Repetitive Sequences Research Articles

Genomic exploration of Sesuvium sesuvioides: comparative study and phylogenetic analysis within the order Caryophyllales from Cholistan desert, Pakistan

BackgroundThe Aizoaceae family’s Sesuvium sesuvioides (Fenzl) Verdc is a medicinal species of the Cholistan desert, Pakistan. The purpose of this study was to determine the genomic features and phylogenetic position of the Sesuvium genus in the Aizoaceae family. We used the Illumina HiSeq2500 and paired-end sequencing to publish the complete chloroplast sequence of S. sesuvioides.ResultsThe 155,849 bp length cp genome sequence of S. sesuvioides has a 36.8% GC content. The Leucine codon has the greatest codon use (10.6%), 81 simple sequence repetitions of 19 kinds, and 79 oligonucleotide repeats. We investigated the phylogeny of the order Caryophyllales’ 27 species from 23 families and 25 distinct genera. The maximum likelihood tree indicated Sesuvium as a monophyletic genus, and sister to Tetragonia. A comparison of S. sesuvioides, with Sesuvium portulacastrum, Mesembryanthemum crystallinum, Mesembryanthemum cordifolium, and Tetragonia tetragonoides was performed using the NCBI platform. In the comparative investigation of genomes, all five genera revealed comparable cp genome structure, gene number and composition. All five species lacked the rps15 gene and the rpl2 intron. In most comparisons with S. sesuvioides, transition substitutions (Ts) were more frequent than transversion substitutions (Tv), producing Ts/Tv ratios larger than one, and the Ka/Ks ratio was lower than one. We determined ten highly polymorphic regions, comprising rpl22, rpl32-trnL-UAG, trnD-GUC-trnY-GUA, trnE-UUC-trnT-GGU, trnK-UUU-rps16, trnM-CAU-atpE, trnH-GUG-psbA, psaJ-rpl33, rps4-trnT-UGU, and trnF-GAA-ndhJ.ConclusionThe whole S. sesuvioides chloroplast will be examined as a resource for in-depth taxonomic research of the genus when more Sesuvium and Aizoaceae species are sequenced in the future. The chloroplast genomes of the Aizoaceae family are well preserved, with little alterations, indicating the family’s monophyletic origin. This study’s highly polymorphic regions could be utilized to build realistic and low-cost molecular markers for resolving taxonomic discrepancies, new species identification, and finding evolutionary links among Aizoaceae species. To properly comprehend the evolution of the Aizoaceae family, further species need to be sequenced.

Characteristics of Amorphophallus konjac as indicated by its genome

Amorphophallus konjac, belonging to the genus Amorphophallus of the Araceae family, is an economically important crop widely used in health products and biomaterials. In the present work, we performed the whole-genome assembly of A. konjac based on the NovaSeq platform sequence data. The final genome assembly was 4.58 Gb with a scaffold N50 of 3212 bp. The genome includes 39,421 protein-coding genes, and 71.75% of the assemblies were repetitive sequences. Comparative genomic analysis showed 1647 gene families have expanded and 2685 contracted in the A. konjac genome. Likewise, genome evolution analysis indicated that A. konjac underwent whole-genome duplication, possibly contributing to the expansion of certain gene families. Furthermore, we identified many candidate genes involved in the tuber formation and development, cellulose and lignification synthesis. The genome of A. konjac obtained in this work provides a valuable resource for the further study of the genetics, genomics, and breeding of this economically important crop, as well as for evolutionary studies of Araceae family.

