Covalent DNA protein crosslinks (DPCs) are common lesions that block replication. We examine here the consequence of DPCs on mutagenesis involving replicational template-switch reactions in Escherichia coli. 5-azacytidine (5azaC) is a potent mutagen for template-switching, dependent on DNA cytosine methylase (Dcm), implicating the trapped Dcm-DNA covalent complex as the initiator for mutagenesis. The leading strand of replication is more mutable than the lagging strand, explained by blocks to the replicative helicase and/or fork regression. We find that template-switch mutagenesis induced by 5-azaC does not require DSB repair via RecABCD. The ability to induce the SOS response is anti-mutagenic by an unknown mechanism. Mutants in recB, but not recA, exhibit high constitutive rates of template-switching and we suggest that RecBCD-mediated DNA degradation prevents template-switching associated with fork regression. A mutation in the DnaB fork helicase also promotes high levels of template-switching. We also find that other DPC-inducers, formaldehyde (a non-specific crosslinker) and ciprofloxacin (a topoisomerase II poison) are also strong mutagens for template-switching. Induction of mutations and genetic rearrangements that occur by template-switching may constitute a previously unrecognized component of the genotoxicity and genetic instability promoted by DPCs.

Viruses with spindle or lemon-shaped virions are rare in the world of viruses, but are common in viruses of archaeal extremophiles, possibly due to the extreme conditions in which they thrive. However, the structural and genetic basis for the unique spindle shape is unknown. The best-studied spindle-shaped virus, SSV1, is composed mostly of the major capsid protein VP1. Similar to many other viruses, proteolytic cleavage of VP1 is thought to be critical for virion formation. Unlike half of the genes in SSV1, including the minor capsid protein gene vp3, the vp1 gene does not tolerate deletion or transposon insertion. In order determine the role of the vp1 gene and its proteolysis for virus function, we developed techniques for site-directed mutagenesis of the SSV1 genome and complemented deletion mutants with vp1 genes from other SSVs. By analyzing these mutants we demonstrate that the N-terminus of the VP1 protein is required, but the N-terminus, or entire SSV1 VP1 protein, can be exchanged with VP1s from other SSVs. However, the conserved glutamate at the cleavage site is not essential for infectivity. Interestingly, viruses containing point mutations at this position generate mostly abnormal virions.

The development of new cultivars is important for the profitability of the floriculture industry. There are a limited number of cultivars of Zantedeschia aethiopica, an iconic ornamental cut flower, garden plant, and potted plant, due to the incompatibility of interspecific crossings within the genus. Most present-day varieties are the result of spontaneous mutations or classical breeding within the species, followed by a long selection process. Breeders are very interested in the development of a time- and cost-effective method for producing new Z. aethiopica cultivars with novel characteristics. Here, Z. aethiopica mutants were generated by treating seeds with 100 Gy of X-ray radiation. The resulting putative mutants were selected based on particular flowering parameters and compared to non-irradiated, control plants. Over two growing seasons, characteristics such as early flowering, flower size and shape, yield and response to soft-rot disease were monitored and considerable variation was observed among the mutated lines. Out of 319 mutants, 20 lines were selected based on their phenotypes and then propagated and further analyzed. Within this group, only two phenotypes displayed at least five improved flowering properties under natural, Mediterranean conditions. The rest displayed two to four desired combinations of flowering traits, some with great commercial potential.

Increasing genetic diversity in crop plants has been used for chemical mutagenesis. Through the application of various mutagenic agents, over 430 new varieties have been derived as rice mutants (Oryza sativa L.) Chemical mutagens such as ethyl methane sulphonate (EMS), diepoxybutane derivative (DEB), sodium azide, and gamma ray, x-ray, and quick neutron irradiation have been commonly used to induce a large number of functional variations in rice and others crops. Among chemical mutagens, ethyl methane sulfonate (EMS) is the alkylating agent most widely used in plants because it induces nucleotide substitutions to be extremely frequent, as detected in various genomes. In this study, seeds of potential genotype of the popular variety, (Oryza sativa L. Super Basmati variety) were treated with EMS at concentrations of 0.25%, 0.50%, 0.75%, 1% and 1.5%. Various measurements on the M1 generation determined EMS sensitivity. As concentration of applied EMS increased, will decrease in germination, shoot length, root length, plant height, productive tillers, Panicle Length, Total Spikelet, sterile spikelet and fertility under field conditions were observed in M1 generation as compared to the non-treatment control. Emergence, shoot length, root length, plant height, productive tillers, Panicle Length, Total Spikelet, sterile spikelet and fertility also decreased with increases in EMS mutagenesis in an approximately linear fashion. The LD₅₀ values were observed based on growth reduction of seedlings after EMS treatment with 0.25% and 0.50% on the rice variety (Oryza sativa L. spp.).

