of m6A sites is thus critical for characterizing the functional roles of m6A in various conditions and dissecting the mechanisms governing its deposition. Here, we design a combined framework of Transformer architecture and recurrent neural network, deepSRAMP, to identify m6A sites using sequence-based and genome-derived features. As a result, deepSRAMP achieves a notably enhanced performance compared to its predecessor, SRAMP, the most-used predictor in this field. Moreover, based on multiple benchmark datasets, deepSRAMP greatly outperforms other state-of-the-art m6A predictors, including WHISTLE and DeepPromise, with an average 16.1% and 18.3% increase in AUROC and a 43.9% and 46.4% increase in AUPRC. Finally, deepSRAMP can be successfully exploited on mammalian m6A epitranscriptome mapping under diverse cellular conditions and can potentially reveal differential m6A sites among transcript isoforms of individual genes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Genes encoded within organelle genomes often evolve at rates different from those in the nuclear genome. Here, we analyzed the relative rates of nucleotide substitution in the mitochondrial, apicoplast, and nuclear genomes in four different lineages of Plasmodium species (malaria parasites) infecting mammals. The rates of substitution in the three genomes exhibit substantial variation among lineages, with the relative rates of nuclear and mitochondrial DNA being particularly divergent between the Laverania (including Plasmodium falciparum) and Vivax lineages (including Plasmodium vivax). Consideration of synonymous and nonsynonymous substitution rates suggests that their variation is largely due to changes in mutation rates, with constraints on amino acid replacements remaining more similar among lineages. Mitochondrial DNA mutation rate variations among lineages may reflect differences in the long-term average lengths of the sexual and asexual stages of the life cycle. These rate variations have far-reaching implications for the use of molecular clocks to date Plasmodium evolution., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Protamines (PRMs) and transition nuclear proteins (TNPs) are two key classes of sperm nuclear basic proteins that regulate chromatin reorganization and condensation in the spermatozoon head, playing crucial roles in mammalian spermatogenesis. In scrotal mammals, such as humans, cryptorchidism, the failure of the testes to descend into the scrotal sac is generally associated with higher rates of defective spermatozoon quality and function. However, ascrotal mammals, such as cetaceans, with naturally undescended testes, produce normal spermatozoa similar to their scrotal counterparts. This study investigates the evolutionary pattern and functional changes in PRMs and TNPs to explore the potential molecular mechanisms underlying spermatogenesis in naturally ascrotal mammals. Although we found a conserved genomic arrangement for PRM and TNP genes across mammals, the coevolutionary loss of intact PRM2 and TNP2 was observed in several species, correlating significantly with diverse testicular positions. Notably, in cetaceans, which lack intact PRM2 and TNP2, we detected enhanced thermostability and DNA binding in PRM1, along with superior DNA repair capability in TNP1. These findings suggest that gene loss of PRM2 and TNP2, combined with functional enhancements in PRM1 and TNP1 proteins, evolved in response to physiological challenges posed by natural cryptorchidism in most ascrotal lineages. This evolutionary strategy enhances chromatin condensation efficiency and promotes DNA repair during spermatogenesis in natural cryptorchid mammals, supporting the Black Queen Hypothesis., Competing Interests: Conflict of Interest Authors declare that they have no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

The totipotent one-cell embryo, or zygote, gives rise to all germ layers and extraembryonic tissues that culminate in the development of a new organism. A zygote is produced at fertilisation by the fusion of differentiated germ cells, egg and sperm. The chromatin of parental genomes is reprogrammed and spatially reorganised in the early embryo. The 3D chromatin organisation is established de novo after fertilisation by a cohesin-dependent mechanism of loop extrusion that forms chromatin loops and topologically associating domains (TADs). Strengthening of TAD insulation is concomitant with the transcriptional 'awakening' of the embryo known as zygotic genome activation (ZGA). Whether and how these processes are causally linked remains poorly understood. In this review, we discuss recent findings of 3D chromatin organisation in mammalian gametes and embryos and how these are potentially related to ZGA., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The PhiC31 integration system allows for targeted and efficient transgene integration and expression by recognizing pseudo attP sites in mammalian cells and integrating the exogenous genes into the open chromatin regions of active chromatin. In order to investigate the regulatory patterns of efficient gene expression in the open chromatin region of PhiC31 integration, this study utilized Ubiquitous Chromatin Opening Element (UCOE) and activating RNA (saRNA) to modulate the chromatin structure in the promoter region of the PhiC31 integration vector. The study analysed the effects of DNA methylation and nucleosome occupancy changes in the integrated promoter on gene expression levels. The results showed that for the OCT4 promoter with moderate CG density, DNA methylation had a smaller impact on expression compared to changes in nucleosome positioning near the transcription start site, which was crucial for enhancing downstream gene expression. On the other hand, for the SOX2 promoter with high CG density, increased methylation in the CpG island upstream of the transcription start site played a key role in affecting high expression, but the positioning and clustering of nucleosomes also had an important influence. In conclusion, analysing the DNA methylation patterns, nucleosome positioning, and quantity distribution of different promoters can determine whether the PhiC31 integration site possesses the potential to further enhance expression or overcome transgene silencing effects by utilizing chromatin regulatory elements.