A chromosome-level genome assembly of Agave hybrid NO.11648 provides insights into the CAM photosynthesis

Abstract The subfamily Agavoideae comprises crassulacean acid metabolism (CAM), C3, and C4 plants with a young age of speciation and slower mutation accumulation, making it a model crop for studying CAM evolution. However, the genetic mechanism underlying CAM evolution remains unclear because of lacking genomic information. This study assembled the genome of Agave hybrid NO.11648, a constitutive CAM plant belonging to subfamily Agavoideae, at the chromosome level using data generated from high-throughput chromosome conformation capture, Nanopore, and Illumina techniques, resulting in 30 pseudo-chromosomes with a size of 4.87 Gb and scaffold N50 of 186.42 Mb. The genome annotation revealed 58 841 protein-coding genes and 76.91% repetitive sequences, with the dominant repetitive sequences being the I-type repeats (Copia and Gypsy accounting for 18.34% and 13.5% of the genome, respectively). Our findings also provide support for a whole genome duplication event in the lineage leading to A. hybrid, which occurred after its divergence from subfamily Asparagoideae. Moreover, we identified a gene duplication event in the phosphoenolpyruvate carboxylase kinase (PEPCK) gene family and revealed that three PEPCK genes (PEPCK3, PEPCK5, and PEPCK12) were involved in the CAM pathway. More importantly, we identified transcription factors enriched in the circadian rhythm, MAPK signaling, and plant hormone signal pathway that regulate the PEPCK3 expression by analysing the transcriptome and using yeast one-hybrid assays. Our results shed light on CAM evolution and offer an essential resource for the molecular breeding program of Agave spp.

Геномные перестройки влияют на устойчивость биопленок почвенных бактерий Azospirillum brasilense к абиотическим стрессам

The bacteria Azospirillum brasilense, used as biofertilizers, have a significant positive effect on the growth and development of plants. The genome of the strain A. brasilense Sp7 is represented by a chromosome and numerous plasmids with molecular weight of 90, 115, and over 300 MDa. Genomic rearrangements that cause changes in the “plasmid profile” can contribute to the formation of subpopulations or phenotypic variants in a bacterial population. There is little data on the role of such rearrangements in the adaptation of A. brasilense to dynamic environmental conditions. The ability of azospirilla to form biofilms also has a determined significance for the successful functioning of the plant-microbial association and the resistance of bacteria and plants to various abiotic stresses. The purpose of this work consisted of the analysis of the genomic rearrangements in spontaneous derivatives of A. brasilense Sp7 and the assessment of the resistance of their biofilms to drying, water stress and oxidative stress. PCR analysis to detect changes in the structure of genomic DNA was performed using primers corresponding to known conservative motifs in repetitive bacterial nucleotide sequences. The relative amount of the biofilm biomass was assessed by measuring the crystal violet A540 desorbed after staining. The level of relative respiratory activity of cells in biofilms was determined by the fluorometric resazurin test. The non-penetrating osmotic agent PEG 6000 was used to create the osmotic/water stress model. It was shown that rearrangements in genomic DNA contribute to the formation of stable phenotypic variants of the Sp7 strain, which form biofilms in different ways under water stress conditions. A derived strain of A. brasilense Sp7.8, the biofilm population of which is more resistant to water stress compared to the parent strain was selected.

Optimized strategy for real-time qPCR detection of Onchocerca volvulus DNA in pooled Simulium sp. blackfly vectors.

Onchocerca volvulus is a filarial parasite that is a major cause of dermatitis and blindness in endemic regions primarily in sub-Saharan Africa. Widespread efforts to control the disease caused by O. volvulus infection (onchocerciasis) began in 1974 and in recent years, following successful elimination of transmission in much of the Americas, the focus of efforts in Africa has moved from control to the more challenging goal of elimination of transmission in all endemic countries. Mass drug administration (MDA) with ivermectin has reached more than 150 million people and elimination of transmission has been confirmed in four South American countries, with at least two African countries having now stopped MDA as they approach verification of elimination. It is essential that accurate data for active transmission are used to assist in making the critical decision to stop MDA, since missing low levels of transmission and infection can lead to continued spread or recrudescence of the disease. Current World Health Organization guidelines for MDA stopping decisions and post-treatment surveillance include screening pools of the Simulium blackfly vector for the presence of O. volvulus larvae using a PCR-ELISA-based molecular technique. In this study, we address the potential of an updated, practical, standardized molecular diagnostic tool with increased sensitivity and species-specificity by comparing several candidate qPCR assays. When paired with heat-stable reagents, a qPCR assay with a mitochondrial DNA target (OvND5) was found to be more sensitive and species-specific than an O150 qPCR, which targets a non-protein coding repetitive DNA sequence. The OvND5 assay detected 19/20 pools of 100 blackfly heads spiked with a single L3, compared to 16/20 for the O150 qPCR assay. Given the improved sensitivity, species-specificity and resistance to PCR inhibitors, we identified OvND5 as the optimal target for field sample detection. All reagents for this assay can be shipped at room temperature with no loss of activity. The qPCR protocol we propose is also simpler, faster, and more cost-effective than the current end-point molecular assays.

