Search Results (558)

Background/Objectives: The highly polymorphic Major Histocompatibility Complex (MHC) genomic region, located on the short arm of chromosome 6, is implicated genetically in Parkinson’s disease (PD), a progressive neurodegenerative disorder with motor and non-motor symptoms. Previously, we reported significant associations between SINE-VNTR-Alu (SVA) expression quantitative trait loci (eQTLs) and Human Leucocyte Antigen (HLA) class I genotypes in PD. In this study, we aimed to evaluate SVA associations and their regulatory effects on transposable element (TE) transcription in the MHC class I region. Methods: Transcriptome data from the peripheral blood cells of 1530 individuals in the Parkinson’s Progression Markers Initiative (PPMI) cohort were reanalyzed for TE and gene expression using publicly available bioinformatics tools, including Salmon and Matrix-eQTL. Results: Four structurally polymorphic SVAs regulated the transcription of 18 distinct clusters of 235 TE loci, comprising LINEs (33%), SINEs (19%), LTRs (35%), and ancient transposon DNA elements (12%) located near HLA genes. The transcribed TEs were predominantly short, with an average length of 445 nucleotides. The regulatory effects of these SVAs varied significantly in terms of TE types, numbers, and transcriptional activation or repression. The SVA-regulated TE RNAs in blood cells appear to function as enhancer-like elements, differentially influencing the expression of HLA class I genes, non-HLA genes, and noncoding RNAs. Conclusions: These findings highlight the roles of SVAs and their associated TEs in the complex regulatory networks governing coding and noncoding gene expression in the MHC class I region, with potential implications for immune function and disease susceptibility. Full article

The successful application of CAR-T cells in the treatment of hematologic malignancies has fundamentally changed cancer therapy. With increasing numbers of registered CAR-T cell clinical trials, efforts are being made to streamline and reduce the costs of CAR-T cell manufacturing while improving their safety. To date, all approved CAR-T cell products have relied on viral-based gene delivery and genomic integration methods. While viral vectors offer high transfection efficiencies, concerns regarding potential malignant transformation coupled with costly and time-consuming vector manufacturing are constant drivers in the search for cheaper, easier-to-use, safer, and more efficient alternatives. In this review, we examine different non-viral gene transfer methods as alternatives for CAR-T cell production, their advantages and disadvantages, and examples of their applications. Transposon-based gene transfer methods lead to stable but non-targeted gene integration, are easy to handle, and achieve high gene transfer rates. Programmable endonucleases allow targeted integration, reducing the potential risk of integration-mediated malignant transformation of CAR-T cells. Non-integrating CAR-encoding vectors avoid this risk completely and achieve only transient CAR expression. With these promising alternative techniques for gene transfer, all avenues are open to fully exploiting the potential of next-generation CAR-T cell therapy and applying it in a wide range of applications. Full article

Plant genomes possess numerous transposable element (TE) insertions that have occurred during evolution. Most TEs are silenced or diverged; therefore, they lose their ability to encode proteins and are transposed in the genome. Knowledge of active plant TEs and TE-encoded proteins essential for transposition and evasion of plant cell transposon silencing mechanisms remains limited. This study investigated active long terminal repeat (LTR) retrotransposons (RTEs) in sunflowers (Helianthus annuus), revealing heterogeneous and phylogenetically distinct RTEs triggered by epigenetic changes and heat stress. Many of these RTEs belong to three distinct groups within the Tekay clade, showing significant variations in chromosomal insertion distribution. Through protein analysis of these active RTEs, it was found that Athila RTEs and Tekay group 2 elements possess additional open reading frames (aORFs). The aORF-encoded proteins feature a transposase domain, a transmembrane domain, and nuclear localization signals. The aORF proteins of the Tekay subgroup exhibited remarkable conservation among over 500 Tekay members, suggesting their functional importance in RTE mobility. The predicted 3D structure of the sunflower Tekay aORF protein showed significant homology with Tekay proteins in rice, maize, and sorghum. Additionally, the structural features of aORF proteins resemble those of plant DRBM-containing proteins, suggesting their potential role in RNA-silencing modulation. These findings offer insights into the diversity and activity of sunflower RTEs, emphasizing the conservation and structural characteristics of aORF-encoded proteins. Full article