Transient gene expression (TGE) in mammalian cells is a well-known approach to the fast expression of recombinant proteins. The human cell line HEK (human embryonic kidney) 293F is widely used in this field, due to its adaptability to grow in suspension to high cell densities in serum-free media, amenability to transfection, and production of recombinant proteins in satisfactory quantities for functional and structural analysis. Amounts of plasmid DNA (pDNA) required in transfections for TGE remain high (usually 1 µg pDNA/mL, or even higher), representing a noticeable proportion of the overall cost. Thus, there is an economic need to reduce amounts of coding pDNA in TGE processes. In this work, amounts of both pDNA and transfecting agent used for TGE in HEK 293F cells have been explored in order to reduce them without compromising (or even improving) the productivity of the process in terms of protein yield. In our hands, minimal polyethyleneimine (PEI) cytotoxicity and optimum protein yields were obtained when transfecting at 0.5 µg pDNA/mL (equal to 0.5 µg pDNA/million cells) and a DNA-to-PEI ratio of 1:3, a trend confirmed for several unrelated recombinant proteins. Thus, carefully tuning pDNA and transfecting agent amounts not only reduces the economic costs but also results in higher recombinant protein yields. These results surely have a direct application and interest for the biopharmaceutical industry, always concerned in increasing productivity while decreasing economic costs. KEY POINTS: • Mammalian cells are widely used to produce recombinant proteins in short times. • Tuning DNA and transfecting agent are of great interest to optimize economic costs. • Reducing DNA and transfecting agent amounts result in higher protein yields., (© 2024. The Author(s).)

Mammalian genome research has conventionally involved mice and rats as model organisms for humans. Given the recent advances in life science research, to understand complex and higher-order biological phenomena and to elucidate pathologies and develop therapies to promote human health and overcome diseases, it is necessary to utilize not only mice and rats but also other bioresources such as standardized genetic materials and appropriate cell lines in order to gain deeper molecular and cellular insights. The Japanese bioresource infrastructure program called the National BioResource Project (NBRP) systematically collects, preserves, controls the quality, and provides bioresources for use in life science research worldwide. In this review, based on information from a database of papers related to NBRP bioresources, we present the bioresources that have proved useful for mammalian genome research, including mice, rats, other animal resources; DNA-related materials; and human/animal cells and microbes., (© 2024. The Author(s).)

Transgenerational epigenetic inheritance in mammals remains a controversial phenomenon. A recent study by Takahashi et al. provides evidence for this mode of inheritance in mice by using a CRISPR/Cas9-based epigenetic editing technique to modify DNA methylation levels at specific promoters and then demonstrating the inheritance of the gain in methylation in offspring. In this technical commentary, we argue that the method used in the original study inherently amplifies the likelihood of genetic changes that thereafter lead to the heritability of epigenetic changes. We provide evidence that genetic changes from multiple sources do indeed occur in these experiments and explore several avenues by which these changes could be causal to the apparent inheritance of epigenetic changes. We conclude a genetic basis of inheritance cannot be ruled out and thus transgenerational epigenetic inheritance has not been adequately established by the original study.

The International Mouse Phenotyping Consortium (IMPC) is a worldwide effort producing and phenotyping knockout mouse lines to expose the pathophysiological roles of all genes in human diseases and make mice and data available and accessible to the global research community. It has created new knowledge on the function of thousands of genes for which little to anything was known. This new knowledge has informed the genetic basis of rare diseases, posited gene product influences on common diseases, influenced research on targeted therapies, revealed functional pleiotropy, essentiality, and sexual dimorphism, and many more insights into the role of genes in health and disease. Its scientific contributions have been many and widespread, however there remain thousands of "dark" genes yet to be illuminated. Nearing the end of its current funding cycle, IMPC is at a crossroads. The vision forward is clear, the path to proceed less so., (© 2024. The Author(s).)