Genetic diversity is the foundation of evolutionary resilience, adaptive potential, and the flourishing vitality of living organisms, serving as the cornerstone for robust ecosystems and the continuous evolution of life on Earth. The landscape of directed evolution, a powerful biotechnological tool inspired by natural evolutionary processes, has undergone a transformative shift propelled by innovative strategies for generating genetic diversity. This shift is fuelled by several factors, encompassing the utilization of advanced toolkits like CRISPR-Cas and base editors, enhanced comprehension of biological mechanisms, cost-effective custom oligo pool synthesis, and the seamless integration of artificial intelligence and automation. This comprehensive review looks into the myriad methodologies employed for constructing gene libraries, both in vitro and in vivo, categorized into three major classes: random mutagenesis, focused mutagenesis, and DNA recombination. The objectives of this review are threefold: firstly, to present a panoramic overview of recent advances in genetic diversity creation, secondly, to inspire novel ideas for further innovation in the genetic diversity generation, and thirdly, to provide a valuable resource for individuals entering the field of directed evolution.

. Colchicine (C22H25NO6) is a substance used for inducing mutations in order to regulate important agronomic traits. The objective of this study was to investigate the effect of different concentrations (0.0, 0.04, 0.06, 0.08 and 0.1%) and treatment time (6, 12 and 24 h) of colchicine on agronomic traits of proso millet (Panicum miliaceum L.) and to assess the genetic diversity of M2 generations using inter simple sequence repeat markers (ISSR). The experiment was conducted in 2021 for the M1-generation and in 2022 for the M2-generation, from May to September. The percentage of the field germination was decreased with the increasing colchicine concentration and exposure duration. The chlorophyll-defective M1-M2 plants were obtained using various concentrations and exposure periods of colchicine. The highest amount of mutational modifications was attained at 0.08-0.1% concentrations colchicine. A total of 248 plant families with chlorophyll-defective mutations based on the research results were selected from 2214 families. The growing season of M1- and M2 plants were diminished under higher colchicine concentration (0.08-0.1%) combined with soaking time. Thus, the highest indicator of growing season (84 days) was observed at six h treatment time for PI 289324, while the fewest (78 days) was recorded at 12 and 24 h. The possibility of obtaining morphological mutations using colchicine has been confirmed. The ISSR primers amplified a total of 1333 fragments, 1281 bands were found to be polymorphic and 52 bands monomorphic. The percentage of polymorphism varied from 80 to 100 % with an average of 96.11%. Our results showed that most of the bands were found at 0.08% colchicine concentration. These positive variations are a great opportunity to use colchicine as a tool for improving agronomic traits in plant breeding.

The development of gamma rays mutant rice lines would be a solution for introducing variability in already farmer using varieties. In vitro gamma (60Co) mutagenesis reduces chimeras and allows a faster selection of desired traits but requires laboratory process optimization. The objective of the present work was the in vitro establishment of a recalcitrant rice embryogenic calli, the determination of its sensitivity to gamma radiation (Co-60), sequencing MATK and Rubisco for identification purposes, as well as generation optimization. The radiosensitivity of embryogenic calli resulted in an LD50 of 110Gy, while the 20% lethal dose was 64Gy. All sequenced genes matched perfectly with already reported MATK and Rubisco O. sativa genes with a clear SNP that identifies the local variety related to the southeast Asia Region. Callus induction improves with an MS with 2mg/L 2,4D, and the regeneration was achieved with an MS medium with 3mg/L BAP and 0,5mg/L NAA. The optimized radiation condition was 60Gy with an 83% regeneration in a semisolid medium, allowing a balance between mutation and regeneration. When increased to 80Gy, the regeneration rate falls to 29%. An immersion system (RITA®) of either 60 or 120 seconds every 8hours allowed a systematic and homogeneous total regeneration of the recalcitrant line, in contrast with the semisolid medium that resulted in positive but irregular regeneration. Other well-known recalcitrant cultivars, CR1821, CR1113 also had an improving regeneration in the immersion system, demonstrating its potential use for recalcitrant materials. To our knowledge, this is the first report on using an immersion system to allow regeneration of gamma-ray mutants from recalcitrant rice materials.

