Passer, a highly active transposon from a fish genome, as a potential new robust genetic manipulation tool.

The discovery of new, active DNA transposons can expand the range of genetic tools and provide more options for genomic manipulation. In this study, a bioinformatics analysis suggested that Passer (PS) transposons, which are members of the pogo superfamily, show signs of recent and current activity in animals and may be active in some species. Cell-based transposition assays revealed that the native PS transposases from Gasterosteus aculeatus and Danio rerio displayed very high activity in human cells relative to the Sleeping Beauty transposon. A typical overproduction inhibition phenomenon was observed for PS, and transposition capacity was decreased by ∼12% with each kilobase increase in the insertion size. Furthermore, PS exhibited a pronounced integration preference for genes and their transcriptional regulatory regions. We further show that two domesticated human proteins derived from PS transposases have lost their transposition activity. Overall, PS may represent an alternative with a potentially efficient genetic manipulation tool for transgenesis and mutagenesis applications.

A native, highly active Tc1/mariner transposon from zebrafish (ZB) offers an efficient genetic manipulation tool for vertebrates.

New genetic tools and strategies are currently under development to facilitate functional genomics analyses. Here, we describe an active member of the Tc1/mariner transposon superfamily, named ZB, which invaded the zebrafish genome very recently. ZB exhibits high activity in vertebrate cells, in the range of those of the widely used transposons piggyBac (PB), Sleeping Beauty (SB) and Tol2. ZB has a similar structural organization and target site sequence preference to SB, but a different integration profile with respect to genome-wide preference among mammalian functional annotation features. Namely, ZB displays a preference for integration into transcriptional regulatory regions of genes. Accordingly, we demonstrate the utility of ZB for enhancer trapping in zebrafish embryos and in the mouse germline. These results indicate that ZB may be a powerful tool for genetic manipulation in vertebrate model species.

The Annotation of Zebrafish Enhancer Trap Lines Generated with PB Transposon.

An enhancer trap (ET) mediated by a transposon is an effective method for functional gene research. Here, an ET system based on a PB transposon that carries a mini Krt4 promoter (the keratin4 minimal promoter from zebrafish) and the green fluorescent protein gene (GFP) has been used to produce zebrafish ET lines. One enhancer trap line with eye-specific expression GFP named EYE was used to identify the trapped enhancers and genes. Firstly, GFP showed a temporal and spatial expression pattern with whole-embryo expression at 6, 12, and 24 hpf stages and eye-specific expression from 2 to 7 dpf. Then, the genome insertion sites were detected by splinkerette PCR (spPCR). The Krt4-GFP was inserted into the fourth intron of the gene itgav (integrin, alpha V) in chromosome 9 of the zebrafish genome, with the GFP direction the same as that of the itgav gene. By the alignment of homologous gene sequences in different species, three predicted endogenous enhancers were obtained. The trapped endogenous gene itgav, whose overexpression is related to hepatocellular carcinoma, showed a similar expression pattern as GFP detected by in situ hybridization, which suggested that GFP and itgav were possibly regulated by the same enhancers. In short, the zebrafish enhancer trap lines generated by the PB transposon-mediated enhancer trap technology in this study were valuable resources as visual markers to study the regulators and genes. This work provides an efficient method to identify and isolate tissue-specific enhancer sequences.

Horizontal transfer of Buster transposons across multiple phyla and classes of animals.

Transposable elements (TEs) are mobile genetic elements in the genome and broadly distributed across both prokaryotes and eukaryotes, and play an important role in shaping the genome evolution of their hosts. hAT elements are thought to be the most widespread cut-and-paste DNA transposon found throughout the tree of life. Buster is a recently recognized family of hAT. However, the evolutionary profile of the Buster family, such as its taxonomic distribution, evolutionary pattern, and activities, remains largely unknown. We conducted a systematic analysis of the evolutionary landscape of the Buster family and found that most Buster transposons are 1.72-4.66 kilobases (kb) in length, encode 500-736-amino acid (aa) transposases and are flanked by short (10-18 bp) terminal inverted repeats (TIRs) and 8 bp target site duplications (TSDs). Buster family is widely distributed in 609 species, involving eight classes of invertebrates and most lineage of vertebrates (including mammals). Horizontal transfer events were detected across multiple phyla and classes of animals, which may have contributed to their wide distribution, and both parasites and invasive species may facilitate HT events of Buster in vertebrates. Our data also suggest that Buster transposons are young, highly active, and appear as intact copies in multiple lineages of animals. High percentages of intact copies (>30%) were identified in some Arthropoda, Actinopterygii, Agnatha, and reptile species, and some of these may be active. These data will help increase understanding of the evolution of the hAT superfamily and its impact on eukaryotic genome evolution.