While transgenerational epigenetic inheritance has been extensively documented in plants, nematodes, and fruit flies, its existence in mammals remains controversial. Several factors have contributed to this debate, including the lack of a clear distinction between intergenerational and transgenerational epigenetic inheritance (TEI), the inconsistency of some studies, the potential confounding effects of in-utero vs. epigenetic factors, and, most importantly, the biological challenge of epigenetic reprogramming. Two waves of epigenetic reprogramming occur: in the primordial germ cells and the developing embryo after fertilization, characterized by global erasure of DNA methylation and remodelling of histone modifications. Consequently, TEI can only occur if specific genetic regions evade this reprogramming and persist through embryonic development. These challenges have revived the long-standing debate about the possibility of inheriting acquired traits, which has been strongly contested since the Lamarckian and Darwinian eras. As a result, coupled with the absence of universally accepted criteria for transgenerational epigenetic studies, a vast body of literature has emerged claiming evidence of TEI. Therefore, the goal of this study is to advocate for establishing fundamental criteria that must be met for a study to qualify as evidence of TEI. We identified five criteria based on the consensus of studies that critically evaluated TEI. To assess whether published original research papers adhere to these criteria, we examined 80 studies that either claimed or were cited as supporting TEI. The findings of this analysis underscore the widespread confusion in this field and highlight the urgent need for a unified scientific consensus on TEI requirements.

When and why did variations in placental structure and function evolve? Such questions cannot be addressed without a reliable version of mammalian phylogeny. Twenty-five years ago, the mammalian tree was reshaped by molecular phylogenetics. Soon it was shown, in contrast to prevailing theories, that the common ancestor of placental mammals had invasive placentation. Subsequently, evolution of many other features of extraembryonic membranes was addressed. This endeavour stimulated research to fill gaps in our knowledge of placental morphology. Last year the mammalian tree was again revised based on a large set of genomic data. With that in mind, this review provides an update on placentation in the nineteen orders of placental mammals, incorporating much recent data. The principal features such as shape, interdigitation, the interhaemal barrier and the yolk sac are summarized in synoptic tables. The evolution of placental traits and its timing is then explored by reference to the revised mammalian tree. Examples are the early appearance of epitheliochorial placentation in the common ancestor of artiodactyls, perissodactyls, pangolins and carnivores (with reversion to invasive forms in the latter) and later refinements such as the binucleate trophoblast cells and placentomes of ruminants. In primates, the intervillous space gradually evolved from the more basic labyrinth whereas trophoblast invasion of the decidua was a late development in humans and great apes. Only seldom can we glimpse the "why" of placental evolution. The best examples concern placental hormones, including some striking examples of convergent evolution such as the chorionic gonadotropins of primates and equids. In concluding, I review current ideas about what drives placental evolution and identify significant gaps in our knowledge of placentation, including several relevant to the evolution of placentation in primates., Competing Interests: Declaration of competing interest The author declares that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author. Published by Elsevier Inc. All rights reserved.)

A largely stable genome is required for normal development, even as genetic change is an integral aspect of reproduction, genetic adaptation and evolution. Recent studies highlight a critical window of mammalian development with intrinsic DNA replication stress and genome instability in the first cell divisions after fertilization. Patterns of DNA replication and genome stability are established very early in mammals, alongside patterns of nuclear organization, and before the emergence of gene expression patterns, and prior to cell specification and germline formation. The study of DNA replication and genome stability in the mammalian embryo provides a unique cellular system due to the resetting of the epigenome to a totipotent state, and the de novo establishment of the patterns of nuclear organization, gene expression, DNA methylation, histone modifications and DNA replication. Studies on DNA replication and genome stability in the early mammalian embryo is relevant for understanding both normal and disease-causing genetic variation, and to uncover basic principles of genome regulation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The impact of negative selection against deleterious mutations in endangered species remains underexplored. Recent studies have measured mutation load by comparing the accumulation of deleterious mutations, however, this method is most effective when comparing within and between populations of phylogenetically closely related species. Here, we introduced new statistics, LDcor, and its standardized form nLDcor, which allows us to detect and compare global linkage disequilibrium of deleterious mutations across species using unphased genotypes. These statistics measure averaged pairwise standardized covariance and standardize mutation differences based on the standard deviation of alleles to reflect selection intensity. We then examined selection strength in the genomes of seven mammals. Tigers exhibited an over-dispersion of deleterious mutations, while gorillas, giant pandas, and golden snub-nosed monkeys displayed negative linkage disequilibrium. Furthermore, the distribution of deleterious mutations in threatened mammals did not reveal consistent trends. Our results indicate that these newly developed statistics could help us understand the genetic burden of threatened species., Competing Interests: Disclosure and competing interests statement. The authors declare no competing interests., (© 2024. The Author(s).)