Expanding horizons of tandem repeats in biology and medicine: Why 'genomic dark matter' matters.

Approximately half of the human genome includes repetitive sequences, and these DNA sequences (as well as their transcribed repetitive RNA and translated amino-acid repeat sequences) are known as the repeatome. Within this repeatome there are a couple of million tandem repeats, dispersed throughout the genome. These tandem repeats have been estimated to constitute ∼8% of the entire human genome. These tandem repeats can be located throughout exons, introns and intergenic regions, thus potentially affecting the structure and function of tandemly repetitive DNA, RNA and protein sequences. Over more than three decades, more than 60 monogenic human disorders have been found to be caused by tandem-repeat mutations. These monogenic tandem-repeat disorders include Huntington's disease, a variety of ataxias, amyotrophic lateral sclerosis and frontotemporal dementia, as well as many other neurodegenerative diseases. Furthermore, tandem-repeat disorders can include fragile X syndrome, related fragile X disorders, as well as other neurological and psychiatric disorders. However, these monogenic tandem-repeat disorders, which were discovered via their dominant or recessive modes of inheritance, may represent the 'tip of the iceberg' with respect to tandem-repeat contributions to human disorders. A previous proposal that tandem repeats may contribute to the 'missing heritability' of various common polygenic human disorders has recently been supported by a variety of new evidence. This includes genome-wide studies that associate tandem-repeat mutations with autism, schizophrenia, Parkinson's disease and various types of cancers. In this article, I will discuss how tandem-repeat mutations and polymorphisms could contribute to a wide range of common disorders, along with some of the many major challenges of tandem-repeat biology and medicine. Finally, I will discuss the potential of tandem repeats to be therapeutically targeted, so as to prevent and treat an expanding range of human disorders.

Assembly of the poorly differentiated Verasper variegatus W chromosome by different sequencing technologies

The assembly of W and Y chromosomes poses significant challenges in vertebrate genome sequencing and assembly. Here, we successfully assembled the W chromosome of Verasper variegatus with a length of 20.48 Mb by combining population and PacBio HiFi sequencing data. It was identified as a young sex chromosome and showed signs of expansion in repetitive sequences. The major component of the expansion was Ty3/Gypsy. The ancestral Osteichthyes karyotype consists of 24 protochromosomes. The sex chromosomes in four Pleuronectiformes species derived from a pair of homologous protochromosomes resulting from a whole-genome duplication event in teleost fish, yet with different sex-determination systems. V. variegatus and Cynoglossus semilaevis adhere to the ZZ/ZW system, while Hippoglossus stenolepis and H. hippoglossus follow the XX/XY system. Interestingly, V. variegatus and H. hippoglossus derived from one protochromosome, while C. semilaevis and H. stenolepis derived from another protochromosome. Our study provides valuable insights into the evolution of sex chromosomes in flatfish and sheds light on the important role of whole-genome duplication in shaping the evolution of sex chromosomes.