The article presents research results of the γ-ray and fast neutrons impact on various rice species, in order to obtain mutant forms resistant to salinity (NaCl) and drought factors (sorbitol. They are going to be used as initial forms in synthetic selection when creating varieties, adapted to the stressful conditions of, both, soil and climate in the Kazakhstan Aral Sea. The average lethal doses (LD50) of γ-rays and fast neutrons, as well as the average NaCl and sorbitol lethal concentrations were established. Such environment is best suited for clear manifestation of the mutagenic effect of ionizing radiation and the resistance of rice plants to salinity and drought. A distinct dependence in the effect of ionizing radiation and stress factors on the number of induced resistant mutant forms has been related to the initial rice sort. The largest number of mutant forms was obtained from the local variety Syr Suluy, followed by Leader and AyKerim varieties. From all varieties, the number of mutants obtained from exposure to γ-rays was 43 pieces out of 4500 grains, and from the impact of fast neutrons - 115 pieces out of 2700 grains. M1 mutant plants significantly differ from the initial forms in terms of morphological features – plant height, panicle length, grain size Most plants are characterized by short growth and even dwarfism (˂80 cm). They are lodging tolerant, have short and highly sterile panicles, indicating that they are mutants and resistant to salinity, drought, or both.

CRISPR-Cas9 technology allows the modification of DNA sequences in vivo at the location of interest. Although CRISPR-Cas9 can produce genomic changes that do not require DNA vector carriers, the use of transgenesis for stable integration of DNA coding for gene-editing tools into plant genomes is still the most used approach and it can generate unintended transgenic integrations, while Cas9 prolonged expression can increase cleavage at off-target sites. In addition, the selection of genetically modified cells from millions of treated cells, especially plant cells, is still challenging. These downfalls can be avoided with the delivery of preassembled ribonucleoprotein complexes (RNPs) composed of purified recombinant enzyme Cas9 and in vitro- transcribed guide RNA (gRNA) molecules in a protoplast system. We therefore aimed to develop the first DNA-free protocol for gene-editing in maize and introduced RNPs into their protoplasts with PEG 4000. We performed effective transformation of maize protoplasts using different gRNAs sequences targeting the inositol phosphate kinase gene and applying two different exposure times to RNPs. Using low-cost Sanger sequencing protocol, we observed an efficiency rate of 0.85 up to 5.85%, which is equivalent to DNA-free protocols used in other plant species. A positive correlation was displayed between exposure time and mutation frequency. Mutation frequency was gRNA sequence- and exposure time-dependent. In summary, we demonstrated the suitability of RNP transfection as an effective screening platform for gene-editing in maize. This efficient and relatively easy assay method for selection of gRNA suitable for editing of gene of interest will be highly useful for genome editing in maize, since genome size and GC-content are large and high in maize genome, respectively. Nevertheless, the large amplitude of mutations at target site requires scrutiny when checking mutations at off-target sites and potential safety concerns.

The article presents the results of the γ-ray and fast neutrons impact on various rice species, in order to obtain mutant forms resistant to salinity (NaCl) and drought factors (sorbite). They are going to be used as initial forms in synthetic selection when creating varieties, adapted to the stressful conditions of, both, soil and climate in the Kazakhstan Aral Sea. The average lethal doses (LD50) of γ-rays and fast neutrons, as well as, the average NaCl and sorbite lethal concentrations were established. A distinct dependence on the effect of ionizing radiation and stress factors on the number of induced resistant mutant forms has been related to the initial rice variety. The largest number of mutant forms was obtained from the local variety Syr Suluy, followed by Leader and Aikerim varieties. Out of all varieties, only 1% of the initially irradiated by γ-rays seeds survived, while after fast neutron irradiation – 4,3%. M1 mutant plants significantly differ from the initial forms in terms of morphological features – plant height, panicle length, and grain size. They are tolerant to lodging, and have short and highly sterile panicles, indicating that they are mutants.

Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although cas9/guide (g)RNA constructs are straightforward to assemble and can be customized to target virtually any site in the plant genome, the implementation of this technology can be cumbersome, especially in species like Triticale that are difficult to transform, for which only limited genome information is available and/or which carry comparatively large genomes. To cope with these challenges, we have pre-validated cas9/gRNA constructs (1) by frameshift restitution of a reporter gene co-introduced by ballistic DNA transfer to barley epidermis cells, and (2) via transfection in Triticale protoplasts followed by either a T7E1-based cleavage assay or by deep-sequencing of target-specific PCR amplicons. For exemplification, we addressed the Triticale ABA 8’-hydroxylase 1 gene, one of the putative determinants of pre-harvest sprouting of grains. We further show that in-del induction frequency in Triticale can be increased by TREX2 nuclease activity, which holds true for both well- and poorly performing gRNAs. The presented results constitute a sound basis for the targeted induction of heritable modifications in Triticale genes.

Abstract: CRISPR/Cas systems are used for genome editing, both in basic science and in bio-technology. At the same time, CRISPR/Cas editors have several limitations, including insufficient specificity leading to "off-targets" and the dependence of activity on chromatin state. A number of highly specific Cas9 variants have been obtained now, but most of them are characterized by reduced activity on eukaryotic chromatin. We identified a spatial cluster of amino acid residues in the PAM-recognizing domain of Streptococcus pyogenes Cas9, whose mutations restore the activity of one of the highly specific forms of SpyCas9 without reducing its activity in Saccharomyces cere-visiae. In addition, one of these new mutations also increases the efficiency of SpyCas9-mediated editing of a site localized on the stable nucleosome. The improved Cas9 variants we obtained, which are capable of editing hard-to-reach regions of the yeast genome, may help in both basic research and yeast biotechnological applications.

Infection with Zika virus (ZIKV), a member of the Flavivirus genus of the Flaviviridae family, typically results in mild self-limited illness, but severe neurological disease occurs in a limited subset of patients. In contrast, serious outcomes commonly occur in pregnancy that affect the developing fetus, including microcephaly and other major birth defects. The genetic similarity of ZIKV to other widespread flaviviruses, such as dengue virus (DENV), presents a challenge to the development of specific ZIKV diagnostic assays. Nonstructural protein 1 (NS1) is established for use in immunodiagnostic assays for flaviviruses. To address the cross-reactivity of ZIKV NS1 with proteins from other flaviviruses we used site-directed mutagenesis to modified putative epitopes. Goat polyclonal antibodies to variant ZIKV NS1 were affinity-purified to remove antibodies binding to the closely related NS1 protein of DENV. An antigen-capture ELISA configured with the affinity-purified polyclonal antibody showed a linear dynamic range between approximately 500 to 30 ng/mL, with a limit of detection of between 1.95 and 7.8 ng/mL. NS1 proteins from DENV, yellow fever virus, St. Louis encephalitis virus and West Nile virus showed significantly reduced reactivity in the ZIKV antigen-capture ELISA. Refinement of approaches similar to those employed here could lead to development of ZIKV-specific immunoassays suitable for use in areas where infections with related flaviviruses are common.

Mouse models of alcohol use disorder (AUD) revealed a subtype of purinergic receptors (P2X4Rs) as a promising target for AUD drug development. We have previously demonstrated that residues at the transmembrane (TM)-ectodomain interface and within TM1 segment contribute to the formation of an ethanol action pocket in P2X4Rs. In the present study, we tested the hypothesis that there are more residues in TM segments, which are important for ethanol sensitivity of P2X4Rs. Using site-directed mutagenesis and two-electrode voltage-clamp electrophysiology in Xenopus oocytes, we found that arginine at position 33 (R33) in the TM1 segment plays a role in ethanol sensitivity of P2X4Rs. Molecular models in both closed and open states provided evidence for interactions between R33 and aspartic acid at position 354 (D354) of the neighboring TM2 segment. Further work with mixtures of wild-type (WT) and reciprocal single (R33D:WT, D354R:WT) and double (R33D-D354R:WT) mutants confirmed the importance of this interaction for ethanol sensitivity, ivermectin action and channel function. Additionally, our findings suggest that valine at TM1 position 49 plays a role in P2X4R function by providing flexibility during channel opening. Collectively, these findings identified new activity sites, and suggest the importance of TM1-TM2 interaction for channel function and ethanol sensitivity of P2X4Rs.

