Transposition Events Research Articles

Required Gene Set for Autotrophic Growth of Clostridium autoethanogenum.

ABSTRACTThe majority of the genes present in bacterial genomes remain poorly characterized, with up to one-third of those that are protein encoding having no definitive function. Transposon insertion sequencing represents a high-throughput technique that can help rectify this deficiency. The technology, however, can only be realistically applied to those species in which high rates of DNA transfer can be achieved. Here, we have developed a number of approaches that overcome this barrier in the autotrophic species Clostridium autoethanogenum by using a mariner-based transposon system. The inherent instability of such systems in the Escherichia coli conjugation donor due to transposition events was counteracted through the incorporation of a conditionally lethal codA marker on the plasmid backbone. Relatively low frequencies of transformation of the plasmid into C. autoethanogenum were circumvented through the use of a plasmid that is conditional for replication coupled with the routine implementation of an Illumina library preparation protocol that eliminates plasmid-based reads. A transposon library was then used to determine the essential genes needed for growth using carbon monoxide as the sole carbon and energy source.IMPORTANCE Although microbial genome sequences are relatively easily determined, assigning gene function remains a bottleneck. Consequently, relatively few genes are well characterized, leaving the function of many as either hypothetical or entirely unknown. High-throughput transposon sequencing can help remedy this deficiency, but is generally only applicable to microbes with efficient DNA transfer procedures. These exclude many microorganisms of importance to humankind either as agents of disease or as industrial process organisms. Here, we developed approaches to facilitate transposon insertion sequencing in the acetogen Clostridium autoethanogenum, a chassis being exploited to convert single-carbon waste gases CO and CO2 into chemicals and fuels at an industrial scale. This allowed the determination of gene essentiality under heterotrophic and autotrophic growth, providing insights into the utilization of CO as a sole carbon and energy source. The strategies implemented are translatable and will allow others to apply transposon insertion sequencing to other microbes where DNA transfer has until now represented a barrier to progress.

TransposonUltimate: software for transposon classification, annotation and detection.

Most genomes harbor a large number of transposons, and they play an important role in evolution and gene regulation. They are also of interest to clinicians as they are involved in several diseases, including cancer and neurodegeneration. Although several methods for transposon identification are available, they are often highly specialised towards specific tasks or classes of transposons, and they lack common standards such as a unified taxonomy scheme and output file format. We present TransposonUltimate, a powerful bundle of three modules for transposon classification, annotation, and detection of transposition events. TransposonUltimate comes as a Conda package under the GPL-3.0 licence, is well documented and it is easy to install through https://github.com/DerKevinRiehl/TransposonUltimate. We benchmark the classification module on the large TransposonDB covering 891,051 sequences to demonstrate that it outperforms the currently best existing solutions. The annotation and detection modules combine sixteen existing softwares, and we illustrate its use by annotating Caenorhabditis elegans, Rhizophagus irregularis and Oryza sativa subs. japonica genomes. Finally, we use the detection module to discover 29 554 transposition events in the genomes of 20 wild type strains of C. elegans. Databases, assemblies, annotations and further findings can be downloaded from (https://doi.org/10.5281/zenodo.5518085).

Novel Putative Transposable Element Associated with the Subtype E5 Botulinum Toxin Gene Cluster of Neurotoxigenic Clostridium butyricum Type E Strains from China.

Previously, a whole-genome comparison of three Clostridium butyricum type E strains from Italy and the United States with different C. botulinum type E strains indicated that the bont/e gene might be transferred between the two clostridia species through transposition. However, transposable elements (TEs) have never been identified close to the bont/e gene. Herein, we report the whole genome sequences for four neurotoxigenic C. butyricum type E strains that originated in China. An analysis of the obtained genome sequences revealed the presence of a novel putative TE upstream of the bont/e gene in the genome of all four strains. Two strains of environmental origin possessed an additional copy of the putative TE in their megaplasmid. Similar putative TEs were found in the megaplasmids and, less frequently, in the chromosomes of several C. butyricum strains, of which two were neurotoxigenic C. butyricum type E strains, and in the chromosome of a single C. botulinum type E strain. We speculate that the putative TE might potentially transpose the bont/e gene at the intracellular and inter-cellular levels. However, the occasional TE occurrence in the clostridia genomes might reflect rare transposition events.

