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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. [ABSTRACT FROM AUTHOR]

Diverse Tc1/mariner elements with the DD37E signature have been detected. However, their evolutionary relationship and profiles are largely unknown. Using bioinformatics methods, we defined the evolution profile of a Tc1/Mariner family, which harbors the catalytic domain with the DD37E signature, and renamed it DD37E/Mosquito (MS). MS transposons form a separate monophyletic clade in the phylogenetic tree, distinct from the other two groups of elements with the DD37E signature, DD37E/L18 and DD37E/TRT (transposon related to Tc1), and represent a very different taxonomic distribution from that of DD37E/TRT. MS is only detected in invertebrate and is mostly present in Arthropoda, as well as in Cnidaria, Ctenophora, Mollusca, Nematoda, and Platyhelminthes, with a total length of about 1.3 kb, containing an open reading frame (ORF) encoding about 340 amino acids transposases, with a conserved DD37E catalytic domain. The terminal inverted repeat (TIR) lengths range from 19 bp to 203 bp, and the target site duplication (TSD) is TA. We also identified few occurrences of MS horizontal transfers (HT) across lineages of diptera. In this paper, the distribution characteristics, structural characteristics, phylogenetic evolution, and horizontal transfer of the MS family are fully analyzed, which is conducive to supplementing and improving the Tc1/Mariner superfamily and excavating active transposons. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Both ZeBrafish (ZB), a recently identified DNA transposon in the zebrafish genome, and SB, a reconstructed transposon originally discovered in several fish species, are known to exhibit high transposition activity in vertebrate cells. Although a similar structural organization was observed for ZB and SB transposons, the evolutionary profiles of their homologs in various species remain unknown. In the present study, we compared their taxonomic ranges, structural arrangements, sequence identities, evolution dynamics, and horizontal transfer occurrences in vertebrates. In total, 629 ZB and 366 SB homologs were obtained and classified into four distinct clades, named ZB, ZB-like, SB, and SB-like. They displayed narrow taxonomic distributions in eukaryotes, and were mostly found in vertebrates, Actinopterygii in particular tended to be the major reservoir hosts of these transposons. Similar structural features and high sequence identities were observed for transposons and transposase, notably homologous to the SB and ZB elements. The genomic sequences that flank the ZB and SB transposons in the genomes revealed highly conserved integration profiles with strong preferential integration into AT repeats. Both SB and ZB transposons experienced horizontal transfer (HT) events, which were most common in Actinopterygii. Our current study helps to increase our understanding of the evolutionary properties and histories of SB and ZB transposon families in animals. [ABSTRACT FROM AUTHOR]

Retrotransposon is an important component of the mammalian genome. Previous studies have shown that the expression of protein-coding genes was affected by the insertion of retrotransposon into the proximal genes, and the phenotype variations would be related to the retrotransposon insertion polymorphisms (RIPs). In this study, leptin (LEP), leptin receptor (LEPR), and leptin receptor overlapping transcript (LEPROT), which play important roles in the regulation of fat synthesis and body weight, were screened to search for the RIPs and their effect on phenotype and gene expression, as well as to further study the function of the insertion. The results showed that three RIPs located in intron 1 of LEPROT and intron 2 and 21 of LEPR were identified, and they were all SINEA1, which was one type of retrotransposon. The SINE insertion at the LEPROT was the dominant allele in native pig breeds. The age of 100 kg body weight of SINE+/+ Large White individuals was significantly higher than those of SINE+/− and SINE−/− individuals (p < 0.05). The LEPROT gene expression in the liver and suet of 30-day-old SINE−/− Sujiang piglets were significantly higher than those of SINE+/+ and SINE+/− piglets (p < 0.01). The dual-luciferase reporter gene assay showed that SINE insertion in PK15 and 3T3-L1 cells significantly reduced the promoter activity of the LEPROT gene (p < 0.01). Therefore, SINE insertion can be a repressor to reduce the expression of LEPROT and could be a useful molecular marker for assisted selection of growth traits in pig breeding. [ABSTRACT FROM AUTHOR]

Retrotransposons account for more than one-third of the pig reference genome. On account of the genome variability in different breeds, structural variation (SV) caused by retrotranspos-on-generated deletion or insertion (indel) may have a function in the genome. Litter size is one of the most important reproductive traits and significantly impacts profitability in terms of pig production. We used the method of bioinformatics, genetics, and molecular biology to make an analysis among different pig genomes. Predicted 100 SVs were annotated as retrotransposon indel in 20 genes related to reproductive performance. The PCR detection based on these predicted SVs revealed 20 RIPs in 20 genes, that most RIPs (12) were generated by SINE indel, and eight RIPs were generated by the ERV indel. We selected 12 RIPs to make the second round PCR detection in 24 individuals among nine pig breeds. The PCR detection results revealed that the RIP-A1CF-4 insertion in the breed of Bama, Large White, and Meishan only had the homozygous genotype but low to moderately polymorphisms were present in other breeds. We found that RIP-CWH43-9, RIP-IDO2-9, RIP-PRLR-6, RIP-VMP1-12, and RIP-OPN-1 had a rich polymorphism in the breed of Large White pigs. The statistical analysis revealed that RIP-CWH43-9 had a SINE insertion profitable to the reproductive traits of TNB and NBA but was significantly affected (p < 0.01) and (p < 0.05) in the reproductive traits of litter birthweight (LW) in Large White. On the other hand, the SINE insertion in IDO2-9 may be a disadvantage to the reproductive traits of LW, which was significantly affected (p < 0.05) in Large White. These two RIPs are significant in pig genome research and could be useful molecular markers in the breeding system. [ABSTRACT FROM AUTHOR]

Although transposon-mediated enhancer trapping (ET) is successfully applied in diverse models, the efficiency of various transposon systems varies significantly, and little information is available regarding efficiency of enhancer trapping by various transposons in zebrafish. Most potential enhancers (Ens) still lack evidence of actual En activity. Here, we compared the differences in ET efficiency between sleeping beauty (SB), piggyBac (PB) and Tol2 transposons. Tol2 represented the highest germline transfer efficiencies at 55.56% (NF0 = 165), followed by SB (38.36%, NF0 = 151) and PB (32.65%, NF0 = 149). ET lines generated by the Tol2 transposon tended to produce offspring with a single expression pattern per line, while PB and SB tended to generate embryos with multiple expression patterns. In our tests, 10 putative Ens (En1–10) were identified by splinkerette PCR and comparative genomic analysis. Combining the GFP expression profiles and mRNA expression patterns revealed that En1 and En2 may be involved in regulation of the expression of dlx1a and dlx2a, while En6 may be involved in regulation of the expression of line TK4 transgene and rps26, and En7 may be involved in the regulation of the expression of wnt1 and wnt10b. Most identified Ens were found to be transcribed in zebrafish embryos, and their regulatory function may involve eRNAs. [ABSTRACT FROM AUTHOR]