Repetitive Sequences Research Articles

Expression of Transposable Elements in the Brain of the Drosophila melanogaster Model for Fragile X Syndrome.

Fragile X syndrome is a neuro-developmental disease affecting intellectual abilities and social interactions. Drosophila melanogaster represents a consolidated model to study neuronal pathways underlying this syndrome, especially because the model recapitulates complex behavioural phenotypes. Drosophila Fragile X protein, or FMRP, is required for a normal neuronal structure and for correct synaptic differentiation in both the peripheral and central nervous systems, as well as for synaptic connectivity during development of the neuronal circuits. At the molecular level, FMRP has a crucial role in RNA homeostasis, including a role in transposon RNA regulation in the gonads of D. m. Transposons are repetitive sequences regulated at both the transcriptional and post-transcriptional levels to avoid genomic instability. De-regulation of transposons in the brain in response to chromatin relaxation has previously been related to neurodegenerative events in Drosophila models. Here, we demonstrate for the first time that FMRP is required for transposon silencing in larval and adult brains of Drosophila "loss of function" dFmr1 mutants. This study highlights that flies kept in isolation, defined as asocial conditions, experience activation of transposable elements. In all, these results suggest a role for transposons in the pathogenesis of certain neurological alterations in Fragile X as well as in abnormal social behaviors.

Functional Characterisation of the Circular RNA, circHTT(2-6), in Huntington's Disease.

Trinucleotide repeat disorders comprise ~20 severe, inherited, human neuromuscular and neurodegenerative disorders, which result from an abnormal expansion of repetitive sequences in the DNA. The most common of these, Huntington's disease (HD), results from expansion of the CAG repeat region in exon 1 of the HTT gene via an unknown mechanism. Since non-coding RNAs have been implicated in the initiation and progression of many diseases, herein we focused on a circular RNA (circRNA) molecule arising from non-canonical splicing (backsplicing) of HTT pre-mRNA. The most abundant circRNA from HTT, circHTT(2-6), was found to be more highly expressed in the frontal cortex of HD patients, compared with healthy controls, and positively correlated with CAG repeat tract length. Furthermore, the mouse orthologue (mmu_circHTT(2-6)) was found to be enriched within the brain and specifically the striatum, a region enriched for medium spiny neurons that are preferentially lost in HD. Transgenic overexpression of circHTT(2-6) in two human cell lines-SH-SY5Y and HEK293-reduced cell proliferation and nuclear size without affecting cell cycle progression or cellular size, or altering the CAG repeat region length within HTT. CircHTT(2-6) overexpression did not alter total HTT protein levels, but reduced its nuclear localisation. As these phenotypic and genotypic changes resemble those observed in HD patients, our results suggest that circHTT(2-6) may play a functional role in the pathophysiology of this disease.

A high-quality chromosomal-level genome assembly of Greater Scaup (Aythya marila)

Aythya marila is one of the few species of Anatidae, and the only Aythya to live in the circumpolar. However, there is a relative lack of research on genetics of this species. In this study, we reported and assembled the first high-quality chromosome-level genome assembly of A. marila. This genome was assembled using Nanopore long reads, and errors corrected using Illumina short reads, with a final genome size of 1.14 Gb, scaffold N50 of 85.44 Mb, and contig N50 of 32.46 Mb. 106 contigs were clustered and ordered onto 35 chromosomes based on Hi-C data, covering approximately 98.28% of the genome. BUSCO assessment showed that 97.0% of the highly conserved genes in aves_odb10 were present intact in the genome assembly. In addition, a total of 154.94 Mb of repetitive sequences were identified. 15,953 protein-coding genes were predicted in the genome, and 98.96% of genes were functionally annotated. This genome will be a valuable resource for future genetic diversity and genomics studies of A. marila.

