Eukaryotic Research Articles

Untacking small RNA profiling and RNA fragment footprinting: Approaches and challenges in library construction.

Small RNAs (sRNAs) with sizes ranging from 15 to 50 nucleotides (nt) are critical regulators of gene expression control. Prior studies have shown that sRNAs are involved in a broad range of biological processes, such as organ development, tumorigenesis, and epigenomic regulation; however, emerging evidence unveils a hidden layer of diversity and complexity of endogenously encoded sRNAs profile in eukaryotic organisms, including novel types of sRNAs and the previously unknown post-transcriptional RNA modifications. This underscores the importance for accurate, unbiased detection of sRNAs in various cellular contexts. A multitude of high-throughput methods based on next-generation sequencing (NGS) are developed to decipher the sRNA expression and their modifications. Nonetheless, distinct from mRNA sequencing, the data from sRNA sequencing suffer frequent inconsistencies and high variations emanating from the adapter contaminations and RNA modifications, which overall skew the sRNA libraries. Here, we summarize the sRNA-sequencing approaches, and discuss the considerations and challenges for the strategies and methods of sRNA library construction. The pros and cons of sRNA sequencing have significant implications for implementing RNA fragment footprinting approaches, including CLIP-seq and Ribo-seq. We envision that this review can inspire novel improvements in small RNA sequencing and RNA fragment footprinting in future. This article is categorized under: RNA Evolution and Genomics > Computational Analyses of RNA RNA Processing > Processing of Small RNAs Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs.

Rules and impacts of nonsense-mediated mRNA decay in the degradation of long noncoding RNAs.

Nonsense-mediated mRNA decay (NMD) is a quality-control process that selectively degrades mRNAs having premature termination codon, upstream open reading frame, or unusually long 3'UTR. NMD detects such mRNAs and rapidly degrades them during initial rounds of translation in the eukaryotic cells. Since NMD is a translation-dependent cytoplasmic mRNA surveillance process, the noncoding RNAs were initially believed to be NMD-resistant. The sequence feature-based analysis has revealed that many putative long noncoding RNAs (lncRNAs) have short open reading frames, most of which have translation potential. Subsequent transcriptome-based molecular studies showed an association of a large set of such putative lncRNAs with translating ribosomes, and some of them produce stable and functionally active micropeptides. The translationally active lncRNAs typically have relatively longer and unprotected 3'UTR, which can induce their NMD-dependent degradation. This review defines the mechanism and regulation of NMD-dependent degradation of lncRNAs and its impact on biological processes related to the functions of lncRNAs or their encoded micropeptides. This article is categorized under: RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA Turnover and Surveillance > Regulation of RNA Stability RNA in Disease and Development > RNA in Disease.

Molecular determinants of Ras-mTORC2 signaling

Recent research has identified the mechanistic Target of Rapamycin Complex 2 (mTORC2) as a conserved direct effector of Ras proteins. While previous studies suggested the involvement of the Switch I (SWI) effector domain of Ras in binding mTORC2 components, the regulation of the Ras-mTORC2 pathway is not entirely understood. In Dictyostelium, mTORC2 is selectively activated by the Ras protein RasC, and the RasC-mTORC2 pathway then mediates chemotaxis to cAMP and cellular aggregation by regulating the actin cytoskeleton and promoting cAMP signal relay. Here, we investigated the role of specific residues in RasC's SWI, C-terminal allosteric domain, and hypervariable region (HVR) related to mTORC2 activation. Interestingly, our results suggest that RasC SWI residue A31, which was previously implicated in RasC-mediated aggregation, regulates RasC’s specific activation by the Aimless RasGEF. On the other hand, our investigation identified a crucial role for RasC SWI residue T36, with secondary contributions from E38 and allosteric domain residues. Finally, we found that conserved basic residues and the adjacent prenylation site in the HVR, which are crucial for RasC’s membrane localization, are essential for RasC-mTORC2 pathway activation by allowing for both RasC’s own cAMP-induced activation and its subsequent activation of mTORC2. Therefore, our findings revealed new determinants of RasC-mTORC2 pathway specificity in Dictyostelium, contributing to a deeper understanding of Ras signaling regulation in eukaryotic cells.