An improved approximation algorithm for the reversal and transposition distance considering gene order and intergenic sizes

BackgroundIn the comparative genomics field, one of the goals is to estimate a sequence of genetic changes capable of transforming a genome into another. Genome rearrangement events are mutations that can alter the genetic content or the arrangement of elements from the genome. Reversal and transposition are two of the most studied genome rearrangement events. A reversal inverts a segment of a genome while a transposition swaps two consecutive segments. Initial studies in the area considered only the order of the genes. Recent works have incorporated other genetic information in the model. In particular, the information regarding the size of intergenic regions, which are structures between each pair of genes and in the extremities of a linear genome.Results and conclusionsIn this work, we investigate the sorting by intergenic reversals and transpositions problem on genomes sharing the same set of genes, considering the cases where the orientation of genes is known and unknown. Besides, we explored a variant of the problem, which generalizes the transposition event. As a result, we present an approximation algorithm that guarantees an approximation factor of 4 for both cases considering the reversal and transposition (classic definition) events, an improvement from the 4.5-approximation previously known for the scenario where the orientation of the genes is unknown. We also present a 3-approximation algorithm by incorporating the generalized transposition event, and we propose a greedy strategy to improve the performance of the algorithms. We performed practical tests adopting simulated data which indicated that the algorithms, in both cases, tend to perform better when compared with the best-known algorithms for the problem. Lastly, we conducted experiments using real genomes to demonstrate the applicability of the algorithms.

Complete Genome Sequences of Klebsiella michiganensis and Citrobacter farmeri, KPC-2-Producers Serially Isolated from a Single Patient.

Carbapenemase-producing Enterobacterales, including KPC-2 producers, have become a major clinical problem. During an outbreak in Quebec City, Canada, KPC-2-producing Klebsiella michiganensis and Citrobacter farmeri were isolated from a patient six weeks apart. We determined their complete genome sequences. Both isolates carried nearly identical IncN2 plasmids with blaKPC-2 on a Tn4401b element. Both strains also carried IncP1 plasmids, but that of C. farmeri did not carry a Beta-lactamase gene, whereas that of K. michiganensis carried a second copy of blaKPC-2 on Tn4401b. These results suggest recent plasmid transfer between the two species and a recent transposition event.

Bacterial Adaptation by a Transposition Burst of an Invading IS Element.

The general importance of transposable elements (TEs) for adaptive evolution remains unclear. This in part reflects a poor understanding of the role of TEs for adaptation in nonmodel systems. Here, we investigated whether insertion sequence (IS) elements are a major source of beneficial mutations during 400 generations of laboratory evolution of the cyanobacterium Acaryochloris marina strain CCMEE 5410, which has experienced a recent or on-going IS element expansion and has among the highest transposase gene contents for a bacterial genome. Most mutations detected in the eight independent experimental populations were IS transposition events. Surprisingly, however, the majority of these involved the copy-and-paste activity of only a single copy of an unclassified element (ISAm1) that has recently invaded the strain CCMEE 5410 genome. ISAm1 transposition was largely responsible for the highly repeatable evolutionary dynamics observed among populations. Notably, this included mutations in multiple targets involved in the acquisition of inorganic carbon for photosynthesis that were exclusively due to ISAm1 activity. These mutations were associated with an increase in linear growth rate under conditions of reduced carbon availability but did not appear to impact fitness when carbon was readily available. Our study reveals that the activity of a single transposase can fuel adaptation for at least several hundred generations but may also potentially limit the rate of adaptation through clonal interference.

Methylation patterns of Tf2 retrotransposons linked to rapid adaptive stress response in the brown planthopper (Nilaparvata lugens)

Transposable elements (TEs) exhibit vast diversity across insect orders and are one of the major factors driving insect evolution and speciation. Presence of TEs can be both beneficial and deleterious to their host. While it is well-established that TEs impact life-history traits, adaptations and survivability of insects under hostile environments, the influence of the ecological niche on TE-landscape remains unclear. Here, we analysed the dynamics of Tf2 retrotransposons in the brown planthopper (BPH), under environmental fluctuations. BPH, a major pest of rice, is found in almost all rice-growing ecosystems. We believe genome plasticity, attributed to TEs, has allowed BPH to adapt and colonise novel ecological niches. Our study revealed bimodal age-distribution for Tf2 elements in BPH, indicating the occurrence of two major transpositional events in its evolutionary history and their contribution in shaping BPH genome. While TEs can provide genome flexibility and facilitate adaptations, they impose massive load on the genome. Hence, we investigated the involvement of methylation in modulating transposition in BPH. We performed comparative analyses of the methylation patterns of Tf2 elements in BPH feeding on resistant- and susceptible-rice varieties, and also under pesticide stress, across different life-stages. Results confirmed that methylation, particularly in non-CG context, is involved in TE regulation and dynamics under stress. Furthermore, we observed differential methylation for BPH adults and nymphs, emphasising the importance of screening juvenile life-stages in understanding adaptive-stress-responses in insects. Collectively, this study enhances our understanding of the role of transposons in influencing the evolutionary trajectory and survival strategies of BPH across generations.