Clustered Regularly Interspaced Short Palindromic Repeats- a new era in Genomic technology and its applications in Theranostics

Clustered Regularly Interspaced Short Palindromic Repeats is termed as CRISPR. Bacteria having "spacer" sequences of deoxyribonucleic acid between the repeats of genomic sequences that resemble genomic sequences in viruses were found to have repetitive DNA sequences known as CRISPR.The CRISPR technology in genetic engineering has revolutionalized the field of medicine in treating various genetically linked diseases that are difficult to treat such as Lebers Congenital amaurosis, in which there is bi-allelic deletion caused by mutation in mitochondrial DNA MT-ND4(NADH ubiquinone oxidoreductase chain4) 1178G are treated by intravitreous injection of AAV2-ND4 produced by CRISPR-Cas9 genomic editing technology.CRISPR-Cas9 genomic editing is used to remove the expression of receptor Enhancer Protein-6 (Reep6 p.Leu135Pro ) gene that causes retinitis pigmentosa. Open angle Glaucoma caused by mutations in myocilin (MYOR) gene was effectively removed out by CRISPR-Cas9 editing technology. Mutation in KRT-12 gene that caused Meesman epithelial corneal dystrophy ( MECD) was alleviated by Cas9/sgRNA injection into the stroma of cornea. CRISPR-Cas9 genomic sequencing used in the treatment of Haemophilia -B , an inherited disease caused by mutation of factor IX gene Y371D was successfully modified by CRISPR-

The ALS/FTD-related C9orf72 hexanucleotide repeat expansion forms RNA condensates through multimolecular G-quadruplexes

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases that exist on a clinico-pathogenetic spectrum, designated ALS/FTD. The most common genetic cause of ALS/FTD is expansion of the intronic hexanucleotide repeat (GGGGCC)n in C9orf72. Here, we investigate the formation of nucleic acid secondary structures in these expansion repeats, and their role in generating condensates characteristic of ALS/FTD. We observe significant aggregation of the hexanucleotide sequence (GGGGCC)n, which we associate to the formation of multimolecular G-quadruplexes (mG4s) by using a range of biophysical techniques. Exposing the condensates to G4-unfolding conditions leads to prompt disassembly, highlighting the key role of mG4-formation in the condensation process. We further validate the biological relevance of our findings by detecting an increased prevalence of G4-structures in C9orf72 mutant human motor neurons when compared to healthy motor neurons by staining with a G4-selective fluorescent probe, revealing signal in putative condensates. Our findings strongly suggest that RNA G-rich repetitive sequences can form protein-free condensates sustained by multimolecular G-quadruplexes, highlighting their potential relevance as therapeutic targets for C9orf72 mutation-related ALS/FTD.

A chromosome-level assembly of the seed beetle Callosobruchus maculatus genome with annotation of its repetitive elements.

Callosobruchus maculatus is a major agricultural pest of legume crops worldwide and an established model system in ecology and evolution. Yet, current molecular biological resources for this species are limited. Here, we employ Hi-C sequencing to generate a greatly improved genome assembly and we annotate its repetitive elements in a dedicated in-depth effort where we manually curate and classify the most abundant unclassified repeat subfamilies. We present a scaffolded chromosome-level assembly, which is 1.01 Gb in total length with 86% being contained within the 9 autosomes and the X chromosome. Repetitive sequences accounted for 70% of the total assembly. DNA transposons covered 18% of the genome, with the most abundant superfamily being Tc1-Mariner (9.75% of the genome). This new chromosome-level genome assembly of C. maculatus will enable future genetic and evolutionary studies not only of this important species but of beetles more generally.

A chromosome-scale assembly of the quinoa genome provides insights into the structure and dynamics of its subgenomes

Quinoa (Chenopodium quinoa Willd.) is an allotetraploid seed crop with the potential to help address global food security concerns. Genomes have been assembled for four accessions of quinoa; however, all assemblies are fragmented and do not reflect known chromosome biology. Here, we use in vitro and in vivo Hi-C data to produce a chromosome-scale assembly of the Chilean accession PI 614886 (QQ74). The final assembly spans 1.326 Gb, of which 90.5% is assembled into 18 chromosome-scale scaffolds. The genome is annotated with 54,499 protein-coding genes, 96.9% of which are located on the 18 largest scaffolds. We also report an updated genome assembly for the B-genome diploid C. suecicum and use it, together with the A-genome diploid C. pallidicaule, to identify genomic rearrangements within the quinoa genome, including a large pericentromeric inversion representing 71.7% of chromosome Cq3B. Repetitive sequences comprise 65.2%, 48.6%, and 57.9% of the quinoa, C. pallidicaule, and C. suecicum genomes, respectively. Evidence suggests that the B subgenome is more dynamic and has expanded more than the A subgenome. These genomic resources will enable more accurate assessments of genome evolution within the Amaranthaceae and will facilitate future efforts to identify variation in genes underlying important agronomic traits in quinoa.