Helicases DDX5 and DDX17 promote heterogeneity in HBV transcription termination in infected human hepatocytes

Helicases DDX5 and DDX17 promote heterogeneity in HBV transcription termination in infected human hepatocytes

Primary Infection and Supernatants Generated by Pseudomonas aeruginosa ExoY Have Lasting Effects on Cyclic Nucleotide Signaling in Pulmonary Endothelial Cells

Rationale: Pseudomonas aeruginosa is an opportunistic pathogen and a common cause of hospital-acquire pneumonia. This gram-negative bacterium has an arsenal of virulence factors including the type three secretion system (TTSS), which encodes for an injection type needle and exotoxins. One of the four exotoxins, ExoY, is a promiscuous nucleotidylyl cyclase that utilizes F-actin as a cofactor to generate both canonical and non-canonical cyclic nucleotides (cAMP, cGMP and cCMP and cUMP respectively). While it is well known that activation of endogenous adenylyl and guanylyl cyclase leads to intracellular increases in cAMP and cGMP and release into the extracellular space, whether ExoY generated cyclic nucleotides are also effluxed out of the cell into the extracellular space is not known. In addition, it is unclear whether presence of the bacteria themselves affects endogenous cAMP signaling. Further, infection with P. aeruginosa expressing ExoY generates virulent cytotoxic amyloids from lung endothelium; however, whether these cytotoxic amyloids disrupt agonist-induced endogenous cAMP generation is unknown. Thus, we tested the hypothesis that during primary infection, cAMP and cGMP are effluxed into the extracellular space, while the primary infection itself and sterile supernatants from the primary infection attenuate isoproterenol induce increases in cAMP in pulmonary microvascular endothelial cells (PMVECs). Methods: During primary infection, PMVECs were inoculated with PBS (vehicle control) or 3 different strains of P. aeruginosa at an moi 20:1. Strains ExoY+ and ExoYK81M have a fully functional TTSS and express either catalytically active or inactive ExoY, respectively, while a third strain, ExoYTM, is unable to translocate ExoY into eukaryotic cells. During the last 15 minutes of the 4-hour infection, cells were stimulated with vehicle or isoproterenol (1 μM) and rolipram (10 μM) and cAMP and cGMP measured in the lysate and media. In separate experiments, supernatants from a primary infection were generated using the same three strains of bacteria at moi 20:1 and 4 hours after inoculation, the supernatants collected and sterilized using a 0.2 μm filter. Sterilized supernatants were added to naïve PMVECs and during the last 15 minutes of the 4-hour inoculation, cells stimulated with vehicle or isoproterenol (1 μM) and rolipram (10 μM) and cAMP and cGMP measured in the lysate and media. Results: Cyclic AMP and cGMP were elevated in both the lysate and media of PMVECs following 4-hour infection with ExoY+ and the total isoproterenol and rolipram-stimulated cAMP response was attenuated by the presence of bacteria, ExoYK81M and ExoYTM, and further blunted with ExoY+ infection. Further, inoculation with ExoYK81M and ExoYTM supernatants attenuated the total isoproterenol and rolipram-stimulated cAMP response, which was further blunted by ExoY+ supernatants. Conclusions: Overall, we demonstrate PMVECs release a large cAMP and cGMP pool into the extracellular space and both primary infection and supernatants derived from the primary infection attenuate agonist induced elevations in cAMP. HL121513, HL66299. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Exploring factors influencing choice of DNA double-strand break repair pathways

DNA double-strand breaks (DSBs) represent one of the most severe threats to genomic integrity, demanding intricate repair mechanisms within eukaryotic cells. A diverse array of factors orchestrates the complex choreography of DSB signaling and repair, encompassing repair pathways, such as non-homologous end-joining, homologous recombination, and polymerase-θ-mediated end-joining. This review looks into the intricate decision-making processes guiding eukaryotic cells towards a particular repair pathway, particularly emphasizing the processing of two-ended DSBs. Furthermore, we elucidate the transformative role of Cas9, a site-specific endonuclease, in revolutionizing our comprehension of DNA DSB repair dynamics. Additionally, we explore the burgeoning potential of Cas9's remarkable ability to induce sequence-specific DSBs, offering a promising avenue for precise targeting of tumor cells. Through this comprehensive exploration, we unravel the intricate molecular mechanisms of cellular responses to DSBs, shedding light on both fundamental repair processes and cutting-edge therapeutic strategies.

