LA-PCR-based quick method for the identification of genes responsible for the complementation of Saccharomyces cerevisiae mutations.
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Cloning, Molecular↗
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Results for “Transposable element identification”
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ISL2, a new mobile genetic element in Lactobacillus helveticus.
Spontaneous, phenotypically stable mutations at the beta-galactosidase locus (lacL-lacM) in Lactobacillus helveticus were identified and analyzed. We found that a significant number of mutations were caused by integration of a new IS element, ISL2, into these lac genes. ISL2 is 858 bp long, flanked by 16-bp perfect inverted repeats and generates 3-bp target duplications upon insertion. It contains one open reading frame, which shows significant homology (40.1% identity) to the putative transposase of IS702 from Cyanobacterium calothrix. ISL2 is present in 4-21 copies in the L. helveticus genome, but it is not found in other lactic acid bacteria. Its divergence in copy number and genomic locations in different L. helveticus strains makes it useful as a tool for strain identification by genetic fingerprinting.
Amino Acid Sequence↗
Long-read sequencing reveals a hidden Alu-mediated splice defect in CPLANE1, causing orofaciodigital syndrome type VI.
Orofaciodigital syndrome type VI (OFD VI) is a recessive ciliopathy characterized by excessive polydactyly, molar tooth sign, cleft lip, and developmental delay, caused by pathogenic variants in CPLANE1. Here, we present a patient with OFD VI that remained genetically unexplained after routine genetic testing, including short-read whole genome sequencing (WGS). Using long-read sequencing, we found two biallelic splice-site variants in CPLANE1, c.8633-4_8633-3del, and an Alu element insertion close to an exon-intron boundary. Transcript analysis showed that each variant independently resulted in exon skipping, and quantitative expression studies revealed reduced total CPLANE1 mRNA levels in patient-derived fibroblasts. Based on these findings, we were able to re-classify the c.8633-4_8633-3del variant from a variant of uncertain significance (VUS) to likely pathogenic. The identification of an Alu element insertion missed by short-read WGS highlights the added diagnostic value of long-read sequencing in uncovering cryptic, transposable element-associated pathogenic variants.
Journal Article↗
From masking repeats to identifying functional repeats in the mouse transcriptome.
The back-to-back release of the mouse genome and the functionally annotated RIKEN mouse full-length cDNA collection was an important milestone in mammalian genomics. Yet much of the data remain to be explored in terms of biological effects and mechanisms. For example, interspersed repeats account for 39 per cent of the mouse genome sequence and 11 per cent of representative transcripts. A considerable number of transposable repeat elements are still active and propagating in mouse compared with human. While existing repeat databases and tools assist the classification of repeats or identification of new repeats, there is little bioinformatic support towards exploring the extent and role of repeats in transcriptional variation, modulation of protein function, or gene regulatory events. Since the mouse is used as a model organism to study human genes and their disease associations, this review focuses on information extraction and collation that captures the functional context of repeats in mouse transcripts to facilitate the biological interpretation and extrapolation of findings to the human.
Animals↗
Similarity of reverse transcriptase-like sequences of viruses, transposable elements, and mitochondrial introns.
Sequences similar to reverse transcriptase (RT) of retroviruses have been found in certain DNA viruses, mitochondrial intron sequences, and a wide variety of transposable elements. While total amino acid similarity between these diverse elements is quite low, we have identified seven regions, consisting of 182 amino acids, that are common to all elements. Highly conserved residues identified in each of these regions are diagnostic for the identification and alignment of these and for future RT-like sequences. Using both the neighbor-joining and the unweighted-pair-group methods, we have derived a probable phylogenetic tree for all RT-containing elements. These elements can be divided into two major groups. Retroviruses and DNA viruses whose propagation involves an RNA intermediate are grouped with a series of transposable elements containing long terminal repeats (LTRs). The second group is made up of RT-containing sequences of fungal mitochondrial introns and a series of transposable elements that lack LTRs. The transposable elements, copia and Ty, were found to be the most difficult to position on the phylogenetic tree, as a result of their higher rate of sequence divergence. The data are most consistent with their being distant members of the LTR group (retroviruses/LTR retrotransposons).
Amino Acid Sequence↗
A transcriptionally active maize MuDR-like transposable element in rice and its relatives.