Phylogenetic Relationships among TnpB-Containing Mobile Elements in Six Bacterial Species.

Some families of mobile elements in bacterial genomes encode not only a transposase but also an accessory TnpB gene. This gene has been shown to encode an RNA-guided DNA endonuclease, co-evolving with Y1 transposase and serine recombinase in mobile elements IS605 and IS607. In this paper, we reveal the evolutionary relationships among TnpB-containing mobile elements (TCMEs) in well-assembled genomes of six bacterial species: Bacillus cereus, Clostridioides difficile, Deinococcus radiodurans, Escherichia coli, Helicobacter pylori and Salmonella enterica. In total, 9996 TCMEs were identified in 4594 genomes. They belonged to 39 different insertion sequences (ISs). Based on their genetic structures and sequence identities, the 39 TCMEs were classified into three main groups and six subgroups. According to our phylogenetic analysis, TnpBs include two main branches (TnpB-A and TnpB-B) and two minor branches (TnpB-C and TnpB-D). The key TnpB motifs and the associated Y1 and serine recombinases were highly conserved across species, even though their overall sequence identities were low. Substantial variation was observed for the rate of invasion across bacterial species and strains. Over 80% of the genomes of B. cereus, C. difficile, D. radiodurans and E. coli contained TCMEs; however, only 64% of the genomes of H. pylori and 44% of S. enterica genomes contained TCMEs. IS605 showed the largest rate of invasion in these species, while IS607 and IS1341 had a relatively narrow distribution. Co-invasions of IS605, IS607 and IS1341 elements were observed in various genomes. The largest average copy number was observed for IS605b elements in C. difficile. The average copy numbers of most other TCMEs were smaller than four. Our findings have important implications for understanding the co-evolution of TnpB-containing mobile elements and their biological roles in host genome evolution.

Apoptotic mechanism of propofol-induced developmental toxicity in zebrafish embryos.

General anesthetics can cause neurological damage and long-term behavioral/cognitive impairment during fetal and early postnatal life. However, the adverse influence on embryo development induced by propofol is unclear. We used embryonic zebrafish to explore the effects of propofol on embryonic and larval growth and development, and the related apoptotic mechanism. Zebrafish embryos were immersed in propofol (1, 2, 3, 4, and 5 µg/ml) dissolved in E3 medium from 6 to 48 hours post fertilization (hpf). The survival rate, locomotion, heart rate, hatchability, deformity rate, and body length were analyzed at defined stages. Terminal deoxynucleotidyl transferase nick-end-labeling was used to detect zebrafish embryo apoptosis, and the expression levels of apoptosis-related genes were determined using quantitative real-time reverse transcription PCR and whole-mount in situ hybridization. Larvae at 48 hpf were anesthetized by immersion in E3 culture medium containing 2 µg/ml propofol, the reasonable anesthetic concentration for zebrafish embryos, which caused significant caudal fin dysplasia, light pigmentation, edema, hemorrhage, and spinal deformity, and decreased the hatchability, body length, and heart rate. The numbers of apoptotic cells in propofol-treated 12, 48 and 72 hpf embryos increased significantly, and the mRNA expression levels of intrinsic apoptosis pathway-related casp3a, casp3b, casp9, and baxb genes were upregulated, mainly in the head and tail. Propofol decreased apoptosis in the head and back of 24 hpf zebrafish, which was consistent with the mRNA expression analysis. Our findings demonstrated that zebrafish embryos and larvae exposed to propofol experienced developmental toxicity, which correlated with the intrinsic apoptosis pathway with casp3a, casp3b, casp9, and baxb as the key genes.

Evolution of piggyBac Transposons in Apoidea.