Fast neutron (FN) radiation mediated mutagenesis is a unique approach among the several induced mutagenesis methods being used in plant science in terms of impacted mutations. The FN mutagenesis usually creates deletions from a few bases to several million bases (Mb). A library of random deletion generated using FN mutagenesis lines can provide indispensable resources for the reverse genetic approaches. In this review, information from several efforts made using FN mutagenesis has been compiled to understand the type of induced mutations, frequency, and genetic stability. Concerns regarding the utilization of FN mutagenesis technique for a plant with different level of ploidy and genome complexity are discussed. We have highlighted the utility of next-generation sequencing techniques which can be efficiently utilized for the characterization of mutant lines as well as for the mapping of causal mutations. Pros and cons of Mut-map, MutChromSeq, exon capture, whole genome sequencing, MutRen-Seq, and different tilling approaches can be used for the detection of FN-induced mutation has also been discussed. Genomic resources developed using the FN mutagenesis have been catalogued wooing to meaningful utilization of the available resources. The information provided here will be helpful for the efficient exploration for the crop improvement programs and for better understanding of genetic regulations.

Minimal plasmids play an essential role in many intermediate steps in molecular biology. They can for example be used to assemble building blocks in synthetic biology or be used as intermediate cloning plasmids that are ideal for PCR-based mutagenesis methods. A small backbone also opens up for additional unique restriction enzyme cloning sites. Here we describe the generation of pICOz, a 1185 bp fully functional high-copy cloning plasmid with an extended multiple cloning site (MCS). To our knowledge, this is the smallest high-copy cloning vector ever described.

Transmembrane carriers of the Slc11 family catalyze proton (H+)-dependent uptake of divalent metal-ions (Me2+) such as manganese and iron - vital elements coveted during infection. Slc11 mechanism of high affinity Me2+ cell import is selective and conserved between prokaryotic (MntH) and eukaryotic (Nramp) homologs, though processes coupling the use of the proton motive force to Me2+ uptake evolved repeatedly. Adding bacterial piracy of Nramp genes spread in distinct environmental niches suggests selective gain of function that may benefit opportunistic pathogens. To better understand Slc11 evolution Alphafold (AF2)/Colabfold (CF) 3D predictions for bacterial sequences from sister clades of eukaryotic descent (MCb and MCg) were compared using both native and mutant templates. AF2/CF model an array of native MCb intermediates spanning the transition from outward open (OO) to inward open (IO) carrier. In silico mutagenesis targeting i) a set of (evolutionary coupled) sites that may define Slc11 function (putative synapomorphy), and ii) residues from networked communities evolving during MCb transition, indicate Slc11 synapomorphy primarily instructs Me2+-selective conformation switch which unlocks carrier inner gate, and contributes to Me2+ binding site occlusion and outer gate locking. Inner gate opening apparently proceeds from interaction between transmembrane helix (h) h5, h8 and h1a. MCg1 xenologs revealed marked differences in carrier shape and plasticity, owing partly to altered intramolecular H+-network. Yet, targeting Slc11 synapomorphy also converted MCg1 IO models to OO state, apparently mobilizing the same residues to control gates. But MCg1 response to mutagenesis differed, extensive divergence within this clade correlating with MCb-like modeling properties. Notably, MCg1 divergent epistasis marks emergence of the genus Bordetella-Achromobacter. Slc11 synapomorphy localizes to the 3D areas that deviate least among MCb and MCg1 models (either IO or OO) implying it constitutes a 3D network of residues articulating Me2+-selective carrier conformation switch which is maintained in fast evolving clades at the cost of divergent epistatic interactions impacting carrier shape and dynamics.