Human rDNA Structure, Expression, and Non-Canonical Functions: the Role of Non-Coding Regions

The review is dedicated to analyzing and summarizing the data on the part of human genome encoding 45S rRNA. The sequences which seem evolutionary conserved on the first glance astonish one with their variability in structure and a variety of functions on closer examination. The major part of rDNA is non-coding and contains regulatory elements, protein binding sites, pseudogenes, repetitive sequences, and microRNA genes. Ribosomal intergenic spacers are not only in charge with the nucleolus morphology and functioning, namely, the rRNA expression and ribosome biogenesis, but also control nuclear chromatin formation thus mediating cell differentiation. Besides, alterations in the expression of these non-coding regions of rDNA in response to environmental stimuli underlies the keen sense of cell to various types of stressors. Malfunctioning of this process may result in a wide range of pathologies from oncology to neurodegenerative disease and mental illness. Here we observe to-date materials on the structure and transcription of the ribosomal intergenic spacer in humans and its role in rRNA expression, in-born disease development, and cancer.

The Role of Immunotherapy in Advanced and Recurrent MMR Deficient and Proficient Endometrial Carcinoma

ABSTRACT The incidence of endometrial cancer has increased approximately 40% in the last decade along with a 60% increase in the number of associated deaths. Although surgery along with possible adjuvant therapy provides good results in patients with early-stage disease, patients with advanced or recurrent disease have limited options and typically undergo chemotherapy with no standard second-line therapy. Immunotherapy with checkpoint inhibitors (ICIs) is a promising option in this setting. This review aims to improve knowledge on the role of ICIs in advanced/recurrent endometrial cancer in different contexts based on molecular and genomic profiling. The DNA mismatch repair (MMR) system is a highly conserved mechanism that acts to restore DNA integrity after mismatching mistakes during replication, recombination, or iatrogenic damage. Somatic or germline mutations or epigenetic silencing can all result in possible inactivation of MMR genes, resulting in an MMR-deficient (dMMR) genotype. Short tandem repeats, called microsatellites, are repetitive DNA sequences that facilitate slippage or replication errors that are generally repaired by MMR; however, in a dMMR context, these can result in repeat mutations called microsatellite instability (MSI). The presence of MSI characterizes a hypermutable state of cell, and MSI tumors have elevated expression of the checkpoint programmed death 1 ligand (PD-L1). Microsatellite instability/dMMR testing may have potential prognostic value as a predictor of response to immunotherapy. Endometrial cancer is the solid tumor characterized by the highest prevalence of MSI−/dMMR, which is present in 30% and 13% to 30% of primary and recurrent endometrial cancer, respectively. Recently, results from the KEYNOTE-158 trial reporting on the efficacy and safety of pembrolizumab (programmed death 1 [PD-1] ICI) in patients with MSI-H/dMMR endometrial cancer who progressed after a prior line of chemotherapy showed an objective response rate of 48%, median progression-free survival of 13.1 months, and a low rate of high-grade toxicity. Results from the GARNET study reporting on the use of dostarlimab (PD-1 ICI) in dMMR advanced endometrial tumors showed that 80% of patients experienced progression-free survival of 18 months and an objective response rate of 44.7%. Several trials have examined the use of ICIs for patients with advanced endometrial carcinoma in MMR-proficient tumors; however, they seem to show a lesser effect in this setting. Results are more promising in MMR-proficient patients when ICIs are combined with other agents such as tyrosine kinase inhibitors; however, the toxicity profile is significantly worse, and efficacy remains significantly higher in the dMMR group. The KEYNOTE-775 study, a phase III randomized controlled trial, found that in patients with advanced or recurrent endometrial cancer pembrolizumab plus lenvatinib (tyrosine kinase inhibitor) increased progression-free survival (6.6 vs 3.8 months) and overall survival (17.4 vs 12 months) compared with conventional chemotherapy; however, treatment discontinuation and dose reduction occurred in 33% and 70% of patients, respectively. This review describes and evaluates the use of new lines of treatment including immunotherapy agents for advanced and recurrent endometrial cancer. Many trials are still ongoing, with several testing ICIs alone or in combination with other agents to improve safety profiles and efficacy.

Exposure to smoky coal combustion emissions and leukocyte Alu retroelement copy number.