Oxidation of the 1‐naphthyl radical C10H7• with oxygen: Thermochemistry, kinetics, and possible reaction pathways

AbstractThe reaction of the 1‐naphthyl radical C10H7• (A2•) with molecular (3O2) and atomic oxygen, as part of the oxidation reactions of naphthalene, is examined using ab‐initio and DFT quantum chemistry calculations. The study focuses on pathways that produce the intermediate final products CO, phenyl and C2H2, which may constitute a repetitive reaction sequence for the successive diminution of six‐membered rings also in larger polycyclic aromatic hydrocarbons. The primary attack of 3O2 on the 1‐naphthyl radical leads to a peroxy radical C10H7OO• (A2OO•), which undergoes further propagation and/or chain branching reactions. The thermochemistry of intermediates and transition state structures is investigated as well as the identification of all plausible reaction pathways for the A2• + O2 / A2• + O systems. Structures and enthalpies of formation for the involved species are reported along with transition state barriers and reaction pathways. Standard enthalpies of formation are calculated using ab initio (CBS‐QB3) and DFT calculations (B3LYP, M06, APFD). The reaction of A2• with 3O2 opens six main consecutive reaction channels with new ones not currently considered in oxidation mechanisms. The reaction paths comprise important exothermic chain branching reactions and the formation of unsaturated oxygenated hydrocarbon intermediates. The primary attack of 3O2 at the A2• radical has a well depth of some 50 kcal mol−1 while the six consecutive channels exhibit energy barriers below the energy of the A2• radical. The kinetic parameters of each path are determined using chemical activation analysis based on the canonical transition state theory calculations. The investigated reactions could serve as part of a comprehensive mechanism for the oxidation of naphthalene. The principal result from this study is that the consecutive reactions of the A2• radical, viz. the channels conducting to a phenyl radical C6H5•, CO2, CO (which oxidized to CO2) and C2H2 are by orders of magnitude faster than the activation of naphthalene by oxygen (A2 + O2 → A2• + HO2).

Mosquitoes Possess Specialized Cuticular Proteins That Are Evolutionarily Related to the Elastic Protein Resilin.

Resilin is an elastic protein that is vital to insects' vigorous movement. Canonical resilin proteins possess the R&R Consensus, a chitin-binding domain conserved in a family of cuticular proteins, and highly repetitive sequences conferring elastic properties. In the malaria vector mosquito, Anopheles gambiae, however, a cuticular protein has been found that has an R&R Consensus resembling that of resilin but lacks the repetitive sequences (here, we call it resilin-related or resilin-r). The relationship between resilin-r and resilin was unclear. It was also unknown whether resilin-r is conserved in mosquitoes. In this paper, phylogenetic and structural analyses were performed to reveal the relationship of resilin homologous proteins from holometabolous insects. Their chitin-binding abilities were also assessed. A resilin-r was found in each mosquito species, and these proteins constitute a clade with resilin from other insects based on the R&R Consensus sequences, indicating an evolutionary relationship between resilin-r and resilin. The resilin-r showed chitin-binding activity as same as resilin, but had distinct structural features from resilin, suggesting that it plays specialized roles in the mosquito cuticle. Another resilin-like protein was found to exist in each holometabolous insect that possesses resilin-like repetitive sequences but lacks the R&R Consensus. These results suggest that similar evolutionary events occurred to create resilin-r and resilin-like proteins.

Structural Reorganization in Two Alfalfa Mitochondrial Genome Assemblies and Mitochondrial Evolution in Medicago Species.