Telomeres are pivotal determinants of cell stemness, organismal aging, and lifespan. Herein, we examined similarities in telomeres of Arabidopsis thaliana, mice, and humans. We report the common traits, which include their composition in multimers of TTAGGG sequences and their protection by specialized proteins. Moreover, given the link between telomeres, on the one hand, and cell proliferation and stemness on the other, we discuss the counterintuitive convergence between plants and mammals in this regard, focusing on the impact of niches on cell stemness. Finally, we suggest that tackling the study of telomere function and cell stemness by taking into consideration both plants and mammals can aid in the understanding of interconnections and contribute to research focusing on aging and organismal lifespan determinants., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Vertebrate development and phylogeny are intimately connected through the vertebral formula, the numerical distribution of vertebrae along the body axis into different categories such as neck and chest. A key window into this relationship is through the conserved Hox gene clusters. Hox gene expression boundaries align with vertebral boundaries, and their manipulation in model organisms often results in the transformation of one vertebral type into its neighbor, a homeotic transformation. If the variety in the vertebrate body plan is produced by homeotic shifts, then the number of adjacent vertebrae will be inversely related when making interspecies comparisons since the gain in one vertebra is due to the loss in its neighbor. To date, such a pattern across species consistent with homeotic transitions has only been found in the thoracolumbar vertebral count of mammals. To further investigate potential homeotic relationships in other vertebrate classes and along the entire body axis, we compiled a comprehensive dataset of complete tetrapod vertebral formulas and systematically searched for patterns by analyzing combinations of vertebrae. We uncovered mammalian homeotic patterns and found balances between distal vertebrae not anticipated by a Hox -vertebral homeotic relationship, including one that emerged during the progression from theropods to birds. We also identified correlations between vertebral counts and intergenic distances in the HoxB gene cluster which do not align with the common picture of a colinear relationship between Hox expression and vertebral categories. This quantitative approach revises our expectations for the diversity of a Hox -mediated vertebrate body plan., Competing Interests: Competing interests statement:The authors declare no competing interest.

High-altitude and marine mammals inhabit distinct ecosystems but share a common challenge: hypoxia. To survive in low-oxygen environments, these species have evolved similar phenotypic pulmonary adaptations, characterized by a high density of elastic fibers. In this study, we explored the molecular mechanisms underlying these adaptations, focusing on pulmonary fibrosis and hypoxia tolerance through comparative genomics and convergent evolution analyses. We observed significant expansions and contractions in certain gene families across both high-altitude and marine mammals, closely associated with processes involved in pulmonary fibrosis. Notably, members of the keratin gene family, such as KRT17 and KRT14 , appear to be associated with the development of the dense elastic fiber phenotype observed in the lungs of hypoxia-tolerant mammals. Through selection pressure and amino acid substitution analyses, we identified multiple genes exhibiting convergent accelerated evolution, positive selection, and amino acid substitution in these species, associated with adaptation to hypoxic environments. Specifically, the convergent evolution of ZFP36L1 , FN1 , and NEDD9 was found to contribute to the high density of elastic fibers in the lungs of both high-altitude and marine mammals, facilitating their hypoxia tolerance. Additionally, we identified convergent amino acid substitutions and gene loss events associated with sperm development, differentiation, and spermatogenesis, such as amino acid substitutions in SLC26A3 and pseudogenization of CFAP47 , as confirmed by PCR. These genetic alterations may be linked to changes in the reproductive capabilities of these animals. Overall, this study offers novel perspectives on the genetic and molecular adaptations of high-altitude and marine mammals to hypoxic environments, with a particular emphasis on pulmonary fibrosis.

PIWI-interacting RNAs (piRNAs), which associate with PIWI clade Argonaute proteins to form piRNA-induced silencing complexes (piRISCs) in germline cells, are responsible for maintaining genomic integrity and reproductive function through transcriptional or post-transcriptional suppression of transposable elements and regulation of protein-coding genes. Recent discoveries of crucial PIWI-piRNA functions in oogenesis and embryogenesis in golden hamsters suggest an indispensable role in female fertility that has been obscured in the predominant mouse model of PIWI-piRNA pathway regulation. In particular, studies of piRNA expression dynamics, functional redundancies, and compositional variations across mammal species have advanced our understanding of piRNA functions in male and, especially, female reproduction. These findings further support the use of hamsters as a more representative model of piRNA biology in mammals. In addition to discussing these new perspectives, the current review also covers emerging directions for piRNA research, its implications for female fertility, and our fundamental understanding of reproductive mechanisms.