SUMO orchestrates multiple alternative DNA-protein crosslink repair pathways.

Endogenous metabolites, environmental agents, and therapeutic drugs promote formation of covalent DNA-protein crosslinks (DPCs). Persistent DPCs compromise genome integrity and are eliminated by multiple repair pathways. Aberrant Top1-DNA crosslinks, or Top1ccs, are processed by Tdp1 and Wss1 functioning in parallel pathways in Saccharomyces cerevisiae. It remains obscure how cells choose between diverse mechanisms of DPC repair. Here, we show that several SUMO biogenesis factors (Ulp1, Siz2, Slx5, and Slx8) control repair of Top1cc or an analogous DPC lesion. Genetic analysis reveals that SUMO promotes Top1cc processing in the absence of Tdp1 but has an inhibitory role if cells additionally lack Wss1. In the tdp1Δ wss1Δ mutant, the E3 SUMO ligase Siz2 stimulates sumoylation in the vicinity of the DPC, but not SUMO conjugation to Top1. This Siz2-dependent sumoylation inhibits alternative DPC repair mechanisms, including Ddi1. Our findings suggest that SUMO tunes available repair pathways to facilitate faithful DPC repair.

Approximation algorithm for rearrangement distances considering repeated genes and intergenic regions

The rearrangement distance is a method to compare genomes of different species. Such distance is the number of rearrangement events necessary to transform one genome into another. Two commonly studied events are the transposition, which exchanges two consecutive blocks of the genome, and the reversal, which reverts a block of the genome. When dealing with such problems, seminal works represented genomes as sequences of genes without repetition. More realistic models started to consider gene repetition or the presence of intergenic regions, sequences of nucleotides between genes and in the extremities of the genome. This work explores the transposition and reversal events applied in a genome representation considering both gene repetition and intergenic regions. We define two problems called Minimum Common Intergenic String Partition and Reverse Minimum Common Intergenic String Partition. Using a relation with these two problems, we show a Theta left( k right)-approximation for the Intergenic Transposition Distance, the Intergenic Reversal Distance, and the Intergenic Reversal and Transposition Distance problems, where k is the maximum number of copies of a gene in the genomes. Our practical experiments on simulated genomes show that the use of partitions improves the estimates for the distances.

PiggyBac transposase and transposon derivatives for gene transfer targeting the ribosomal DNA loci of CHO cells

Integrative non-viral vectors such as transposons engineered to mediate targeted gene transfer into safe harbor sites in the genome may be a promising approach for the production of therapeutic proteins or for gene therapy in an efficient and secure way. In this context, we designed and evaluated two strategies for targeting the nuclear ribosomal DNA (rDNA) loci. One approach relied on the co-location of the transposase and transposon near transcriptionally active rDNA copies using a nucleolar localization signal (NoLS). Another one consisted of targeting the 18S-coding region in the rDNA loci using a NoLS-FokI-dCas9 endonuclease to perform targeted transgene knock-in. We show that integration into the rDNA of Chinese hamster ovary (CHO) cells can be achieved at a high frequency using the piggyBac transposon system, indicating that the rDNA is highly accessible for transposition. Consistently, rDNA-targeted transposition events were most frequently obtained when both the piggyBac transposon DNA and the transposase were nucleoli-targeted, yielding cells displaying stable and homogeneous expression of the transgene. This approach thus provides an alternative strategy to improve targeted transgene delivery and protein expression using CHO cells.

MuA-based Molecular Indexing for Rare Mutation Detection by Next-Generation Sequencing

Detection of low-frequency mutations in cancer genomes or other heterogeneous cell populations requires high-fidelity sequencing. Molecular barcoding is one of the key technologies that enables the differentiation of true mutations from errors, which can be caused by sequencing or library preparation processes. However, current approaches where barcodes are introduced via primer extension or adaptor ligation do not utilize the full power of barcoding, due to complicated library preparation workflows and biases. Here we demonstrate the remarkable tolerance of MuA transposase to the presence of multiple replacements in transposon sequence, and explore this unique feature to engineer the MuA transposome complex with randomised nucleotides in 12 transposon positions, which can be introduced as a barcode into the target molecule after transposition event. We applied the approach of Unique MuA-based Molecular Indexing (UMAMI) to assess the power of rare mutation detection by shortgun sequencing on the Illumina platform. Our results show that UMAMI allows detection of rare mutations readily and reliably, and in this paper we report error rate values for the number of thermophilic DNA polymerases measured by using UMAMI.