Amaranthus cruentus L. and Amaranthus hypochondriacus L. are valuable and promising food crops for multi-purpose use that are distributed worldwide in temperate, subtropical, and tropical zones. However, their karyotypes and genomic relationships still remain insufficiently studied. For the first time, a comparative repeatome analysis of A. cruentus and A. hypochondriacus was performed based on the available NGS data; bioinformatic analyses using RepeatExplorer/TAREAN pipelines; and chromosome FISH mapping of 45S rDNA, 5S rDNA, and the most abundant satellite DNAs. In the repeatomes of these species, interspecific variations in the amount of Ty3/Gypsy and Ty1/Copia retroelements, DNA transposons, ribosomal, and satellite DNA were detected. In the repeatomes of both species, shared satDNAs with high sequence similarity were identified. The chromosome distribution patterns of four effective molecular markers, 45S rDNA, 5S rDNA, AmC4, and AmC9, allowed us to identify all chromosome pairs in the species karyotypes, construct unique karyograms of A. cruentus and A. hypochondriacus, and confirm the close relationship between their genomes. These results are important for comparative karyotypic studies within the genus Amaranthus. Our findings demonstrated that cytogenomic analyses might provide important data on genomic relationships within Amaranthus and increase knowledge on genome organization in these valuable crops. Full article

Insertions of the transposable element IS5 into its target sites in response to stressful environmental conditions, DNA structures, and DNA-binding proteins are well studied, but how the genomic contexts near IS5′s native loci impact its transpositions is largely unknown. Here, by examining the roles of all 11 copies of IS5 within the genome of E. coli strain BW25113 in transposition, we reveal that the most significant copy of IS5 is one nested within and oriented in the same direction as the nmpC gene, while two other copies of IS5 harboring point mutations are hardly transposed. Transposition activity is heavily reliant on the upstream nmpC promoter that drives IS5 transposase gene ins5A, with more transpositions resulting from greater promoter activity. The IS5 element at nmpC but not at other loci transcribed detectable amounts of ins5A mRNA. By increasing expression of the ins5CB operon harbored in IS5, we demonstrate that Ins5B and Ins5C appear to exert a stimulatory role in IS5 transposition, suggesting that the downstream genomic regions near the native loci are involved in overall IS5 transposition as well. Using a strain that carries IS5 only at the nmpC locus, we confirm that IS5 primarily uses a copy/paste mechanism for transposition, although we cannot rule out the cut/paste mechanism. Full article

Alternative splicing of ddx4 (DEAD-box helicase 4), a key germline marker gene, has been reported to generate sex-specific transcripts in zebrafish gonads. The biological functions and regulatory mechanisms of the ddx4 ovary-specific transcript (ddx4-L) during oogenesis remain unclear. In this study, we found that ddx4-L mutants, in which ddx4-L was specifically deleted, had enlarged ovaries but laid fewer eggs, along with having a lower fertilization rate compared to WT controls. RNA-seq analysis was performed to detect the changes in gene expression between WT and ddx4-L mutant ovaries. A total of 524 upregulated and 610 downregulated DEGs were identified. GO and GSEA enrichment analyses showed that genes involved in fertilization and reproduction biological processes were significantly downregulated. More specifically, we observed a remarkable reduction in Sycp1, a core component of synaptonemal complex, in ddx4-L mutant ovaries at both the mRNA and protein levels. In addition, the expressions of transposon elements, as well as the events of alternative splicing, alternative polyadenylation, and RNA editing, were analyzed based on the RNA-seq data. We found that the deletion of ddx4-L resulted in derepression of DNA transposons in zebrafish ovaries, possibly causing genome instability. In conclusion, our work demonstrates that the ovary-specific ddx4 transcript plays important roles in oocyte meiosis and DNA transposon repression, which extends our understanding of the biological functions and regulatory mechanisms of sex-specific alternative splicing in zebrafish oogenesis and reproduction. Full article