Two Mu-like transposable elements were cloned from a rice genomic library using a partial cDNA clone that exhibits high homology to the mudrA gene of the maize element MuDR. Database searches led to the identification of six other sequences that carried highly homologous terminal inverted repeats (TIRs). All the rice elements possessed approximately 200-bp TIRs, and four were flanked by 9-bp target-site duplications (TSDs). The longer of the two cloned elements, OsMu4-2, could potentially encode a protein colinear with a MURA-like transposase, but it had stop codons in the coding region indicating that it is a pseudogene. All the other elements had large internal deletions. Direct dinucleotide repeats were found in two elements at positions flanking the deleted regions, suggesting that the deletions arose via the interrupted-gap-repair mechanism. Sequences related to empty sites of insertion were found in OsMu4-2 and one of the elements identified in the databases. These results provide evidence that the rice OsMu element was active and transposed in the past. Analysis of OsMu4-2 cDNAs revealed two types of transcripts produced by alternative splicing. Genomic Southern analysis suggested that OsMu4-2 was conserved in rice species with the A genome, but a deleted version was unique to japonica subspecies. Some wild rice species harbored paralogous copies of the OsMu element.
Amino Acid Sequence↗
Identification of the Tol2 transposase of the medaka fish Oryzias latipes that catalyzes excision of a nonautonomous Tol2 element in zebrafish Danio rerio.
The Tol2 element is found in the genome of the medaka fish, Oryzias latipes, and contains DNA sequences similar to those of transposons of the hAT family. Previously, we have developed a transient embryonic excision assay in zebrafish, in which zebrafish embryos were injected with a plasmid DNA harboring the Tol2 element, and have shown that the Tol2 element is excisable from the injected plasmid DNA (Kawakami, K., Koga, A., Hori, H., Shima, A., 1998. Excision of the Tol2 transposable element of the medaka fish, Oryzias latipes, in zebrafish, Danio rerio. Gene 225, 17-22). Although the Tol2 element is thought to be autonomous, an active transposase encoded by the Tol2 element has not been identified. Here we report the identification and analysis of mRNA transcribed from the Tol2 element in zebrafish embryos. The Tol2 transcript has the capacity to encode a protein of 649 amino acids, whose amino acid sequence is similar to those of transposases of the hAT family. To determine whether the transcript encodes an active enzyme, we developed a novel transient embryonic excision assay in which zebrafish fertilized eggs were co-injected with RNA transcribed in vitro using the Tol2 cDNA as a template and a plasmid DNA harboring a nonautonomous Tol2 element, which has a deletion in the transposase coding region. The nonautonomous Tol2 element could be efficiently excised in the zebrafish only when co-injected with the Tol2 RNA. This result indicates that the Tol2 transcript encodes an active enzyme, a probable transposase, that can catalyze the excision reaction in trans. Further, by the co-injection analysis, we found that the Tol2 sequence lacking the first intron sequence of the transposase gene could not be excised, suggesting that it may contain essential cis-elements.
Amino Acid Sequence↗
A U-rich tract enhances usage of an alternative 3' splice site in yeast.
There has been a long-standing belief that the mechanisms of mammalian and yeast splicing differ fundamentally in their requirement for a pyrimidine-rich motif preceding the 3' splice site. Using an in vivo assay, we have tested the influence of uridine content on competition between alternative 3' splice sites in yeast. We find that a uridine-rich tract preceding a PyAG greatly enhances its ability to compete as a splice acceptor. Moreover, a proximal PyAG is often overlooked if a more distal PyAG occurs in a superior sequence context; this observation cannot be accounted for by simple scanning models. Finally, we show that a distal (greater than 30 nucleotide) 3' splice site that is not preceded by uridines is a poor substrate for the second step of splicing; this argues that recognition of a uridine-rich motif is required for effective identification and utilization of distant splice sites.
Actins↗
Characterization of IS1501 mutants of Leptospira interrogans serovar pomona.
Leptospira interrogans is a diverse species in which individual serovars have distinctive restriction fragment length polymorphisms that are useful in strain identification. Many of these polymorphisms can be detected using hybridization probes derived from insertion sequences; an observation that suggests these IS elements are active and can transpose in L. interrogans. Two spontaneous mutants of L. interrogans serovar Pomona strain RZ11 were isolated by immune selection and characterized. Changes in the size and antigenicity of LPS from these mutants were detected. Genetic analysis showed that both mutants have additional copies of an IS3-like element, designated IS1501, that are not present in the parental strain. One mutant, GT211, has a single additional copy of IS1501, whereas the other mutant, GT210 has three additional copies of IS1501 relative to strain RZ11. IS1501 transposition generated 3-bp direct repeats from target sequences flanking the insertion site. RT-PCR analysis of transcripts at altered loci showed IS1501 transcripts extended into adjacent sequences. These data are the first to show spontaneous transposition of an endogenous Leptospira insertion sequence, and suggest that IS1501 may be capable of gene activation.