LINE-1 vectors mediate recombinant antibody gene transfer by retrotransposition in Chinese hamster ovary cells.

Retrotransposons, such as long interspersed element-1 (LINE-1), can copy themselves to other genomic loci via a transposition event (termed retrotransposition). Retrotransposons, therefore, have potential use as an efficient gene delivery tool to integrate multiple copies of a target gene into a host genome. Here, we developed a retrotransposon vector based on LINE-1 that achieves target gene integration of multiple transgene copies. The retrotransposon vector contains a neomycin resistance gene split by an intron as a marker gene, and a gene encoding an antibody single-chain variable fragment (Fv) fused with the constant antibody region (Fc) (scFv-Fc) as a model target gene. G418-resistant Chinese hamster ovary cells were generated using this retrotransposon vector, and scFv-Fc was produced in the culture medium. To regulate retrotransposition, we developed a retrotransposon vector system that separately expressed the two open reading frames (ORF1 and ORF2) of LINE-1. Genomic PCR analysis detected the transgene sequence in almost all tested clones. Compared with clones established using the intact LINE-1 vector, clones generated with the split ORF1 and ORF2 system showed similar specific scFv-Fc productivity and retrotransposition efficiency. This approach of using a retrotransposon-based vector system has the potential to provide a new gene delivery tool for mammalian cells.

Comparative mitogenomics of Agaricomycetes: Diversity, abundance, impact and coding potential of putative open-reading frames

The mitochondrion is an organelle found in eukaryote organisms, and it is vital for different cellular pathways. The mitochondrion has its own DNA molecule and, because its genetic content is relatively conserved, despite the variation of size and structure, mitogenome sequences have been widely used as a promising molecular biomarker for taxonomy and evolution in fungi. In this study, the mitogenomes of two fungal species of Agaricomycetes class, Phellinotus piptadeniae and Trametes villosa, were assembled and annotated for the first time. We used these newly sequenced mitogenomes for comparative analyses with other 55 mitogenomes of Agaricomycetes available in public databases. Mitochondrial DNA (mtDNA) size and content are highly variable and non-coding and intronic regions, homing endonucleases (HEGs), and unidentified ORFs (uORFs) significantly contribute to the total size of the mitogenome. Furthermore, accessory genes (most of them as HEGs) are shared between distantly related species, most likely as a consequence of horizontal gene transfer events. Conversely, uORFs are only shared between taxonomically related species, most probably as a result of vertical evolutionary inheritance. Additionally, codon usage varies among mitogenomes and the GC content of mitochondrial features may be used to distinguish coding from non-coding sequences. Our results also indicated that transposition events of mitochondrial genes to the nuclear genome are not common. Despite the variation of size and content of the mitogenomes, mitochondrial genes seemed to be reliable molecular markers in our time-divergence analysis, even though the nucleotide substitution rates of mitochondrial and nuclear genomes of fungi are quite different. We also showed that many events of mitochondrial gene shuffling probably happened amongst the Agaricomycetes during evolution, which created differences in the gene order among species, even those of the same genus. Altogether, our study revealed new information regarding evolutionary dynamics in Agaricomycetes.

The C-terminal Domain of piggyBac Transposase Is Not Required for DNA Transposition

PiggyBac(PB)-like elements (pble) are members of a eukaryotic DNA transposon family. This family is of interest to evolutionary genomics because pble transposases have been domesticated at least 9 times in vertebrates. The amino acid sequence of pble transposases can be split into three regions: an acidic N-terminal domain (~100 aa), a central domain (~400 aa) containing a DD[D/E] catalytic triad, and a cysteine-rich domain (CRD; ~90 aa). Two recent reports suggested that a functional CRD is required for pble transposase activity. Here we found that two CRD-deficient pble transposases, a PB variant and an isoform encoded by the domesticated PB-derived vertebrate transposase gene 5 (pgbd5) trigger transposition of the Ifp2 pble. When overexpressed in HeLa cells, these CRD-deficient transposases can insert Ifp2 elements with proper and improper transposon ends, associated with deleterious effects on cells. Finally, we found that mouse CRD-deficient transposase Pgbd5, as well as PB, do not insert pbles at random into chromosomes. Transposition events occurred more often in genic regions, in the neighbourhood of the transcription start sites and were often found in genes predominantly expressed in the human central nervous system.