Spermatogenesis is a highly coordinated process that requires the precise expression of specific subsets of genes in different types of germ cells, controlled both temporally and spatially. Among these genes, those that can exert an indispensable influence in spermatogenesis via participating in alternative splicing make up the overwhelming majority. mRNA alternative-splicing (AS) events can generate various isoforms with distinct functions from a single DNA sequence, based on specific AS codes. In addition to enhancing the finite diversity of the genome, AS can also regulate the transcription and translation of certain genes by directly binding to their cis-elements or by recruiting trans-elements that interact with consensus motifs. The testis, being one of the most complex tissue transcriptomes, undergoes unparalleled transcriptional and translational activity, supporting the dramatic and dynamic transitions that occur during spermatogenesis. Consequently, AS plays a vital role in producing an extensive array of transcripts and coordinating significant changes throughout this process. In this review, we summarize the intricate functional network of alternative splicing in spermatogenesis based on the integration of current research findings.

Advances in synthetic biology allow the design and manipulation of DNA from the scale of genes to genomes, enabling the engineering of complex genetic information for application in biomanufacturing, biomedicine and other areas. The transfer and subsequent maintenance of large DNA are two core steps in large scale genome rewriting. Compared to small DNA, the high molecular weight and fragility of large DNA make its transfer and maintenance a challenging process. This review outlines the methods currently available for transferring and maintaining large DNA in bacteria, fungi, and mammalian cells. It highlights their mechanisms, capabilities and applications. The transfer methods are categorized into general methods (e.g., electroporation, conjugative transfer, induced cell fusion-mediated transfer, and chemical transformation) and specialized methods (e.g., natural transformation, mating-based transfer, virus-mediated transfection) based on their applicability to recipient cells. The maintenance methods are classified into genomic integration (e.g., CRISPR/Cas-assisted insertion) and episomal maintenance (e.g., artificial chromosomes). Additionally, this review identifies the major technological advantages and disadvantages of each method and discusses the development for large DNA transfer and maintenance technologies., Competing Interests: Declaration of competing interest All the authors declare that they have no competing interests in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Originating 30 million years ago, shrews (Soricidae) have diversified into around 400 species worldwide. Shrews display a wide array of adaptations, with some species having developed distinctive traits such as echolocation, underwater diving, and venomous saliva. Accordingly, these tiny insectivores are ideal to study the genomic mechanisms of evolution and adaptation. We conducted a comparative genomic analysis of four shrew species and 16 other mammals to identify genomic variations unique to shrews. Using two existing shrew genomes and two de novo assemblies for the maritime (Sorex maritimensis) and smoky (Sorex fumeus) shrews, we identified mutations in conserved regions of the genomes, also known as accelerated regions, gene families that underwent significant expansion, and positively selected genes. Our analyses unveiled shrew-specific genomic variants in genes associated with the nervous, metabolic, and auditory systems, which can be linked to unique traits in shrews. Notably, genes suggested to be under convergent evolution in echolocating mammals exhibited accelerated regions in shrews, and pathways linked to putative body size plasticity were detected. These findings provide insight into the evolutionary mechanisms shaping shrew species, shedding light on their adaptation and divergence over time., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Obesity and anorexia are life-threatening diseases that are still poorly understood at the genetic and neuronal levels. Patients suffering from these conditions experience disrupted regulation of food consumption, leading to extreme weight gain or loss and, in severe situations, death from metabolic dysfunction. Despite the development of various behavioral and pharmacological interventions, current treatments often yield limited and short-lived success. To address this, a deeper understanding of the genetic and neural mechanisms underlying food perception and appetite regulation is essential for identifying new drug targets and developing more effective treatment methods. This review summarizes the progress of past research in understanding the genetic and neural mechanisms controlling food consumption and appetite regulation, focusing on two key model organisms: the fruit fly Drosophila melanogaster and the mouse Mus musculus . These studies investigate how the brain senses energy and nutrient deficiency, how sensory signals trigger appetitive behaviors, and how food intake is regulated through interconnected neural circuits in the brain.