Household air pollution (HAP) from indoor combustion of solid fuel is a global health burden that has been linked to multiple diseases including lung cancer. In Xuanwei, China, lung cancer rate for non-smoking women is among the highest in the world and largely attributed to high levels of polycyclic aromatic hydrocarbons (PAHs) that are produced from combustion of smoky (bituminous) coal. Alu retroelements, repetitive mobile DNA sequences that can somatically multiply and promote genomic instability have been associated with risk of lung cancer and diesel engine exhaust exposure. We conducted analyses for 160 non-smoking women in an exposure assessment study in Xuanwei, China with a repeat sample from 49 subjects. Quantitative PCR was used to measure Alu repeat copy number relative to albumin gene copy number (Alu/ALB ratio). Associations between clusters derived from predicted levels of 43 HAP constituents, 5-methylchrysene (5-MC), a PAH previously associated with lung cancer in Xuanwei and was selected a priori for analysis, and Alu repeats were analyzed using generalized estimating equations. A cluster of 31 PAHs reflecting current exposure was associated with increased Alu copy number (β:0.03 per standard deviation change; 95% confidence interval (CI):0.01,0.04; P-value = 2E-04). One compound within this cluster, 5-MC, was also associated with increased Alu copy number (P-value = 0.02). Our findings suggest that exposure to PAHs due to indoor smoky coal combustion may contribute to genomic instability. Additionally, our study provides further support for 5-MC as a prominent carcinogenic component of smoky coal emissions. Further studies are needed to replicate our findings.

A new type of tandem repeats in Myotis petax (Chiroptera, Vespertilionidae) mitochondrial control region.

Tandem repeats in mitochondrial DNA control region are known to different animal taxa, including bat species of the family Vespertilionidae. The long R1-repeats in the bat ETAS-domain are often presented in a variable copy number and may exhibit both inter-individual and intra-individual sequence diversity. The function of repeats in the control region is still unclear, but it has been shown that repetitive sequences in some animal groups (shrews, cats and sheep) may include parts of ETAS1 and ETAS2 conservative blocks of mitochondrial DNA. Analysis of the control region sequences for 31 Myotis petax specimens allowed the identification of the inter-individual variability and clarification of the composition of the R1-repeats. The copy number of the R1-repeats varies from 4 to 7 in individuals. The specimens examined do not exhibit a size heteroplasmy previously described for Myotis species. The unusual short 30bp R1-repeats have been detected in M. petax for the first time. The ten specimens from Amur Region and Primorsky Territory have one or two copies of these additional repeats. It was determined that the R1-repeats in M. petax control region consist of parts of the ETAS1 and ETAS2 blocks. The origin of the additional repeats seems to be related to the 51bp deletion in the central part of the R1-repeat unit and subsequent duplication. Comparison of repetitive sequences in the control region of closely-related Myotis species identified the occurrence of incomplete repeats also resulting from the short deletions, but distinct from additional repeats of M. petax.

Дискурсивные маркеры категории адресованности в англоязычных новостных медиатекстах

The aim of the research is to determine the discursive markers of the category of addressing of modern English-language news media texts within the framework of the discursive-dictemic model of communication. The research is novel in that it is the first to identify the discursive markers of the category of addressing on the basis of the proposed discursive-dictemic model of media communication, which consists of three equally dependent, repetitive sequences of components: linguistic personality – discourse – dicteme (text) – linguistic personality – ... The use of this model in the analysis of practical material made it possible to expand the boundaries of linguistic research methods and take into account both textual (dictemic) and discursive characteristics of news media texts, as well as the specifics of speech formation (speech generation). As a result, it has been found that the category of addressing, inextricably linked with the linguistic personality of both the addressee and the addresser of the message, is represented in the communicative strategy of bringing closer / distancing (identification) and is expressed in media texts (dictemes) by explicit, implicit and explicit-implicit discursive markers. Explicit markers include conversational elements of communication, implicit discursive markers are represented by certain grammatical constructions, while iconic components and graphic language means serve as explicit-implicit discursive markers.