Antigens, Bacterial↗
Functional genomics of Plasmodium falciparum through transposon-mediated mutagenesis.
Plasmodium falciparum is the causative agent for the most lethal form of human malaria, killing millions annually. Genetic analyses of P. falciparum have been relatively limited due to the lack of robust techniques to manipulate this parasite. Development of transfection technologies and whole genome analyses have helped in understanding the complex biology of this parasite. Even with this wealth of information functional genomics approaches are still very limited in P. falciparum due to the cumbersome and inefficient methods of genetic manipulation. This review focuses on a recently developed, highly efficient method for transposon-based mutagenesis and transgene expression in P. falciparum that will allow functional genomics studies to be performed proficiently on this deadly malaria parasite. By using a piggyBac-based transposition system, multiple random integrations have been obtained into the genome of the parasite. This technique could hence be employed to set up several biological screens in this lethal protozoan parasite that may lead to identification of novel drug targets and vaccine candidates.
Animals↗
[Molecular bases for brucella virulence].
The authors review published reports on the molecular bases of Brucella virulence, including type IV secretion proteins, S-lipopolysaccharide biosynthesis enzymes (O-antigen), regulatory proteins of various systems, and cellular metabolism proteins. High efficiency of modified transposon mutagenesis technique (selective labeled transposon mutagenesis) in search for virulence genes is shown. Analysis of DNA sequences of Brucella genome promotes identification of new virulence factors.
Bacterial Proteins↗
A novel IS element, ISMpa1, in Mycobacterium avium subsp. paratuberculosis.
A novel insertion element belonging to the IS110 family was identified in Mycobacterium avium subsp. paratuberculosis. The IS element, ISMpa1, is 1500 bp and has one ORF encoding a putative transposase. Three copies of ISMpa1 were identified in the M. avium subsp. paratuberculosis genome. The element had inserted into the 3' end of the highly conserved mycobacterial genes prrB and a homologue of M. tuberculosis Rv1593c, and between a putative cytochrome p450 oxygenase and a putative hydrolase. The IS element was present in all (n = 11) M. avium subsp. paratuberculosis strains but not detected in most other mycobacterial species examined, including 10 M. avium subsp. avium isolates of human, avian and porcine origin. However two porcine isolates of M. avium subsp. avium and the reference strain IWGMT49 did harbour ISMpa1. These three strains belong to a previously described subgroup of M. avium subsp. avium based on IS1245 restriction fragment length polymorphism (RFLP) pattern and serovars. All of the M. avium subsp. paratuberculosis strains examined had an identical RFLP pattern when probed with sequences corresponding to the 5' end of ISMpa1, whereas a different pattern was seen in the positive M. avium subsp. avium strains. This novel IS element might be a useful tool in strain classification of M. avium subsp. avium and also for the identification of M. avium subsp. paratuberculosis when used in combination with IS900.
DNA Transposable Elements↗
NextLongIso: a comprehensive Nextflow pipeline for multi-dimensional long-read RNA-seq analysis.
SUMMARY: Long-read RNA sequencing technologies, including Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), enable direct characterization of full-length transcripts and transcriptome complexity. However, analysis of long-read RNA-seq data remains fragmented across multiple tools, limiting the ability to obtain a unified view of transcript structure, expression, and regulatory variation in long-read transcriptomes. We present NextLongIso, a scalable and reproducible Nextflow pipeline that enables coordinated analysis of multiple layers of transcript regulation. Rather than focusing solely on transcript reconstruction, NextLongIso integrates transcript discovery with downstream regulatory analyses to jointly characterize alternative splicing, isoform switching, transcript boundary dynamics (including alternative promoters and polyadenylation), and transposable element-associated transcription from both PacBio and ONT datasets. By eliminating complex cross-tool data harmonization, this unified framework facilitates the transition from transcript identification to functional interpretation of transcriptomic variation. AVAILABILITY AND IMPLEMENTATION: NextLongIso is implemented in Nextflow and is freely available at github: https://github.com/YidanSunResearchLab/nf-LongIso.git and Zenodo: https://doi.org/10.5281/zenodo.21049837.
Software↗
Hinf family: a novel repeated DNA family of the human genome.