Life at the interface: Engineering bio-nanomaterials through interfacial molecular self-assembly.

Interfacial self-assembly describes the directed organization of molecules and colloids at phase boundaries. Believed to be fundamental to the inception of primordial life, interfacial assembly is exploited by a myriad of eukaryotic and prokaryotic organisms to execute physiologic activities and maintain homeostasis. Inspired by these natural systems, chemists, engineers, and materials scientists have sought to harness the thermodynamic equilibria at phase boundaries to create multi-dimensional, highly ordered, and functional nanomaterials. Recent advances in our understanding of the biophysical principles guiding molecular assembly at gas-solid, gas-liquid, solid-liquid, and liquid-liquid interphases have enhanced the rational design of functional bio-nanomaterials, particularly in the fields of biosensing, bioimaging and biotherapy. Continued development of non-canonical building blocks, paired with deeper mechanistic insights into interphase self-assembly, holds promise to yield next generation interfacial bio-nanomaterials with unique, and perhaps yet unrealized, properties. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Therapeutic Approaches and Drug Discovery > Emerging Technologies.

Effects of nitrogen deficiency and drought stresses on miRNA expressions in Arabidopsis thaliana

MicroRNAs are now known to have an important role in regulating gene expression of eukaryotic organisms. miRNA research in plants gained importance after the discovery that several stress factors alter certain miRNA expressions, which subsequently regulate their target gene expressions and affect development and growth of plants. In this study, two of the widely studied abiotic stress conditions for plants, nitrogen deficiency and drought were used individually and as a combined stress treatment on Arabidopsis thaliana callus tissues to observe the change of expressions in certain miRNAs, when multiple stressors are encountered. Combined stress strongly inhibited callus growth compared to other conditions, while strongly altering certain miRNA expressions. Compared to control, in 7-day stress treated callus, miR165a-3p,b, miR319a,b, miR396b-5p, miR399d and miR827 showed significant downregulation for all stress treatments, while 7-day N deficiency caused miR167 upregulation. Stress treatments for 7 days mostly downregulated miR167c-5p, miR319c, miR399a, miR399e expressions except for the N deficient samples. After 14 days of stress, miR165a-3p,b, miR396a-5p, miR399b, miR399d were downregulated. During 14-day drought and combined stress, miR399a and miR396b-5p expressions were upregulated, respectively. The differences observed in this study between stress responses of 7 and 14-day-long treatments are believed to be valuable to further elucidate the associated molecular mechanisms of miRNAs, determination and validation of miRNA targets, and how plants respond to stress conditions via various genetic regulations.

Discrete-to-continuum models of pre-stressed cytoskeletal filament networks

We introduce a mathematical model for the mechanical behaviour of the eukaryotic cell cytoskeleton. This discrete model involves a regular array of pre-stressed protein filaments that exhibit resistance to enthalpic stretching, joined at cross-links to form a network. Assuming that the inter-cross-link distance is much shorter than the length scale of the cell, we upscale the discrete force balance to form a continuum system of governing equations and deduce the corresponding macroscopic stress tensor. We use these discrete and continuum models to analyse the imposed displacement of a bead placed in the domain, characterizing the cell rheology through the force–displacement curve. We further derive an analytical approximation to the stress and strain fields in the limit of small bead radius, predicting the net force required to generate a given deformation and elucidating the dependency on the microscale properties of the filaments. We apply these models to networks of the intermediate filament vimentin and demonstrate good agreement between predictions of the discrete, continuum and analytical approaches. In particular, our model predicts that the network stiffness increases sublinearly with the filament pre-stress and scales logarithmically with the bead size.