E. urophylla × E. grandis high-quality genome and comparative genomics provide insights on evolution and diversification of eucalyptus

BackgroundEucalyptus urophylla × Eucalyptus grandis, an economically important forest tree, provides important raw material for energy and reduces damage to native forests. However, the absence of a high-quality E. urophylla × E. grandis reference genome has significantly hindered its evolution and genetic analysis.ResultsWe successfully presented a high-quality reference genome of E. urophylla × E. grandis (545.75 Mb; scaffold N50, 51.62 Mb) using a combination of the Illumina, PacBio HiFi, and Hi-C sequencing platforms. A total of 34,502 genes and 58.56% of the repetitive sequences in this genome were annotated. Using genome evolution analyses, we identified a recent whole-genome duplication (WGD) event in E. urophylla × E. grandis. We further found that gene families associated with starch and sucrose metabolism, flavonoid biosynthesis, and plant–pathogen interaction were significantly expanded in E. urophylla × E. grandis. Moreover, comparative genomic and evolutionary analyses showed large structural variations among the different chromosomes of the 34 Eucalyptus accessions, which were divided into six clades.ConclusionsOverall, our findings provide a valuable resource for expanding our understanding of the E. urophylla × E. grandis genome evolution, genetic improvement, and its comparative biology.

The first released available genome of the common ice plant (Mesembryanthemum crystallinum L.) extended the research region on salt tolerance, C3-CAM photosynthetic conversion, and halophism

Background: The common ice plant (Mesembryanthemum crystallinum L.) is an annual herb belonging to the genus Mesembryanthemum of the family Aizoaceae, native to Southern Africa. Methods: We performed shotgun genome paired-end sequencing using the Illumina platform to determine the genome sequence of the ice plants. We assembled the whole genome sequences using the genome assembler “ALGA” and “Redundans”, then released them as available genomic information. Finally, we mainly estimated the potential genomic function by the homology search method. Results: A draft genome was generated with a total length of 286 Mb corresponding to 79.2% of the estimated genome size (361 Mb), consisting of 49,782 contigs. It encompassed 93.49% of the genes of terrestrial higher plants, 99.5% of the ice plant transcriptome, and 100% of known DNA sequences. In addition, 110.9 Mb (38.8%) of repetitive sequences and untranslated regions, 971 tRNA, and 100 miRNA loci were identified, and their effects on stress tolerance and photosynthesis were investigated. Molecular phylogenetic analysis based on ribosomal DNA among 26 kinds of plant species revealed genetic similarity between the ice plant and poplar, which have salt tolerance. Overall, 35,702 protein-coding regions were identified in the genome, of which 56.05% to 82.59% were annotated and submitted to domain searches and gene ontology (GO) analyses, which found that eighteen GO terms stood out among five plant species. These terms were related to biological defense, growth, reproduction, transcription, post-transcription, and intermembrane transportation, regarded as one of the fundamental results of using the utilized ice plant genome. Conclusions: The information that we characterized is useful for elucidation of the mechanism of growth promotion under salinity and reversible conversion of the photosynthetic type from C3 to Crassulacean Acid Metabolism (CAM).

Pseudomonas petrae sp. nov. isolated from regolith samples in Antarctica

A polyphasic taxonomic approach was used to characterize the four strains P2653T, P2652, P2498, and P2647, isolated from Antarctic regolith samples. Initial genotype screening performed by PCR fingerprinting based on repetitive sequences showed that the isolates studied formed a coherent cluster separated from the other Pseudomonas species. Identification results based on 16S rRNA gene sequences showed the highest sequence similarity with Pseudomonas graminis (99.7%), which was confirmed by multilocus sequence analysis using the rpoB, rpoD, and gyrB genes. Genome sequence comparison of P2653T with the most related P. graminis type strain DSM 11363T revealed an average nucleotide identity of 92.1% and a digital DNA-DNA hybridization value of 46.6%. The major fatty acids for all Antarctic strains were C16:0, Summed Feature 3 (C16:1ω7c/C16:1ω6c) and Summed Feature 8 (C18:1ω7c/C18:1ω6c). The predominant respiratory quinone was Q-9, and the major polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, and phosphatidylglycerol. The regolith strains could be differentiated from related species by the absence of arginine dihydrolase, ornithine and lysine decarboxylase and by negative tyrosine hydrolysis. The results of this polyphasic study allowed the genotypic and phenotypic differentiation of four analysed strains from the closest related species, which confirmed that the strains represent a novel species within the genus Pseudomonas, for which the name Pseudomonas petrae sp. nov. is proposed with P2653T (CCM 8850T = DSM 112068T = LMG 30619T) as the type strain.