Satellite cells are an important cellular model for studying muscle growth and development and mammalian locomotion-related molecular mechanisms. In this study, we investigated the effects of voltage, pulse duration, and DNA dosage on horse skeletal muscle satellite cells' electroporation transfection efficiency using the eukaryotic expression plasmid Td Tomato-C1 (5.5 kb) encoding the red fluorescent protein gene mainly based on fluorescence-positive cell rate and cell survival rate. By comparison of different voltages, pulse durations, and DNA doses, horse skeletal muscle satellite cells have nearly 80% transfection efficiency under the condition of voltage 120 V, DNA dosage 7 µg/ml, and pulse duration 30 ms. This optimized electroporation condition would facilitate the application of horse skeletal muscle satellite cells in genetic studies of muscle function and related diseases.

The quality of Recombination signal sequences (RSSs), location, and genetics of mammalian V, D, and J genes synergistically affect the recombination frequency of genes; however, the specific regulatory mechanism and efficiency have not been elucidated. By taking advantage of single-cell RNA-sequencing (scRNA-seq) and high-throughput sequencing (HTS) to investigate V(D)J rearrangement characteristics in the CDR3 repertoire, we found that the distal and proximal V genes (or J genes) "to D" gene were involved in rearrangement significantly more frequently than the middle V genes (or J genes) in the TRB locus among various species, including Primates (human and rhesus monkey), Rodentia (BALB/c, C57BL/6, and Kunming mice), Artiodactyla (buffalo), and Chiroptera (Rhinolophus affinis). The RSS quality of the V and J genes affected their frequency in rearrangement to varying degrees, especially when the V-RSSs with recombination signal information content (RIC) score < -45 significantly reduced the recombination frequency of the V gene. The V and J genes that were "away from D" had the dual advantages of recombinant structural accessibility and relatively high-quality RSSs, which promoted their preferential utilization in rearrangement. The quality of J-RSSs formed during mammalian evolution was apparently greater than that of V-RSSs, and the D-J distance was obviously shorter than that of V-D, which may be one of the reasons for guaranteeing that the "D-to-J preceding V-to-DJ rule" occurred when rearranged. This study provides a novel perspective on the mechanism and efficiency of V-D-J rearrangement in the mammalian TRB locus, as well as the biased utilization characteristics and application of V and J genes in the initial CDR3 repertoire., (© 2024. The Author(s).)

Endosperm in angiosperms and placenta in eutherians are convergent innovations for efficient embryonic nutrient transfer. Despite advantages, this reproductive strategy incurs metabolic costs that maternal parents disproportionately shoulder, leading to potential inter-parental conflict over optimal offspring investment. Genomic imprinting-parent-of-origin-biased gene expression-is fundamental for endosperm and placenta development and has convergently evolved in angiosperms and mammals, in part, to resolve parental conflict. Here, we review the mechanisms of genomic imprinting in these taxa. Despite differences in the timing and spatial extent of imprinting, these taxa exhibit remarkable convergence in the molecular machinery and genes governing imprinting. We then assess the role of parental conflict in shaping evolution within angiosperms and eutherians using four criteria: 1) Do differences in the extent of sibling relatedness cause differences in the inferred strength of parental conflict? 2) Do reciprocal crosses between taxa with different inferred histories of parental conflict exhibit parent-of-origin growth effects? 3) Are these parent-of-origin growth effects caused by dosage-sensitive mechanisms and do these loci exhibit signals of positive selection? 4) Can normal development be restored by genomic perturbations that restore stoichiometric balance in the endosperm/placenta? Although we find evidence for all criteria in angiosperms and eutherians, suggesting that parental conflict may help shape their evolution, many questions remain. Additionally, myriad differences between the two taxa suggest that their respective biologies may shape how/when/where/to what extent parental conflict manifests. Lastly, we discuss outstanding questions, highlighting the power of comparative work in quantifying the role of parental conflict in evolution., (© The Author(s) 2024. Published by Oxford University Press on behalf of The American Genetic Association. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The XX/XY sex chromosome system is deeply conserved in therian mammals, as is the role of Sry in testis determination, giving the impression of stasis relative to other taxa. However, the long tradition of cytogenetic studies in mammals documents sex chromosome karyotypes that break this norm in myriad ways, ranging from fusions between sex chromosomes and autosomes to Y chromosome loss. Evolutionary conflict, in the form of sexual antagonism or meiotic drive, is the primary predicted driver of sex chromosome transformation and turnover. Yet conflict-based hypotheses are less considered in mammals, perhaps because of the perceived stability of the sex chromosome system. To address this gap, we catalog and characterize all described sex chromosome variants in mammals, test for family-specific rates of accumulation, and consider the role of conflict between the sexes or within the genome in the evolution of these systems. We identify 152 species with sex chromosomes that differ from the ancestral state and find evidence for different rates of ancestral to derived transitions among families. Sex chromosome-autosome fusions account for 79% of all variants whereas documented sex chromosome fissions are limited to three species. We propose that meiotic drive and drive suppression provide viable explanations for the evolution of many of these variant systems, particularly those involving autosomal fusions. We highlight taxa particularly worthy of further study and provide experimental predictions for testing the role of conflict and its alternatives in generating observed sex chromosome diversity., (© The Author(s) 2024. Published by Oxford University Press on behalf of The American Genetic Association. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