Codon Usage Characteristics and Evolutionary Analysis of Mitochondrial Genome of Winter Squash (Cucurbita maxima Duch.)

Mitochondrial genomes are essential components of eukaryotic cells, understanding their structural characteristics and codon usage bias is critical. Winter squash (Cucurbita maxima Duch ex Poiret) is an economically important cucurbit crop. To clarify its structural characteristics and codon usage bias of mitochondrial genome, as well as the evolutionary characteristics in the Cucurbitaceae family, whole genome sequencing analysis of C. maxima mitochondrial genome was carried out. The results revealed that the total length of the C. maxima mitochondrial genome was 640,814 bp, containing 9,162,318,300 bp of bases. There were 68 annotated genes identified. The mitochondrial coding genes of C. maxima exhibited an average GCall content of 42.57%, with the last position enriched in A/T bases. Codon usage analysis indicated weak codon bias, with the effective codon number (ENC) ranging from 44.22 to 61.00. Correlation analysis and neutrality plot analysis demonstrated a nonsignificant correlation between GC3 and GC12. PR2-plot and ENC-plot analysis further supported the influence of natural selection on the codon preference in the C. maxima mitochondrial genome. Relative synonymous codon usage (RSCU) analysis identified 19 optimal codons, with a preference for U or A as the final base. The analysis of scattered repeat sequences in the genome indicated a high frequency of forward and palindromic repeats in the C. maxima mitochondrial genome. Nucleic acid diversity analysis highlighted rpl5 as the gene with the highest variability. Comparative analysis of mitochondrial structure and phylogenetic tree analysis revealed a close evolutionary relationship between C. maxima and Cucurbita pepo, showing synteny in evolution. Homologous fragment analysis demonstrated a similarity of over 70% between the C. maxima mitochondrial genome and chloroplast genome fragments. These findings provide a foundation for C. maxima identification and phylogenetic studies.

1H, 15N and 13C resonance backbone and side-chain assignments and secondary structure determination of the BRCT domain of Mtb LigA.

The BRCA1 carboxyl-terminal (BRCT) domain, an evolutionarily conserved structural motif, is ubiquitous in a multitude of proteins spanning prokaryotic and eukaryotic organisms. In Mycobacterium tuberculosis (Mtb), BRCT domain plays a pivotal role in the catalytic activity of the NAD+-dependent DNA ligase (LigA). LigA is pivotal in DNA replication, catalyzing the formation of phosphodiester bonds in Okazaki fragments and repairing single-strand breaks in damaged DNA, essential for the survival of Mtb. Structural and functional aspects of LigA unveil its character as a highly modular protein, undergoing substantial conformational changes during its catalytic cycle. Although the BRCT domain of Mtb LigA plays an essential role in DNA binding and protein-protein interactions, the precise mechanism of action remains poorly understood. Unravelling the structure of the BRCT domain holds the promise of advancing our understanding of this pivotal domain. Additionally, it will facilitate further exploration of the protein-protein interactions and enhance our understanding of inter domain interactions within LigA, specifically between BRCT and the Adenylation domain. In this study, we demonstrate the overexpression of the BRCT domain of Mtb LigA and conduct its analysis using solution NMR spectroscopy, revealing a well-folded structure and we present the nearly complete chemical shift assignments of both backbone and sidechains. In addition, a secondary structure prediction by TALOS N predicts BRCT consisting of 3 α-helices and 4 β-sheets, closely resembling the typical structural topology of most BRCT domains.