Pseudomonas cannabina pv. alisalensis (Pcal) causes bacterial blight on cabbage. In a previous study, we screened for reduced virulence using Tn5 transposon mutants and identified a LysR-type transcriptional regulator (LTTR) as a potential virulence factor in Pcal. However, the role of LTTR in Pcal virulence has not been thoroughly investigated. In this study, we demonstrated that the Pcal NN14 mutant (with Tn5 insertion in the LTTR-encoding gene) showed reduced disease symptoms and bacterial populations in cabbage, indicating that LTTR contributes to Pcal virulence. RNA-seq analysis identified 39 LTTR-dependent genes. Genes associated with 13 of the type three secretion system (T3SS), two of flagellar apparatus, ABC transporters, and transcription factors were expressed at lower levels in the NN14 mutant compared to the wild type. Conversely, tssH and hcp, type six secretion system (T6SS)-related genes, showed higher expression in NN14. Furthermore, these differences in gene expression were observed in minimal medium, but not in nutrient-rich medium, suggesting that LTTR acts as a global regulator responsive to nutrient conditions. Additionally, LTTR activated the expression of T3SS-related genes during Pcal infection. We also demonstrated that NN14 showed a reduced ability to induce hypersensitive reaction (HR) cell death in non-host plants. Collectively, these results suggest that LTTR contributes to Pcal virulence by regulating T3SS in response to environmental changes. Full article

Mycoplasma bovis (M. bovis) is characterized by a reduced genomic size and limited synthetic capacity, including the inability to synthesize nucleotides de novo, relies on nucleases for nutrient acquisition and survival. A number of nucleases have been implicated in M. bovis pathogenicity, facilitating substrate degradation and contributing to DNA repair mechanisms that enhance bacterial persistence. The present study confirmed that the T5.808 mutant, in which a novel nuclease gene (Mbov_0701) was disrupted by the mini-Tn4001 transposon, exhibits a growth defect when co-cultured with EBL cells. However, the restoration of Mbov_0701 resulted in the resumption of growth in the mutant. The characterization of MbovP701 revealed that it had high activity in hydrolyzing dsDNA with 5′- to 3′- polarity. Furthermore, the substrates of MbovP701 were extended to include linear dsDNA, ssDNA, RNA, and plasmid DNA. The exonuclease activity is dependent on the presence of Mn²⁺ and/or Mg²⁺ ions, with an optimal pH and temperature of 8.3 and 43 °C, respectively. The truncation experiments of rMbovP701 revealed that YqaJ (41–185 aa) is the key functional domain of MbovP701 exonuclease. In conclusion, the present study identified a novel nuclease in M. bovis that plays an essential role in the proliferation of this minimal organism. This finding elucidates the survival strategy and pathogenesis of M. bovis, suggesting a potential therapeutic strategy for the treatment of M. bovis through targeting the inhibition of MbovP701. Moreover, it provides a foundation for future investigations into the interactions between MbovP701 and other nucleases involved in M. bovis biology. Full article

Morels (Morchella sp.) are important edible fungi cultivated mainly in China. Although the relevant culture technology for Morchella is now fundamentally mature, it is limited to the Elata and Rufobrunnea clades, and the artificial culture technology for the Esculenta clade, which also has economic value, has not been extensively studied. In this study, we selected a wild morel belonging to the Esculenta clade as the research material and performed de novo sequencing and assembly of the Morchella sp. (Mosp) genome using second- and third-generation sequencing. The whole-genome size of Mosp was 55.17 Mb with a contig N50 of 1.89 Mb, and the GC content was 47.49%. A total of 10,896 protein-coding genes were identified. The non-coding RNA prediction results showed that there were 329 tRNAs, 65 rRNAs, and 37 snRNAs in the Mosp genome. The functional annotation of the Mosp genes showed that most of the genes were related to the reproductive and metabolic processes of the cells and participated in nutrient digestion, absorption, utilization, and catabolism in morels. There was a high degree of repetition (21.58%) in the Mosp genome, and the sizes of the DNA transposons and the long terminal repeats were 0.55 Mb and 5.85 Mb, respectively. The phylogeny analysis showed that Mosp clusters together with four other Morchella species: Morchella importuna, Morchella conica, Morchella sextelata, and Morchella snyderi. Molecular dating indicated that the differentiation of Mosp and the black morels group occurred about 147.0 million years ago (MYA). In addition, the evolutionary analysis showed that 296 gene families were contracted and 96 gene families were expanded in Mosp versus the related morel species. The results of this study provide new insights into the genome evolution of Mosp and lay the foundation for future in-depth research into the molecular biology and breeding of the genus Morchella. Full article