PIWI-interacting RNAs (piRNAs) play a crucial role in safeguarding genome integrity by silencing mobile genetic elements. From flies to humans, piRNAs originate from long single-stranded precursors encoded by genomic piRNA clusters. How piRNA clusters form to adapt to genomic invaders and evolve to maintain protection remain key outstanding questions. Here, we generate a roadmap of piRNA clusters across seven species that highlights both similarities and variations. In mammals, we identify transcriptional readthrough as a mechanism to generate piRNAs from transposon insertions (piCs) downstream of genes (DoG). Together with the well-known stress-dependent DoG transcripts, our findings suggest a molecular mechanism for the formation of piRNA clusters in response to retroviral invasion. Finally, we identify a class of dynamic piRNA clusters in humans, underscoring unique features of human germ cell biology. Our results advance the understanding of conserved principles and species-specific variations in piRNA biology and provide tools for future studies., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

One of the two X-chromosomes in female mammals is epigenetically silenced in embryonic stem cells by X-chromosome inactivation. This creates a mosaic of cells expressing either the maternal or the paternal X allele. The X-chromosome inactivation ratio, the proportion of inactivated parental alleles, varies widely among individuals, representing the largest instance of epigenetic variability within mammalian populations. While various contributing factors to X-chromosome inactivation variability are recognized, namely stochastic and/or genetic effects, their relative contributions are poorly understood. This is due in part to limited cross-species analysis, making it difficult to distinguish between generalizable or species-specific mechanisms for X-chromosome inactivation ratio variability. To address this gap, we measure X-chromosome inactivation ratios in ten mammalian species (9531 individual samples), ranging from rodents to primates, and compare the strength of stochastic models or genetic factors for explaining X-chromosome inactivation variability. Our results demonstrate the embryonic stochasticity of X-chromosome inactivation is a general explanatory model for population X-chromosome inactivation variability in mammals, while genetic factors play a minor role., (© 2024. The Author(s).)

CRISPR-dCas9 (dead Cas9 protein) technology, combined with chemical molecules and light-triggered genetic switches, offers customizable control over gene perturbation. However, these simple ON/OFF switches cannot precisely determine the sophisticated perturbation process. Here, we developed a resveratrol and protocatechuic acid-programmed CRISPR-mediated gene remodeling biocomputer (REPA CRISPR ) for conditional endogenous transcriptional regulation of genes in vitro and in vivo. Two REPA CRISPR variants, REPA CRISPRi and REPA CRISPRa , were designed for the logic control of gene inhibition and activation, respectively. We successfully demonstrated the digital computations of single or multiplexed endogenous gene transcription by using REPA CRISPRa . We also established mathematical models to predict the dose-responsive transcriptional levels of a target endogenous gene controlled by REPA CRISPRa . Moreover, high levels of endogenous gene activation in mice mediated by the AND logic gate demonstrated computational control of CRISPR-dCas9-based epigenome remodeling in mice. This CRISPR-based biocomputer expands the synthetic biology toolbox and can potentially advance gene-based precision medicine. A record of this paper's transparent peer review process is included in the supplemental information., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Mammalian cells are suitable hosts for producing recombinant therapeutic proteins, with Chinese hamster ovary (CHO) and human embryonic kidney 293 (HEK293) cells being the most commonly used cell lines. Mammalian cell expression system includes stable and transient gene expression (TGE) system, with the TGE system having the advantages of short cycles and simple operation. By optimizing the TGE system, the expression of recombinant proteins has been significantly improved. Here, the TGE system and the detailed and up-to-date improvement strategies of mammalian cells, including cell line, expression vector, culture media, culture processes, transfection conditions, and co-expression of helper genes, are reviewed. KEY POINTS: • Detailed improvement strategies of transient gene expression system of mammalian cells are reviewed • The composition of transient expression system of mammalian cell are summarized • Proposed optimization prospects for transient gene expression systems., (© 2024. The Author(s).)