Telomere-to-telomere genome assembly of matsutake (Tricholoma matsutake)

Here, we report the first telomere-to-telomere genome assembly of matsutake (Tricholoma matsutake), which consists of 13 sequences (spanning 161.0Mb) and a 76kb circular mitochondrial genome. All the 13 sequences were supported with telomeric repeats at the ends. GC-rich regions are located at the middle of the sequences and are enriched with long interspersed nuclear elements (LINEs). Repetitive sequences including long-terminal repeats (LTRs) and LINEs occupy 71.6% of the genome. A total of 21,887 potential protein-coding genes were predicted. The genomic data reported in this study served not only matsutake gene sequences but also genome structures and intergenic sequences. The information gained would be a great reference for exploring the genetics, genomics, and evolutionary study of matsutake in the future, and ultimately facilitate the conservation of this vulnerable genetic resource.

Electronegative clusters modulate folding status and RNA binding of unstructured RNA-binding proteins.

Electronegative clusters (ENCs) made up of acidic residues and/or phosphorylation sites are the most abundant repetitive sequences in RNA-binding proteins. Previous studies have indicated that ENCs inhibit RNA binding for structured RNA-binding domains (RBDs). However, this is not the case for the unstructured RBD in histone pre-mRNA stem-loop binding protein (SLBP). The SLBP RBD contains 70 amino acids and is followed by a phosphorylatable ENC. ENC phosphorylation increases RNA-binding affinity of SLBP to the sub-picomolar range. In this study, we use NMR and molecular dynamics simulations to elucidate the mechanism for this tight binding. Our NMR data demonstrate that the ENC transiently folds apo SLBP into an RNA-bound resembling state. We find that in the RNA-bound state, the phosphorylated ENC interacts with the loop region opposite to the RNA-binding site. This allosteric interaction stabilizes the complex and therefore enhances RNA binding. To evaluate the generality of our findings, we graft an ENC onto endoribonuclease homolog 1's first double-stranded RNA-binding motif (DRBM1), an unstructured RBD that shares no homology with SLBP. We find that the engineered ENC increases the folded species of DRBM1 and inhibits RNA binding. On the contrary, introducing basic residues to DRBM1 makes the domain more unfolded, enhances RNA binding, and mitigates the inhibitory effect of the engineered ENC. In summary, our study suggests that ENCs promote folding of unstructured RNA-binding domains, and their effects on RNA binding depend on the electropositive charges on the RBD surface.

Deep Intronic LINE-1 Insertions in NF1: Expanding the Spectrum of Neurofibromatosis Type 1-Associated Rearrangements.

Neurofibromatosis type 1 is an autosomal-dominant condition caused by NF1 gene inactivation. Clinical diagnosis is corroborated by genetic tests on gDNA and cDNA, which are inconclusive in approximately 3-5% of cases. Genomic DNA approaches may overlook splicing-affecting intronic variants and structural rearrangements, especially in regions enriched in repetitive sequences. On the other hand, while cDNA-based methods provide direct information about the effect of a variant on gene transcription, they are hampered by non-sense-mediated mRNA decay and skewed or monoallelic expression. Moreover, analyses on gene transcripts in some patients do not allow tracing back to the causative event, which is crucial for addressing genetic counselling, prenatal monitoring, and developing targeted therapies. We report on a familial NF1, caused by an insertion of a partial LINE-1 element inside intron 15, leading to exon 15 skipping. Only a few cases of LINE-1 insertion have been reported so far, hampering gDNA studies because of their size. Often, they result in exon skipping, and their recognition of cDNA may be difficult. A combined approach, based on Optical Genome Mapping, WGS, and cDNA studies, enabled us to detect the LINE-1 insertion and test its effects. Our results improve knowledge of the NF1 mutational spectrum and highlight the importance of custom-built approaches in undiagnosed patients.

Optimization of Performance Parameters of the TAGGG Telomere Length Assay.