Plant mitochondria are crucial for species evolution, phylogenetics, classification, and identification as maternal genetic material. However, the presence of numerous repetitive sequences, complex structures, and a low number of genes in the mitochondrial genome has hindered its complete assembly and related research endeavors. In this study, we assembled two mitochondrial genomes of alfalfa varieties of Zhongmu No.1 (299,123 bp) and Zhongmu No.4 (306,983 bp), based on a combination of PacBio, Illumina, and Hi-C sequences. The comparison of genome assemblies revealed that the same number of mitochondrial genes, including thirty-three protein-coding genes, sixteen tRNA genes, and three rRNA genes existed in the two varieties. Additionally, large fragments of repetitive sequences were found underlying frequent mitochondrial recombination events. We observed extensive transfer of mitochondrial fragments into the nuclear genome of Zhongmu No.4. Analysis of the cox1 and rrn18s genes in 35 Medicago accessions revealed the presence of population-level deletions and substitutions in the rrn18s gene. We propose that mitochondrial structural reorganizations may contribute to alfalfa evolution.

Complete mitochondrial genome of the endangered Prunus pedunculata (Prunoideae, Rosaceae) in China: characterization and phylogenetic analysis.

Prunus pedunculata (Prunoideae: Rosaceae), a relic shrub with strong resistance and multiple application values, is endangered in China. Extensive research had been devoted to gene expression, molecular markers, plastid genome analysis, and genetic background investigations of P. pedunculata. However, the mitochondrial genome of this species has not been systematically described, owing to the complexity of the plant mitogenome. In the present research, the complete mitochondrial genome of P. pedunculata was assembled, annotated, and characterized. The genomic features, gene content and repetitive sequences were analyzed. The genomic variation and phylogenetic analysis have been extensively enumerated. The P. pedunculata mitogenome is a circular molecule with a total length of 405,855 bp and a GC content of 45.63%, which are the smallest size and highest GC content among the known Prunus mitochondrial genomes. The mitogenome of P. pedunculata encodes 62 genes, including 34 unique protein-coding genes (PCGs, excluding three possible pseudogenes), three ribosomal RNA genes, and 19 transfer RNA genes. The mitogenome is rich in repetitive sequences, counting 112 simple sequence repeats, 15 tandem repeats, and 50 interspersed repetitive sequences, with a total repeat length of 11,793 bp, accounting for 2.91% of the complete genome. Leucine (Leu) was a predominant amino acid in PCGs, with a frequency of 10.67%, whereas cysteine (Cys) and tryptophan (Trp) were the least adopted. The most frequently used codon was UUU (Phe), with a relative synonymous codon usage (RSCU) value of 1.12. Selective pressure was calculated based on 20 shared PCGs in the mitogenomes of the 32 species, most of which were subjected to purifying selection (Ka/Ks < 1), whereas ccmC and ccmFn underwent positive selection. A total of 262 potential RNA editing sites in 26 PCGs were identified. Furthermore, 56 chloroplast-derived fragments were ascertained in the mitogenome, ranging from 30 to 858 bp, and were mainly located across IGS (intergenic spacer) regions or rRNA genes. These findings verify the occurrence of intracellular gene transfer events from the chloroplast to the mitochondria. Furthermore, the phylogenetic relationship of P. pedunculata was supported by the mitogenome data of 30 other taxa of the Rosaceae family. Understanding the mitochondrial genome characteristics of P. pedunculata is of great importance to promote comprehension of its genetic background and this study provides a basis for the genetic breeding of Prunus.

Assembly and comparative analysis of the complete mitochondrial genome of Trigonella foenum-graecum L.