Congenital Disorders of Glycosylation (CDG) are a heterogenous group of primarily autosomal recessive mendelian diseases caused by disruptions in the synthesis of lipid linked oligosaccha-rides and their transfer to proteins. CDGs affect multiple organ systems and vary in presentation, even within families. Here we describe a chemically induced mouse mutant, tvrm76, with early onset photoreceptor degeneration. The recessive mutation was mapped to Chromosome 9 and as-sociated with a missense mutation in the Dpagt1 gene encoding UDP-N-acetyl-D-glucosamine:dolichyl-phosphate N-acetyl-D-glucosaminephosphotransferase (EC 2.7.8.15). The mutation is predicted to cause a substitution of aspartic acid with glycine at residue 166 of DPAGT1. Increased expression of Ddit3, and elevated levels of HSPA5 (BiP) sug-gest the presence of early-onset endoplasmic reticulum (ER) stress. These changes were associated with induction of photoreceptor apoptosis in tvrm76 retinas. Mutations in human DPAGT1 cause Myasthenic Syndrome 13 and severe forms of Congenital Disorder of Glycosylation Type Ij. In contrast, Dpagt1tvrm76 homozygous mice present with congenital photoreceptor degeneration without overt muscle or muscular junction involvement. Our results suggest the possibility of DPAGT1 mutations in human patients that present primarily with retinitis pigmentosa with little or no muscle disease. Variants in DPAGT1 should be considered when evaluating cases of non-syndromic retinal degeneration.

Here, I introduce a concept called autonomous selection to refer to a source of selection that is part of the individuals upon which it acts. The concept is motivated by a set of phenomena with the following characteristics: Natural selection shaped a variant (e.g., gene, epigenetic mark, or combination thereof) to act in a manner that reduces the frequency of one or more heritable traits of the individual in which it is located if those traits are detrimental to individual or group fitness. Phenomena with these characteristics are peculiar to traditional evolutionary theory but have been identified rather frequently in recent decades. They are also relevant to adaptive evolution: By reducing the frequency of a trait detrimental to fitness, the variant accelerates the evolution of adaptations, which allows its holders to adapt better to constantly changing environments. The variant is shaped by (natural) selection, but also does (autonomous) selection. Several phenomena with these characteristics have been invoked by proponents of the extended evolutionary synthesis (EES). The concept of autonomous selection helps resolve some of the controversy surrounding the EES: EES proponents call attention to the incompleteness of contemporary theory, emphasizing individuals’ processes that influence which adaptations those individuals evolve. I argue for the special importance of individuals’ processes that do not just influence those individuals’ adaptations, but also accelerate the adaptive evolution of those individuals. All known phenomena that fit this description are examples of autonomous selection. Other phenomena raised by EES proponents do not meet this threshold.

The cardiac sodium ion channel (NaV1.5) is a protein with four domains (DI-DIV), each with six transmembrane segments. Its opening and subsequent inactivation results in the brief rapid influx of Na+ ions resulting in the depolarization of cardiomyocytes. The neurotoxin veratridine (VTD) inhibits NaV1.5 inactivation resulting in longer channel opening times, and potentially fatal action potential prolongation. VTD is predicted to bind at the channel pore, but alternative binding sites have not been ruled out. To determine the binding site of VTD on NaV1.5, we performed docking calculations and high-throughput electrophysiology experiments. The docking calculations identified two distinct binding regions. The first site was in the pore, close to the binding site of NaV1.4 and NaV1.5 blocking drugs in experimental structures. The second site was at the “mouth” of the pore at the cytosolic side, partly solvent-exposed. Mutations at this site (L409, E417, and I1466) had large effects on VTD binding, while residues deeper in the pore had no effect, consistent with VTD binding at the mouth site. Overall, our results suggest a VTD binding site close to the cytoplasmic mouth of the channel pore. Binding at this alternative site might indicate an allosteric inactivation mechanism for VTD at NaV1.5.

Ostreococcus tauri is a simple unicellular green alga representing an ecologically important group of phytoplankton in oceans worldwide. Modern molecular techniques must be developed in order to understand the mechanisms that permit adaptation of microalgae to their environment. We present for the first time in O. tauri a detailed characterization of individual genomic integration events of foreign DNA of plasmid origin after PEG-mediated transformation. Vector integration appears to be random, occurring mainly at a single locus, and thus confirming the utility of this technique for insertional mutagenesis. While the mechanism of double-stranded DNA repair in the O. tauri model remains to be elucidated, we clearly demonstrate by genome resequencing that the integration of the vector leads to frequent structural variations (deletions/insertions and duplications) and some chromosomal rearrangements in the genome at the insertion loci, and often within the vector sequence itself. From these observations, we speculate that a non-homologous end joining-like mechanism is required during random insertion events, as described in plants and other freshwater algal models. PEG-mediated transformation is therefore a promising molecular biology tool, not only for functional genomic studies, but also for biotechnological research in ecologically important marine algae.