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening occurs due to a problem with end replication and the absence of the telomerase enzyme, which is responsible for maintaining telomere length. Interestingly, telomeres also shorten in response to various internal physiological processes, like oxidative stress and inflammation, which may be impacted due to extracellular agents like pollutants, infectious agents, nutrients, or radiation. Thus, telomere length serves as an excellent biomarker of aging and various physiological health parameters. The TAGGG telomere length assay kit is used to quantify average telomere lengths using the telomere restriction fragment (TRF) assay and is highly reproducible. However, it is an expensive method, and because of this, it is not employed routinely for large sample numbers. Here, we describe a detailed protocol for an optimized and cost-effective measurement of telomere length using Southern blots or TRF analysis and non-radioactive chemiluminescence-based detection.

Haplotype-resolved genomes of two buckwheat crops provide insights into their contrasted rutin concentrations and reproductive systems

BackgroundTwo widely cultivated annual buckwheat crops, Fagopyrum esculentum and F. tataricum, differ from each other in both rutin concentration and reproductive system. However, the underlying genetic mechanisms remain poorly elucidated.ResultsHere, we report the first haplotype-resolved chromosome-level genome assemblies of the two species. Two haplotype genomes of F. esculentum were assembled as 1.23 and 1.19 Gb with N50 = 9.8 and 12.4 Mb, respectively; the two haplotype genomes of F. tataricum were 453.7 and 446.2 Mb with N50 = 50 and 30 Mb, respectively. We further annotated protein-coding genes of each haplotype genome based on available gene sets and 48 newly sequenced transcriptomes. We found that more repetitive sequences, especially expansion of long terminal repeat retrotransposons (LTR-RTs), contributed to the large genome size of F. esculentum. Based on the well-annotated sequences, gene expressions, and luciferase experiments, we identified the sequence mutations of the promoter regions of two key genes that are likely to have greatly contributed to the high rutin concentration and selfing reproduction in F. tartaricum.ConclusionsOur results highlight the importance of high-quality genomes to identify genetic mutations underlying phenotypic differences between closely related species. F. tataricum may have been experienced stronger selection than F. esculentum through choosing these two non-coding alleles for the desired cultivation traits. These findings further suggest that genetic manipulation of the non-coding promoter regions could be widely employed for breeding buckwheat and other crops.

Genomic and transcriptomic analysis of sacred fig (Ficus religiosa)

BackgroundPeepal/Bodhi tree (Ficus religiosa L.) is an important, long-lived keystone ecological species. Communities on the Indian subcontinent have extensively employed the plant in Ayurveda, traditional medicine, and spiritual practices. The Peepal tree is often thought to produce oxygen both during the day and at night by Indian folks. The goal of our research was to produce molecular resources using whole-genome and transcriptome sequencing techniques.ResultsThe complete genome of the Peepal tree was sequenced using two next-generation sequencers Illumina HiSeq1000 and MGISEQ-2000. We assembled the draft genome of 406 Mb, using a hybrid assembly workflow. The genome annotation resulted in 35,093 protein-coding genes; 53% of its genome consists of repetitive sequences. To understand the physiological pathways in leaf tissues, we analyzed photosynthetically distinct conditions: bright sunny days and nights. The RNA-seq analysis supported the expression of 26,479 unigenes. The leaf transcriptomic analysis of the diurnal and nocturnal periods revealed the expression of the significant number of genes involved in the carbon-fixation pathway.ConclusionsThis study presents a draft hybrid genome assembly for F. religiosa and its functional annotated genes. The genomic and transcriptomic data-derived pathways have been analyzed for future studies on the Peepal tree.

Chromosome-scale genome assembly of Prunus pusilliflora provides novel insights into genome evolution, disease resistance, and dormancy release in Cerasus L.