In this study, we investigated the presence of piggyBac (PB) transposons in 44 bee genomes from the Apoidea order, which is a superfamily within the Hymenoptera, which includes a large number of bee species crucial for pollination. We annotated the PB transposons in these 44 bee genomes and examined their evolution profiles, including structural characteristics, distribution, diversity, activity, and abundance. The mined PB transposons were divided into three clades, with uneven distribution in each genus of PB transposons in Apoidea. The complete PB transposons we discovered are around 2.23-3.52 kb in length and encode transposases of approximately 580 aa, with terminal inverted repeats (TIRs) of about 14 bp and 4 bp (TTAA) target-site duplications. Long TIRs (200 bp, 201 bp, and 493 bp) were also detected in some species of bees. The DDD domains of the three transposon types were more conserved, while the other protein domains were less conserved. Generally, most PB transposons showed low abundance in the genomes of Apoidea. Divergent evolution dynamics of PB were observed in the genomes of Apoidea. PB transposons in some identified species were relatively young, whiles others were older and with some either active or inactive. In addition, multiple invasions of PB were also detected in some genomes of Apoidea. Our findings highlight the contribution of PB transposons to genomic variation in these species and suggest their potential as candidates for future gene transfer tools.

Comparative Analysis and Phylogenetic Insights of Cas14-Homology Proteins in Bacteria and Archaea.

Type-V-F Cas12f proteins, also known as Cas14, have drawn significant interest within the diverse CRISPR-Cas nucleases due to their compact size. This study involves analyzing and comparing Cas14-homology proteins in prokaryotic genomes through mining, sequence comparisons, a phylogenetic analysis, and an array/repeat analysis. In our analysis, we identified and mined a total of 93 Cas14-homology proteins that ranged in size from 344 aa to 843 aa. The majority of the Cas14-homology proteins discovered in this analysis were found within the Firmicutes group, which contained 37 species, representing 42% of all the Cas14-homology proteins identified. In archaea, the DPANN group had the highest number of species containing Cas14-homology proteins, a total of three species. The phylogenetic analysis results demonstrate the division of Cas14-homology proteins into three clades: Cas14-A, Cas14-B, and Cas14-U. Extensive similarity was observed at the C-terminal end (CTD) through a domain comparison of the three clades, suggesting a potentially shared mechanism of action due to the presence of cutting domains in that region. Additionally, a sequence similarity analysis of all the identified Cas14 sequences indicated a low level of similarity (18%) between the protein variants. The analysis of repeats/arrays in the extended nucleotide sequences of the identified Cas14-homology proteins highlighted that 44 out of the total mined proteins possessed CRISPR-associated repeats, with 20 of them being specific to Cas14. Our study contributes to the increased understanding of Cas14 proteins across prokaryotic genomes. These homologous proteins have the potential for future applications in the mining and engineering of Cas14 proteins.

Revisiting the Tigger Transposon Evolution Revealing Extensive Involvement in the Shaping of Mammal Genomes.

The data of this study revealed that Tigger was found in a wide variety of animal genomes, including 180 species from 36 orders of invertebrates and 145 species from 29 orders of vertebrates. An extensive invasion of Tigger was observed in mammals, with a high copy number. Almost 61% of those species contain more than 50 copies of Tigger; however, 46% harbor intact Tigger elements, although the number of these intact elements is very low. Common HT events of Tigger elements were discovered across different lineages of animals, including mammals, that may have led to their widespread distribution, whereas Helogale parvula and arthropods may have aided Tigger HT incidences. The activity of Tigger seems to be low in the kingdom of animals, most copies were truncated in the mammal genomes and lost their transposition activity, and Tigger transposons only display signs of recent and current activities in a few species of animals. The findings suggest that the Tigger family is important in structuring mammal genomes.

Prokaryotic and Eukaryotic Horizontal Transfer of Sailor (DD82E), a New Superfamily of IS630-Tc1-Mariner DNA Transposons.

Here, a new superfamily of IS630-Tc1-mariner (ITm) DNA transposons, termed Sailor, is identified, that is characterized by a DD82E catalytic domain and is distinct from all previously known superfamilies of the ITm group. Phylogenetic analyses revealed that Sailor forms a monophyletic clade with a more intimate link to the clades of Tc1/mariner and DD34E/Gambol. Sailor was detected in both prokaryotes and eukaryotes and invaded a total of 256 species across six kingdoms. Sailor is present in nine species of bacteria, two species of plantae, four species of protozoa, 23 species of Chromista, 12 species of Fungi and 206 species of animals. Moreover, Sailor is extensively distributed in invertebrates (a total of 206 species from six phyla) but is absent in vertebrates. Sailor transposons are 1.38-6.98 kb in total length and encoded transposases of ~676 aa flanked by TIRs with lengths between 18, 1362 and 4 bp (TATA) target-site duplications. Furthermore, our analysis provided strong evidence of Sailor transmissions from prokaryotes to eukaryotes and internal transmissions in both. These data update the classification of the ITm group and will contribute to the understanding of the evolution of ITm transposons and that of their hosts.