BackgroundTrigonella foenum-graecum L. is a Leguminosae plant, and the stems, leaves, and seeds of this plant are rich in chemical components that are of high research value. The chloroplast (cp) genome of T. foenum-graecum has been reported, but the mitochondrial (mt) genome remains unexplored.ResultsIn this study, we used second- and third-generation sequencing methods, which have the dual advantage of combining high accuracy and longer read length. The results showed that the mt genome of T. foenum-graecum was 345,604 bp in length and 45.28% in GC content. There were 59 genes, including: 33 protein-coding genes (PCGs), 21 tRNA genes, 4 rRNA genes and 1 pseudo gene. Among them, 11 genes contained introns. The mt genome codons of T. foenum-graecum had a significant A/T preference. A total of 202 dispersed repetitive sequences, 96 simple repetitive sequences (SSRs) and 19 tandem repetitive sequences were detected. Nucleotide diversity (Pi) analysis counted the variation in each gene, with atp6 being the most notable. Both synteny and phylogenetic analyses showed close genetic relationship among Trifolium pratense, Trifolium meduseum, Trifolium grandiflorum, Trifolium aureum, Medicago truncatula and T. foenum-graecum. Notably, in the phylogenetic tree, Medicago truncatula demonstrated the highest level of genetic relatedness to T. foenum-graecum, with a strong support value of 100%. The interspecies non-synonymous substitutions (Ka)/synonymous substitutions (Ks) results showed that 23 PCGs had Ka/Ks < 1, indicating that these genes would continue to evolve under purifying selection pressure. In addition, setting the similarity at 70%, 23 homologous sequences were found in the mt genome of T. foenum-graecum.ConclusionsThis study explores the mt genome sequence information of T. foenum-graecum and complements our knowledge of the phylogenetic diversity of Leguminosae plants.

Integrase-associated niche differentiation of endogenous large DNA viruses in crustaceans.

Crustacean genomes harbor sequences originating from a family of large DNA viruses called nimaviruses, but it is unclear why they are present. We show that endogenous nimaviruses selectively insert into repetitive sequences within the host genome, and this insertion specificity was correlated with different types of integrases, which are DNA recombination enzymes encoded by the nimaviruses themselves. This suggests that endogenous nimaviruses have colonized various genomic niches through the acquisition of integrases with different insertion specificities. Our results point to a novel survival strategy of endogenous large DNA viruses colonizing the host genomes. These findings may clarify the evolution and spread of nimaviruses in crustaceans and lead to measures to control and prevent the spread of pathogenic nimaviruses in aquaculture settings.

Chromosome-scale genome assembly of sweet tea (Lithocarpus polystachyus Rehder)

Lithocarpus, with >320 species, is the second largest genus of Fagaceae. However, the lack of a reference genome limits the molecular biology and functional study of Lithocarpus species. Here, we report the chromosome-scale genome assembly of sweet tea (Lithocarpus polystachyus Rehder), the first Lithocarpus species to be sequenced to date. Sweet tea has a 952-Mb genome, with a 21.4-Mb contig N50 value and 98.6% complete BUSCO score. In addition, the per-base consensus accuracy and completeness of the genome were estimated at 60.6 and 81.4, respectively. Genome annotation predicted 37,396 protein-coding genes, with repetitive sequences accounting for 64.2% of the genome. The genome did not undergo whole-genome duplication after the gamma (γ) hexaploidy event. Phylogenetic analysis showed that sweet tea diverged from the genus Quercus approximately at 59 million years ago. The high-quality genome assembly and gene annotation resources enrich the genomics of sweet tea, and will facilitate functional genomic studies in sweet tea and other Fagaceae species.

Telomere length is not a useful tool for chronological age estimation in animals.

Telomeres are short repetitive DNA sequences capping the ends of chromosomes. Telomere shortening occurs during cell division and may be accelerated by oxidative damage or ameliorated by telomere maintenance mechanisms. Consequently, telomere length changes with age, which was recently confirmed in a large meta-analysis across vertebrates. However, based on the correlation between telomere length and age, it was concluded that telomere length can be used as a tool for chronological age estimation in animals. Correlation should not be confused with predictability, and the current data and studies suggest that telomeres cannot be used to reliably predict individual chronological age. There are biological reasons for why there is large individual variation in telomere dynamics, which is mainly due to high susceptibility to a wide range of environmental, but also genetic factors, rendering telomeres unfeasible as a tool for age estimation. The use of telomeres for chronological age estimation is largely a misguided effort, but its occasional reappearance in the literature raises concerns that it will mislead resources in wildlife conservation.