Prunus pusilliflora is a wild cherry germplasm resource distributed mainly in Southwest China. Despite its ornamental and economic value, a high-quality assembled P. pusilliflora genome is unavailable, hindering our understanding of its genetic background, population diversity, and evolutionary processes. Here, we de novo assembled a chromosome-scale P. pusilliflora genome using Oxford Nanopore, Illumina, and chromosome conformation capture sequencing. The assembled genome size was 309.62Mb, with 76 scaffolds anchored to eight pseudochromosomes. We predicted 33 035 protein-coding genes, functionally annotated 98.27% of them, and identified repetitive sequences covering 49.08% of the genome. We found that P. pusilliflora is closely related to Prunus serrulata and Prunus yedoensis, having diverged from them ~41.8 million years ago. A comparative genomic analysis revealed that P. pusilliflora has 643 expanded and 1128 contracted gene families. Furthermore, we found that P. pusilliflora is more resistant to Colletotrichum viniferum, Phytophthora capsici, and Pseudomonas syringae pv. tomato (Pst) DC3000 infections than cultivated Prunus avium. P. pusilliflora also has considerably more nucleotide-binding site-type resistance gene analogs than P. avium, which explains its stronger disease resistance. The cytochrome P450 and WRKY families of 263 and 61 proteins were divided into 42 and 8 subfamilies respectively in P. pusilliflora. Furthermore, 81 MADS-box genes were identified in P. pusilliflora, accompanying expansions of the SVP and AGL15 subfamilies and loss of the TM3 subfamily. Our assembly of a high-quality P. pusilliflora genome will be valuable for further research on cherries and molecular breeding.

Chromosome-Level Genome Assembly of the Blue Mussel Mytilus chilensis Reveals Molecular Signatures Facing the Marine Environment.

The blue mussel Mytilus chilensis is an endemic and key socioeconomic species inhabiting the southern coast of Chile. This bivalve species supports a booming aquaculture industry, which entirely relies on artificially collected seeds from natural beds that are translocated to diverse physical-chemical ocean farming conditions. Furthermore, mussel production is threatened by a broad range of microorganisms, pollution, and environmental stressors that eventually impact its survival and growth. Herein, understanding the genomic basis of the local adaption is pivotal to developing sustainable shellfish aquaculture. We present a high-quality reference genome of M. chilensis, which is the first chromosome-level genome for a Mytilidae member in South America. The assembled genome size was 1.93 Gb, with a contig N50 of 134 Mb. Through Hi-C proximity ligation, 11,868 contigs were clustered, ordered, and assembled into 14 chromosomes in congruence with the karyological evidence. The M. chilensis genome comprises 34,530 genes and 4795 non-coding RNAs. A total of 57% of the genome contains repetitive sequences with predominancy of LTR-retrotransposons and unknown elements. Comparative genome analysis of M. chilensis and M. coruscus was conducted, revealing genic rearrangements distributed into the whole genome. Notably, transposable Steamer-like elements associated with horizontal transmissible cancer were explored in reference genomes, suggesting putative relationships at the chromosome level in Bivalvia. Genome expression analysis was also conducted, showing putative genomic differences between two ecologically different mussel populations. The evidence suggests that local genome adaptation and physiological plasticity can be analyzed to develop sustainable mussel production. The genome of M. chilensis provides pivotal molecular knowledge for the Mytilus complex.

Phage N15-Based Vectors for Gene Cloning and Expression in Bacteria and Mammalian Cells.

Bacteriophage N15 is the first virus known to deliver linear prophage into Escherichia coli. During its lysogenic cycle, N15 protelomerase (TelN) resolves its telomerase occupancy site (tos) into hairpin telomeres. This protects the N15 prophage from bacterial exonuclease degradation, enabling it to stably replicate as a linear plasmid in E. coli. Interestingly, purely proteinaceous TelN can retain phage DNA linearization and hairpin formation without involving host- or phage-derived intermediates or cofactors in the heterologous environment. This unique feature has led to the advent of synthetic linear DNA vector systems derived from the TelN-tos module for the genetic engineering of bacterial and mammalian cells. This review will focus on the development and advantages of N15-based novel cloning and expression vectors in the bacterial and mammalian environments. To date, N15 is the most widely exploited molecular tool for the development of linear vector systems, especially the production of therapeutically useful miniDNA vectors without a bacterial backbone. Compared to typical circular plasmids, linear N15-based plasmids display remarkable cloning fidelity in propagating unstable repetitive DNA sequences and large genomic fragments. Additionally, TelN-linearized vectors with the relevant origin of replication can replicate extrachromosomally and retain transgenes functionality in bacterial and mammalian cells without compromising host cell viability. Currently, this DNA linearization system has shown robust results in the development of gene delivery vehicles, DNA vaccines and engineering mammalian cells against infectious diseases or cancers, highlighting its multifaceted importance in genetic studies and gene medicine.