The isolation of a mutant adenovirus carrying an insertion of cellular DNA has led to the identification of a new family of human repetitive sequences, which are found tandemly arranged in the genome. The sequence of the viral insert resembles that of eukaryotic transposable elements.
Adenoviruses, Human↗
oskar gene expression in the vector mosquitoes, Anopheles gambiae and Aedes aegypti.
A disease control strategy based on the introduction into mosquito populations of a gene conferring a pathogen-refractory phenotype is currently under investigation. This population replacement approach requires a drive system that will quickly spread and fix antipathogen effector genes in target populations. Modified transposable elements containing the control sequences of developmentally regulated genes may provide the basis for a gene drive system that regulates gene mobilization in a sex- and stage-restrictive manner. Screening of a Drosophila melanogaster database for genes whose products localize exclusively in the future germ cells during early embryonic development resulted in the identification of several candidate genes. The regulatory sequences of these genes could be used to drive transposition. Mosquito orthologous genes of oskar were identified based on sequence homology and characterized further. The tissue- and sex-specific expression profiles and hybridizations in situ show that oskar orthologous transcripts in Anopheles gambiae and Aedes aegypti accumulate in developing oocytes of adult females and localize to the posterior poles of early embryos. These characteristics potentiate the use of the regulatory sequences of mosquito oskar genes for the control of modified transposable elements.
Aedes↗
Functionality of mutations at conserved nucleotides in eukaryotic SECIS elements is determined by the identity of a single nonconserved nucleotide.
In eukaryotes, the specific cotranslational insertion of selenocysteine at UGA codons requires the presence of a secondary structural motif in the 3' untranslated region of the selenoprotein mRNA. This selenocysteine insertion sequence (SECIS) element is predicted to form a hairpin and contains three regions of sequence invariance that are thought to interact with a specific protein or proteins. Specificity of RNA-binding protein recognition of cognate RNAs is usually characterized by the ability of the protein to recognize and distinguish between a consensus binding site and sequences containing mutations to highly conserved positions in the consensus sequence. Using a functional assay for the ability of wild-type and mutant SECIS elements to direct cotranslational selenocysteine incorporation, we have investigated the relative contributions of individual invariant nucleotides to SECIS element function. We report the novel finding that, for this consensus RNA motif, mutations at the invariant nucleotides are tolerated to different degrees in different elements, depending on the identity of a single nonconserved nucleotide. Further, we demonstrate that the sequences adjacent to the minimal element, although not required for function, can affect function through their propensity to base pair. These findings shed light on the specific structure these conserved sequences may form within the element. This information is crucial to the design of strategies for the identification of SECIS-binding proteins, and hence the elucidation of the mechanism of selenocysteine incorporation in eukaryotes.
Base Sequence↗
The mobile element IS1207 of Brevibacterium lactofermentum ATCC21086: isolation and use in the construction of Tn5531, a versatile transposon for insertional mutagenesis of Corynebacterium glutamicum.
IS1207 is the insertion most frequently found among the spontaneous mutations that abolish the activity of an Escherichia coli phage lambda cI gene integrated in the Corynebacterium Brevibacterium lactofermentum ATCC21086 genome. We examined the transposition of transposon-like structures composed of a selective kanamycin resistance gene (aph3), and one or two IS1207 sequences. One of these, the Tn5531 transposon, transposed efficiently in Corynebacterium glutamicum. A replicative and a non-replicative Tn5531 delivery vector were used in Tn5531 mutagenesis. As IS1207, transposon Tn5531 shows a high frequency of transposition and mutagenesis, and a low target specificity. These features make of Tn5531 an adequate choice for gene identification and gene tagging experiments.
Brevibacterium↗
Microbial pathogen genomes - new strategies for identifying therapeutic and vaccine targets.
Efficient mining of genomic sequence information from multiple pathogens for therapeutic and vaccine targets requires efficient tools. Fortunately, robust methods applicable to whole genomes have been developed and applied in the past few years to identify genes essential for growth or virulence and to detect potential vaccine targets. Successful approaches to identify potential therapeutic targets include a variety of ingenious uses of nearly random transposon insertions, more directed methods such as antisense and insertion-duplication mutagenesis, and expression profiling facilitated by microarrays. Vaccine targets have been identified by gene fusion and expression experiments to discover gene products that are immunogenic in humans or animal models. All genome-wide methods require focused secondary assays to validate the findings, but these genomic methods excel at reducing to a manageable number the genes to be examined further. This editorial reviews the latest developments in genome-wide target identification tools.
Anti-Infective Agents↗