Abstract C086: NP1867, a potent, selective, covalent small molecule inhibitor of DNA Mismatch Repair (MMR) protein PMS2, functionally inhibits MMR in cells and elicits COSMIC mutational signatures consistent with MMR-deficient patient samples

Abstract Two complementary mechanisms safeguard genome fidelity during DNA replication: DNA polymerase proofreading and DNA mismatch repair (MMR). DNA polymerase proofreading or MMR deficiencies can occur independently or, more rarely, simultaneously; loss of function of either or both pathways leads to the accumulation of DNA mutations. Lynch Syndrome is caused by mono-allelic germline mutations in MMR genes MLH1, MSH2, MSH6 or PMS2. Gene expression from the wild-type allele elicits sufficient MMR activity until a second hit somatic mutation inactivates the wild-type allele leading to MMR deficiency (MMR-d). Constitutional mismatch repair deficiency (CMMRD) syndrome is an inherited early onset cancer predisposition syndrome resulting from biallelic germline mutations in MLH1, MSH2, MSH6 or PMS2. MMR-d cancers are commonly and typically characterized by accumulation of single nucleotide variants (SNVs) and insertions or deletions (indels) amongst repetitive DNA sequences - microsatellite instability (MSI); importantly, such mutations occur at higher rates in genetically unstable tumour cells under replicative stress than in normal cells. Cancers that harbour &amp;gt;40% microsatellite variations (positive for two or more of five clinically tested microsatellite markers) are described as MSI-high (MSI-H). Importantly, MMR-d, MSI-H tumours are now eligible for immune checkpoint therapy, irrespective of tissue type, providing substantial patient benefit and a major advance in cancer treatment. This clinical context is consistent with the hypothesis that MMR-d elicits an immunogenic state. However, there is an unmet need for chemical tools to explore the role of therapeutic MMR inhibition alongside immune checkpoint blockade. Here, for the first time, we demonstrate that NP1867 is a potent, selective small molecule inhibitor of the Gyrase, Hsp90, Histidine Kinase, MutL (GHKL) family endonuclease PMS1 Homolog 2, Mismatch Repair System Component (PMS2). We demonstrate by biochemical, biophysical, and protein-ligand X-ray crystallographic methods that NP1867 binds irreversibly to the ATP binding site of the N-terminal ATPase domain of PMS2. NP1867 displays potent binding to PMS2 in a NanoBRET cellular target engagement assay and inhibits MMR activity in cell-based assays of MMR-dependent DNA repair. NP1867 displays minimal off-target binding in broad biochemical screening panels including kinases and ATPases. Treatment of MMR-proficient, colorectal cancer cell lines with NP1867 increases tumour mutational burden and generates COSMIC mutational signatures consistent with MMR-d patient samples demonstrating, for the first time, that acute inhibition of PMS2 with a small molecule phenocopies the epigenetic silencing and/or genetic loss of MMR observed in patients. We characterise cell- and tissue-based target engagement, and in vivo pharmacokinetic and pharmacodynamic properties of NP1867 to inform optimal use by the research community for further exploring PMS2 and MMR function. Citation Format: Julian Blagg, Philippe Riou, Alexia Hervieu, Eleonora Piumatti, Giuseppe Rospo, Sasi Arunachalam, Bettina Meier, Sam Weeks, Maria Rodriguez, Kalpesh Parmar, Pradip Patel, Adam Peall, Paige Tongue, Tess McLaren, Robert Workman, Ruzica Bago, Stuart Robjohns, Rebecca Beaumont, Mike Briggs, Gareth Langley, Charles Nichols, David Clark, Paul Winship, Benoit Rousseau, Matthew Baker, Martin Drysdale, Giovanni Germano, Alberto Bardelli. NP1867, a potent, selective, covalent small molecule inhibitor of DNA Mismatch Repair (MMR) protein PMS2, functionally inhibits MMR in cells and elicits COSMIC mutational signatures consistent with MMR-deficient patient samples [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C086.