Transcriptional enhancers orchestrate cell type- and time point-specific gene expression programs. Genetic variation within enhancer sequences is an important contributor to phenotypic variation including evolutionary adaptations and human disease. Certain genes and pathways may be more prone to regulatory evolution than others, with different patterns across diverse organisms, but whether such patterns exist has not been investigated at a sufficient scale. To address this question, we identified signatures of accelerated sequence evolution in conserved enhancer elements throughout the mammalian phylogeny at an unprecedented scale. While different genes and pathways were enriched for regulatory evolution in different parts of the tree, we found a striking overall pattern of pleiotropic genes involved in gene regulatory and developmental processes being enriched for accelerated enhancer evolution. These genes were connected to more enhancers than other genes, which was the basis for having an increased amount of sequence acceleration over all their enhancers combined. We provide evidence that sequence acceleration is associated with turnover of regulatory function. Detailed study of one acceleration event in an enhancer of HES1 revealed that sequence evolution led to a new activity domain in the developing limb that emerged concurrently with the evolution of digit reduction in hoofed mammals. Our results provide evidence that enhancer evolution has been a frequent contributor to regulatory innovation at conserved developmental signaling genes in mammals., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Cetaceans have evolved unique limb structures, such as flippers, due to genetic changes during their transition to aquatic life. However, the full understanding of the genetic and evolutionary mechanisms behind these changes is still developing. By examining 25 limb-related protein-coding genes across various mammalian species, we compared genetic changes between aquatic mammals, like whales, and other mammals with unique limb structures such as bats, rodents and elephants. Our findings revealed significant modifications in limb-related genes, including variations in the Hox, GDF5 and Evx genes. Notably, a relaxed selection in several key genes was observed, suggesting a lifting of developmental constraints, which might have facilitated the emergence of morphological innovations in cetacean limb morphology. We also uncovered non-synonymous changes, insertions and deletions in these genes, particularly in the polyalanine tract of HOXD13 , which are distinctive to cetaceans or convergent with other aquatic mammals. These genetic variations correlated with the diverse and specialized limb structures observed in cetaceans, indicating a complex interplay of relaxed selection and specific mutations in mammalian limb evolution.

The DNA dependent protein kinase (DNA-PK) initiates non-homologous recombination (NHEJ), the predominate DNA double-strand break (DSBR) pathway in higher vertebrates. It has been known for decades that the enzymatic activity of DNA-PK [that requires its three component polypeptides, Ku70, Ku80 (that comprise the DNA-end binding Ku heterodimer), and the catalytic subunit (DNA-PKcs)] is present in humans at 10-50 times the level observed in other mammals. Here, we show that the high level of DNA-PKcs protein expression appears evolutionarily in mammals between prosimians and higher primates. Moreover, the RNAs encoding the three component polypeptides of DNA-PK are present at similarly high levels in hominids, new-, and old-world monkeys, but expression of these RNAs in prosimians is ∼5-50 fold less, analogous to the levels observed in other non-primate species. This is reminiscent of the appearance of Alu repeats in primate genomes -- abundant in higher primates, but present at much lower density in prosimians. Alu repeats are well-known for their capacity to promote non-allelic homologous recombination (NAHR) a process known to be inhibited by DNA-PK. Nanopore sequence analyses of cultured cells proficient or deficient in DNA-PK revealed an increase of inter-chromosomal translocations caused by NAHR. Although the high levels of DNA-PK in primates may have many functions, we posit that high levels of DNA-PK may function to restrain deleterious NAHR events between Alu elements., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

DNA barcoding and environmental DNA (eDNA) represent significant advances for biomonitoring the world's biodiversity and its threats. However, these methods are highly dependent on the presence of species sequences on molecular databases. Brazil is one of the world's largest and most biologically diverse countries. However, many knowledge gaps still exist for describing, identifying, and monitoring of mammalian biodiversity using molecular methods. We aimed to unravel the patterns of the presence of Brazilian mammal species on molecular databases to improve our understanding of how effectively it would be to monitor them using DNA barcoding and environmental DNA, and contribute to mammalian conservation. We foundt many gaps in molecular databases, with many taxa being poorly represented, particularly from Amazonia, the order Lagomorpha, and arboreal, gomivorous, near extinct, and illegally traded species. Moreover, our analyses revealed that species description year was the most important factor determining the probability of a species to being sequenced. Primates are the group with the highest number of species considered a priority for sequencing due to their high level of combined threats. We highlight where investments are needed to fill knowledge gaps and increase the representativity of species on molecular databases to enable a better monitoring ability of Brazilian mammals encompassing different traits using DNA barcoding and environmental DNA., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Hernani Fernandes Magalhaes de Oliveira reports financial support was provided by Coordenação de Aperfeiçoamento de Ensino Superior (CAPES). If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)