Background: Transposable elements (TEs) and noncoding sequences are major components of the genome, yet their functional contributions to long noncoding RNAs (lncRNAs) are not well understood. Although many lncRNAs originating from TEs (TE-lncRNAs) have been identified across various organisms, their characteristics and regulatory roles, particularly in insects, remain largely unexplored. This study integrated multi-omics data to investigate TE-lncRNAs in D. melanogaster, focusing on the influence of transposons across different omics levels. Results: We identified 16,118 transposons overlapping with lncRNA sequences that constitute 2119 TE-lncRNAs (40.4% of all lncRNAs) using 256 public RNA-seq samples and 15 lncRNA-seq samples of Drosophila S2 cells treated with heavy metals. Of these, 67.2% of TE-lncRNAs contain more than one TE. The LTR/Gypsy family was the most common transposon insertion. Transposons preferred to insert into promoters, transcription starting sites, and intronic regions, especially in chromosome ends. Compared with lncRNAs, TE-lncRNAs showed longer lengths, a lower conservation, and lower levels but a higher specificity of expression. Multi-omics data analysis revealed positive correlations between transposon insertions and chromatin openness at the pre-transcriptional level. Notably, a total of 516 TE-lncRNAs provided transcriptional factor binding sites through transposon insertions. The regulatory network of a key transcription factor was rewired by transposons, potentially recruiting other transcription factors to exert regulatory functions under heavy metal stress. Additionally, 99 TE-lncRNAs were associated with m⁶A methylation modification sites, and 115 TE-lncRNAs potentially provided candidate small open reading frames through transposon insertions. Conclusions: Our data analysis demonstrated that TEs contribute to the regulation of lncRNAs. TEs not only promote the transcriptional regulation of lncRNAs, but also facilitate their post-transcriptional and epigenetic regulation. Full article

Bile salts possess innate antibacterial properties and can cause significant damage to bacteria. To survive in the mammalian gut, Klebsiella pneumoniae has developed mechanisms to tolerate bile salts; however, the specific mechanisms remain unclear. Transposon library screening revealed that the efflux pump AcrAB is involved in bile salt resistance. acrA and acrB mutants exhibited high sensitivity not only to bile salts but also to SDS and various antibiotics, with a switch-loop, comprising residues G615, F616, A617, and G618, proving to be crucial in this process. A colonization defect of acrA and acrB mutants was demonstrated to be located in the mouse small intestine, where the bile salt concentration is higher compared to the large intestine. Additionally, both acrA and acrB mutants displayed reduced virulence in the Galleria mellonella model. In conclusion, our results suggest that the Resistance-Nodulation-Cell Division efflux pump serves as a critical determinant in the pathogenesis of K. pneumoniae through various aspects. Full article

Spider silk is part of a special class of natural protein fibers that have high strength and toughness: these materials have excellent comprehensive properties that are not found in other natural fibers (including silk) or most synthetic fibers. Spider egg case filaments have good hardness, can resist water, can protect spider eggs from external threats, have a significantly high initial modulus and high moisture absorption rate, and are expected to be used as a new generation of environmentally friendly natural polymer fibers and biomaterials. However, spiders are predatory and difficult to rear in large numbers, and it is also difficult to obtain spider egg case filaments in large quantities. Silkworms and spiders have a similar spinning system, and the use of transgenic technology in silkworms can obtain stable and high-yield exogenous gene proteins for a long time, representing an ideal bioreactor for the production of spider silk. In this study, the eukaryotic bioreactor and piggyBac transposon system were employed to recombinantly introduce the egg case silk protein of Nephila clavata (Nc-CYSP1) into the silkworm in the silkworm heavy-chain expression system. The results revealed that the silk glands produced a new type of transgenic silk with a significantly high initial modulus and high moisture absorption. In summary, this study provides an experimental reference for future research on the large-scale production and application of spider egg case filamentous protein, with great application prospects in the development of new environmentally friendly materials. Full article

The CRISPR-Cas system functions as an adaptive immune mechanism in archaea and bacteria, providing defense against the invasion of foreign nucleic acids. Most CRISPR-Cas systems are classified into class 1 or class 2, with further subdivision into several subtypes. The primary distinction between class 1 and class 2 systems lies in the assembly of their effector modules. In class 1 systems, the effector complex consists of multiple proteins with distinct functions, whereas in class 2 systems, the effector is associated with a single protein. Class 1 systems account for approximately 90% of the CRISPR-Cas repertoire and are categorized into three types (type I, type IV, and type III) and 12 subtypes. To date, various CRISPR-Cas systems have been widely employed in the field of genetic engineering as essential tools and techniques for genome editing. Type I CRISPR-Cas systems remain a valuable resource for developing sophisticated application tools. This review provides a comprehensive review of the characteristics, mechanisms of action, and applications of class 1 type I CRISPR-Cas systems, as well as transposon-associated systems, offering effective approaches and insights for future research on the mechanisms of action, as well as the subsequent development and application of type I CRISPR-Cas systems. Full article