Design and analysis of novel microelectromechanical system based microgripper for manipulating microbiological species and micro objects

Design and analysis of a pair of novel microelectromechanical system (MEMS) microgrippers having single and multiple sets of jaws respectively, incorporated with displacement amplification mechanism and electrostatic force sensor is presented in this research work. The novelty of the proposed microgrippers is their unique and efficient designs, having the versatility of gripping ranges. The size of the microgripper is 3.2 × 3.7 mm2 in both cases and has one and three sets of jaws with gripping ranges of 30–64 µm, 175–200 µm, and 350–380 µm respectively. The three jaw sets are application-specific to the multicellular small organism, eukaryotic cells, and microcells, respectively. The microgrippers are being actuated by an optimized V-shaped thermal chevron actuator whose input is amplified by using a pantograph displacement amplification mechanism with an amplification factor of 54. The force applied to the gripping object is sensed by a rotary comb electrostatic force sensor. Special attention has been paid to the structural design, and detailed stress analysis has been discussed. This research work includes static, electrostatic, parametric, and electrothermal analysis carried out using Finite Element Method (FEM) techniques and analytical modeling. The results show that the operating voltage range of the devices is from 4 to 9.25 V, having a maximum operational temperature of 380 °C with a factor of safety of 1.026. The maximum capacitance change with the full operational range of microgrippers is 0.63 µf, having a capacitive sensitivity of 19.6 nf/µm. The microgrippers can be used as an instrument to grip not only the microbiological species but also can be beneficial to carry out manipulations at the micro-level like micro-assembly. The proposed microgripper has a substantial structural stability, has a high amplification factor and accommodates manipulation of a wide range of microorganism.

Identification and expression profiles of Tubby‑like proteins coding genes in walnut (Juglans regia L.) in response to stress and hormone treatments

Tubby-like proteins (TLPs) constitute a diverse multigene family found across a broad range of eukaryotic organisms, from single-celled to multicellular eukaryotes. Despite their importance, the existence of TLPs in walnuts (Juglans regia L.) has not been reported until now. In this study, we identified 12 members of the JrTLP gene family in the walnut genome, revealing a pattern of gene replication events. Notably, all JrTLPs exhibited a split gene structure, containing two to eight introns, and these genes could be divided into five distinct categories based on their evolutionary relationships. The majority of TLP proteins possess two signature domains: the F-box and Tubby domains. Subcellular localization prediction and experiments confirmed that JrTLP2 was located in the nucleus and membrane. Expression profiling revealed that multiple JrTLPs respond to a range of stimuli, including pathogen infection, hormonal treatment (such as auxin, ABA, MeJA, and GA), and abiotic stress conditions such as cold, heat, and salt. This study represents the first comprehensive identification of the walnut TLP family, providing valuable insights into the function and regulation of TLP in walnut and laying the foundation for future functional dissection, particularly in the context of stress tolerance.

Evolutionary formation of melatonin and vitamin D in early life forms: insects take centre stage.

Melatonin, a product of tryptophan metabolism via serotonin, is a molecule with an indole backbone that is widely produced by bacteria, unicellular eukaryotic organisms, plants, fungi and all animal taxa. Aside from its role in the regulation of circadian rhythms, it has diverse biological actions including regulation of cytoprotective responses and other functions crucial for survival across different species. The latter properties are also shared by its metabolites including kynuric products generated by reactive oxygen species or phototransfomation induced by ultraviolet radiation. Vitamins D and related photoproducts originate from phototransformation of ∆5,7 sterols, of which 7-dehydrocholesterol and ergosterol are examples. Their ∆5,7 bonds in the B ring absorb solar ultraviolet radiation [290-315 nm, ultraviolet B (UVB) radiation] resulting in B ring opening to produce previtamin D, also referred to as a secosteroid. Once formed, previtamin D can either undergo thermal-induced isomerization to vitamin D or absorb UVB radiation to be transformed into photoproducts including lumisterol and tachysterol. Vitamin D, as well as the previtamin D photoproducts lumisterol and tachysterol, are hydroxylated by cyochrome P450 (CYP) enzymes to produce biologically active hydroxyderivatives. The best known of these is 1,25-dihydroxyvitamin D (1,25(OH)2D) for which the major function in vertebrates is regulation of calcium and phosphorus metabolism. Herein we review data on melatonin production and metabolism and discuss their functions in insects. We discuss production of previtamin D and vitamin D, and their photoproducts in fungi, plants and insects, as well as mechanisms for their enzymatic activation and suggest possible biological functions for them in these groups of organisms. For the detection of these secosteroids and their precursors and photoderivatives, as well as melatonin metabolites, we focus on honey produced by bees and on body extracts of Drosophila melanogaster. Common biological functions for melatonin derivatives and secosteroids such as cytoprotective and photoprotective actions in insects are discussed. We provide hypotheses for the photoproduction of other secosteroids and of kynuric metabolites of melatonin, based on the known photobiology of ∆5,7 sterols and of the indole ring, respectively. We also offer possible mechanisms of actions for these unique molecules and summarise differences and similarities of melatoninergic and secosteroidogenic pathways in diverse organisms including insects.