Hemolysin II (HlyII) – one of the pathogenic factors of Bacillus cereus, a pore-forming β-barrel toxin – possesses a C-terminal extension of 94 amino acid residues, designated as the C-terminal domain of HlyII (HlyIICTD), which plays an important role in the functioning of the toxin. Our previous work described a monoclonal antibody (HlyIIC-20), capable of strain-specific inhibi-tion of hemolysis caused by HlyII, and demonstrated the dependence of the efficiency of hemol-ysis on the presence of proline at position 324 in HlyII outside the conformational antigenic de-terminant. In this work, we studied 16 mutant forms of HlyIICTD. Each of the mutations, ob-tained by multiple site-directed mutagenesis leading to the replacement of amino acid residues lying on the surface of the 3D structure of HlyIICTD, led to a decrease in the interaction of HlyIIC-20 with the mutant form of the protein. Changes in epitope structure confirm the high conformational mobility of HlyIICTD required for the functioning of HlyII. Comparison of the effect of the introduced mutations on the effectiveness of interaction between HlyIICTD and HlyIIC-20 and a control antibody recognizing a non-overlapping epitope enabled identifying the amino acid residues N339, K340 included in the conformational antigenic determinant rec-ognized by HlyIIC-20.

Proprotein convertases activate various envelope glycoproteins and participate in cellular entry of many viruses. We recently showed that the convertase furin is critical for the infectivity of SARS-CoV-2. This study investigated the implication of the two cholesterol-regulating convertases SKI-1 and PCSK9 in SARS-CoV-2 entry. We used cell-to-cell fusion assays in HeLa cells and pseudoparticle entry into Calu-3 cells. SKI-1 increases cell-to-cell fusion by enhancing the activation of SREBP-2, whereas PCSK9 reduces cell-to-cell fusion by promoting the cellular degradation of ACE2. Metalloprotease activation is sensitive to enhanced cholesterol levels resulting from SKI-1-activated SREBP-2 that leads to enhanced S2’ formation. However, high metalloprotease activity results in S2’ shedding into a new C-terminal fragment (S2”), leading to reduced cell-to-cell fusion. Indeed, S-mutants that increase S2’’ formation, abolish S2’ and cell-to-cell fusion, as well as pseudoparticles entry, indicating that the formation of S2’’ prevents SARS-CoV-2 cell-to-cell fusion and entry. We next demonstrated that PCSK9 enhanced the cellular degradation of ACE2, thereby reducing cell-to-cell fusion. However, different from the LDLR, a canonical target of PCSK9, the C-terminal CHRD domain of PCSK9 is dispensable for the PCSK9-induced degradation of ACE2. Molecular modeling suggested binding of ACE2 to the Pro/Catalytic domains of mature PCSK9. Thus, both cholesterol-regulating convertases SKI-1 and PCSK9 can modulate SARS-CoV-2 entry via two independent mechanisms.

Foot-and-mouth disease virus (FMDV) causes the highly contagious foot-and-mouth disease, which is characterized by the appearance of vesicles in and around the mouth and feet of cloven-hoofed animals. BHK21 cells are the cell line of choice for the propagation of FMDV for vaccine production world-wide but vary in their susceptibility for different FMDV strains. Previous studies showed that the FMDV resistance of a certain BHK cell line can be overcome by using a closely related but permissive cell line for the pre-adaptation of the virus, but the adapted strains were found to harbor several capsid mutations. In this study, these adaptive mutations were introduced into the original Asia-1 Shamir isolate individually or in combination to create a panel of 17 Asia-1 mutants by reverse genetics and examine the effects of the mutations on receptor usage, viral growth, immunogenicity and stability. A single amino acid exchange from glutamic acid to lysine at position 202 in VP1 turned out to be of major importance for productive infection of the suspension cell line BHK-2P. In consequence, two traditionally passage-derived strains and two recombinant viruses with a minimum set of mutations were tested in vivo. While the passaged-derived viruses showed a reduced particle stability, the genetically modified viruses were more stable but did not confer a protective immune response against the original virus isolate.