Erysipelas is a significant problem in the waterfowl farming in Poland, and information on the characteristics of the Erysipelothrix rhusiopathiae strains causing this disease is limited. In this study, we determined the serotypes, antimicrobial susceptibility, and potential mechanisms of resistance gene transfer in E. rhusiopathiae isolates (n = 60) from domestic geese and ducks. We also developed a multiplex PCR for the detection of resistance genes. The antimicrobial susceptibility of the isolates was assessed using the broth microdilution method. Resistance genes, integrative conjugative element (ICE)-specific genes, phage-specific genes, and serotype determinants were detected by PCR. Multilocus sequence typing (MLST) was performed for selected resistant strains. The comparative analyses included 260 E. rhusiopathiae strains whose whole genome sequences (WGSs) are publicly available. E. rhusiopathiae isolates represented 7 serotypes, among which serotypes 5 (38.3%) and 1b (28.3%) were the most common. All strains were susceptible to β-lactams, and the vast majority of them were resistant to tetracycline (85%) and enrofloxacin (80%). The percentages of isolates resistant to other antimicrobials used ranged from 3.3% to 16.7%. Ten isolates (16.7%) were found to be multidrug resistant (MDR). The genotypic resistance profiles of the E. rhusiopathiae strains corresponded to their phenotypic resistance, and the amplification patterns obtained using the 10-plex PCR developed in this study were fully consistent with the results of single PCRs. The most prevalent resistance gene was tetM. In enrofloxacin-resistant strains, nonsynonymous mutations in the gyrA and parC genes were identified. The presence of ICE-specific genes was confirmed in resistant strains, and in MDR isolates of serotype 8 that represented sequence type (ST) 113, prophage DNA (Javan630-like) linked to the lsaE gene was additionally detected. The results indicate that β-lactam antibiotics should be the first choice for the treatment of waterfowl erysipelas in Poland. ICEs, including a transposon from the Tn916/Tn1545 family, and bacteriophages are most likely responsible for the transfer of resistance genes in E. rhusiopathiae. Full article

Infection by retroviruses and the mobilization of transposable elements cause DNA damage that can be catastrophic for a cell. If the cell survives, the mutations generated by retrotransposition may confer a selective advantage, although, more commonly, the effect of new integrants is neutral or detrimental. If retrotransposition occurs in gametes or in the early embryo, it introduces genetic modifications that can be transmitted to the progeny and may become fixed in the germline of that species. PIWI-interacting RNAs (piRNAs) are single-stranded, 21–35 nucleotide RNAs generated by the PIWI clade of Argonaute proteins that maintain the integrity of the animal germline by silencing transposons. The sequence specific manner by which piRNAs and germline-encoded PIWI proteins repress transposons is reminiscent of CRISPR, which retains memory for invading pathogen sequences. piRNAs are processed preferentially from the unspliced transcripts of piRNA clusters. Via complementary base pairing, mature antisense piRNAs guide the PIWI clade of Argonaute proteins to transposon RNAs for degradation. Moreover, these piRNA-loaded PIWI proteins are imported into the nucleus to modulate the co-transcriptional repression of transposons by initiating histone and DNA methylation. How retroviruses that invade germ cells are first recognized as foreign by the piRNA machinery, as well as how endogenous piRNA clusters targeting the sequences of invasive genetic elements are acquired, is not known. Currently, koalas (Phascolarctos cinereus) are going through an epidemic due to the horizontal and vertical transmission of the KoRV-A gammaretrovirus. This provides an unprecedented opportunity to study how an exogenous retrovirus becomes fixed in the genome of its host, and how piRNAs targeting this retrovirus are generated in germ cells of the infected animal. Initial experiments have shown that the unspliced transcript from KoRV-A proviruses in koala testes, but not the spliced KoRV-A transcript, is directly processed into sense-strand piRNAs. The cleavage of unspliced sense-strand transcripts is thought to serve as an initial innate defense until antisense piRNAs are generated and an adaptive KoRV-A-specific genome immune response is established. Further research is expected to determine how the piRNA machinery recognizes a new foreign genetic invader, how it distinguishes between spliced and unspliced transcripts, and how a mature genome immune response is established, with both sense and antisense piRNAs and the methylation of histones and DNA at the provirus promoter. Full article