The Phytophthora parasitica effector AVH195 interacts with ATG8, attenuates host autophagy, and promotes biotrophic infection

BackgroundPlant pathogens secrete effector proteins into host cells to suppress immune responses and manipulate fundamental cellular processes. One of these processes is autophagy, an essential recycling mechanism in eukaryotic cells that coordinates the turnover of cellular components and contributes to the decision on cell death or survival.ResultsWe report the characterization of AVH195, an effector from the broad-spectrum oomycete plant pathogen, Phytophthora parasitica. We show that P. parasitica expresses AVH195 during the biotrophic phase of plant infection, i.e., the initial phase in which host cells are maintained alive. In tobacco, the effector prevents the initiation of cell death, which is caused by two pathogen-derived effectors and the proapoptotic BAX protein. AVH195 associates with the plant vacuolar membrane system and interacts with Autophagy-related protein 8 (ATG8) isoforms/paralogs. When expressed in cells from the green alga, Chlamydomonas reinhardtii, the effector delays vacuolar fusion and cargo turnover upon stimulation of autophagy, but does not affect algal viability. In Arabidopsis thaliana, AVH195 delays the turnover of ATG8 from endomembranes and promotes plant susceptibility to P. parasitica and the obligate biotrophic oomycete pathogen Hyaloperonospora arabidopsidis.ConclusionsTaken together, our observations suggest that AVH195 targets ATG8 to attenuate autophagy and prevent associated host cell death, thereby favoring biotrophy during the early stages of the infection process.

Vacuolar H+-ATPase in Diabetes, Hypertension, and Atherosclerosis.

Vacuolar H+-ATPase (V-ATPase) is a multisubunit protein complex which, along with its accessory proteins, resides in almost every eukaryotic cell. It acts as a proton pump and as such is responsible for regulating pH in lysosomes, endosomes, and the extracellular space. Moreover, V-ATPase has been implicated in receptor-mediated signaling. Although numerous studies have explored the role of V-ATPase in cancer, osteoporosis, and neurodegenerative diseases, research on its involvement in vascular disease remains limited. Vascular diseases pose significant challenges to human health. This review aimed to shed light on the role of V-ATPase in hypertension and atherosclerosis. Furthermore, given that vascular complications are major complications of diabetes, this review also discusses the pathways through which V-ATPase may contribute to such complications. Beginning with an overview of the structure and function of V-ATPase in hypertension, atherosclerosis, and diabetes, this review ends by exploring the pharmacological potential of targeting V-ATPase.

Viral RNA capping: Mechanisms and antiviral therapy.

RNA capping is an essential trigger for protein translation in eukaryotic cells. Many viruses have evolved various strategies for initiating the translation of viral genes and generating progeny virions in infected cells via synthesizing cap structure or stealing the RNA cap from nascent host messenger ribonucleotide acid(mRNA). In addition to protein translation, a new understanding of the role of the RNA cap in antiviral innate immunity has advanced the field of mRNA synthesis in vitro and therapeutic applications. Recent studies on these viral RNA capping systems have revealed startlingly diverse ways and molecular machinery. A comprehensive understanding of how viruses accomplish the RNA capping in infected cells is pivotal for designing effective broad-spectrum antiviral therapies. Here we systematically review the contemporary insights into the RNA-capping mechanisms employed by viruses causing human and animal infectious diseases, while also highlighting its impact on host antiviral innate immune response. The therapeutic applications of targeting RNA capping against viral infections and the development of RNA-capping inhibitors are also summarized.

Circular RNA PRKCI (hsa_circ_0067934): a potential target in the pathogenesis of human malignancies.

Circular RNAs (circRNAs) are a new type of endogenous non-coding RNA formed by a covalent closed loop. CircRNAs are characterized by specificity, universality, conservation, and stability. They are abundant in eukaryotic cells and have biological regulatory roles at various transcriptional and post-transcriptional levels. The upregulation of circPRKCI has been observed in a variety of tumors and is directly related to the clinicopathological characteristics of tumors and prognosis. More importantly, circPRKCI can participate in the tumorigenesis, progression, recurrence, and metastasis of various tumors through many functional mechanisms, including the activation of signaling pathways, such as the phosphatidylinositol-3-kinase (PI3K)/AKT pathway, and sponging of many microRNAs (miRNAs). This review summarizes the progress achieved in understanding the biological functions of circRNA PRKCI in various tumors. The goal is to inform the discovery of more functional mechanisms and new anticancer molecular targets.

Exploration of Genome-Wide Recombination Rate Variation Patterns at Different Scales in Pigs.

Meiotic recombination is a prevalent process in eukaryotic sexual reproduction organisms that plays key roles in genetic diversity, breed selection, and species evolution. However, the recombination events differ across breeds and even within breeds. In this study, we initially computed large-scale population recombination rates of both sexes using approximately 52 K SNP genotypes in a total of 3279 pigs from four different Chinese and Western breeds. We then constructed a high-resolution historical recombination map using approximately 16 million SNPs from a sample of unrelated individuals. Comparative analysis of porcine recombination events from different breeds and at different resolutions revealed the following observations: Firstly, the 1Mb-scale pig recombination maps of the same sex are moderately conserved among different breeds, with the similarity of recombination events between Western pigs and Chinese indigenous pigs being lower than within their respective groups. Secondly, we identified 3861 recombination hotspots in the genome and observed medium- to high-level correlation between historical recombination rates (0.542~0.683) and estimates of meiotic recombination rates. Third, we observed that recombination hotspots are significantly far from the transcription start sites of pig genes, and the silico-predicted PRDM9 zinc finger domain DNA recognition motif is significantly enriched in the regions of recombination hotspots compared to recombination coldspots, highlighting the potential role of PRDM9 in regulating recombination hotspots in pigs. Our study analyzed the variation patterns of the pig recombination map at broad and fine scales, providing a valuable reference for genomic selection breeding and laying a crucial foundation for further understanding the molecular mechanisms of pig genome recombination.

Zymolyase Treatment of Saccharomyces cerevisiae Affects Cellular Proteins and Degrades Tyrosyl-DNA Phosphodiesterase I.

Saccharomyces cerevisiae is a genetically tractable, affordable, and extensively documented eukaryotic single-cell model organism. This budding yeast is amenable for the development of genetic and biochemical experiments and is frequently used to investigate the function, activity, and mechanism of mammalian proteins. However, yeast contains a cell wall that hinders select assays including organelle isolation. Lytic enzymes, with Zymolyase as the most effective and frequently used tool, are utilized to weaken the yeast cell wall resulting in yeast spheroplasts. Spheroplasts are easily lysed by, for example, osmotic-shock conditions to isolate yeast nuclei or mitochondria. However, during our studies of the DNA repair enzyme tyrosyl-DNA phosphodiesterase I (Tdp1), we encountered a negative effect of Zymolyase. We observed that Zymolyase treatment affected the steady-state protein levels of Tdp1. This was revealed by inconsistencies in technical and biological replicate lysates of plasmid-born galactose-induced expression of Tdp1. This off-target effect of Zymolyase is rarely discussed in articles and affects a select number of intracellular proteins, including transcription factors and assays such as chromatin immunoprecipitations. Following extensive troubleshooting, we concluded that the culprit is the Ser-protease, Zymolyase B, component of the Zymolyase enzyme mixture that causes the degradation of Tdp1. In this study, we report the protocols we have used, and our final protocol with an easy, affordable adaptation to any assay/protocol involving Zymolyase.