Detection of Transposition Events from Next-Generation Sequencing Data.

Detection of transposition events of a transposon from short reads of next-generation sequencing (NGS) is challenging because transposons are repetitive and difficult to be distinguished from already existing transposons in the genome. Many transposons generate target site duplication (TSD) as the result of chromosomal integration. Since TSDs flanking the 5'-end (head) and 3'-end (tail) of a transposon has the identical sequences which are absent from the reference copy, the short reads containing the head or tail sequences of the transposon following the same TSD sequence may reveal the evidence of transposition. Transposon Insertion Finder (TIF) focuses on the TSD with flanking sequence of transposon and detects transposition events from NGS data. TIF software is available at https://github.com/akiomiyao/tif .

Expression, Interaction, and Role of Pseudogene Adh6-ps1 in Cancer Phenotypes.

Pseudogenes have been classified as functionless and their annotation is an ongoing problem. The Adh6-ps1—a mouse pseudogene belonging to the alcohol dehydrogenase gene complex (Adh) was analyzed to review the conservation, homology, expression, and interactions and identify any role it plays in disease phenotypes using bioinformatics databases. Results showed that Adh6-ps1 have 2 transcripts (processed and unprocessed) which may have emerged from a transposition and duplication event, respectively, and that induced inversions (Uox gene, In(3)11Rk) involving gene complexes associated with Adh6-ps1 have been implicated in a diverse range of diseases. Adh6-ps1 is highly conserved in vertebrates particularly rodents and expressed in the liver. The top 5 MirRNA targets were Mir455, Mir511, Mir1903, Mir361, and Mir669o markers. While much is unknown about Mir1903 and Mir669o, the silencing of Mir455 and Mir511 is linked with hepatocellular carcinoma (HCC), and Mir361 is implicated in endometrial cancers. Given the identified MirRNA interactions with Adh6-ps1 and its expression in HCC and reproductive systems, it may well have a role in tumorigenesis and disease phenotypes. Nonetheless, further studies are required to establish these facts to add to the growing efforts to understand pseudogenes and their potential involvement in disease conditions.

Genome Plasticity by Insertion Sequences Learned From a Case of Radiation-Resistant Bacterium Deinococcus geothermalis.

The genome of the radiation-resistant bacterium Deinococcus geothermalis contains 19 types of insertion sequences (ISs), including 93 total transposases (Tpases) in 73 full-length ISs from the main chromosome and 2 mega plasmids. In this study, 68 ISs from the D. geothermalis genome were extracted to implicate the earlier genome before its mutation by transposition of ISs. The total size of eliminated ISs from genome was 78.85 kb. From these in silico corrections of mutation by the ISs, we have become aware of some bioinformatics factualness as follows: (1) can reassemble the disrupted genes if the exact IS region was eliminated, (2) can configure the schematic clustering of major DDE type Tpases, (3) can determine IS integration order across multiple hot spots, and (4) can compare genetic relativeness by the partial synteny analysis between D. geothermalis and Deinococcus strain S9. Recently, we found that several IS elements actively transferred to other genomic sites under hydrogen peroxide-induced oxidative stress conditions, resulting in the inactivation of functional genes. Therefore, the single species genome’s mobilome study provides significant support to define bacterial genome plasticity and molecular evolution from past and present progressive transposition events.

Identification of a Chromosomal Integrated DNA Fragment Containing the rmpA2 and iucABCDiutA Virulence Genes in Klebsiella pneumoniae.

Klebsiella pneumoniae is increasingly being regarded as a reservoir of diverse antibiotic resistance genes and a pathogen that causes severe infections in both the hospital and the community. In this study, we performed molecular characterization of a carbapenem-resistant and highly virulent K. pneumoniae strain recovered from a hospital patient. The virulence-encoding genes rmpA2 and iucABCDiutA were found to be located in the chromosome and are flanked by IS26 elements. These chromosomally located virulence genes, if not incurring fitness costs, are expected to be much more stable than plasmid-located genes. Detailed analysis of the fragment in which these virulence genes are located showed that the fragment can readily form a circular intermediate that may promote integration of this virulence-encoding fragment into various plasmid backbones and other chromosomal regions. Findings in this work provide new insights into mechanisms of transmission of virulence-encoding genes in K. pneumoniaeIMPORTANCE This study reported for the first time and characterized in detail the genetic features of a mobile virulence-encoding fragment located in the chromosome of a clinical virulent K. pneumoniae strain and revealed the occurrence of a transposition event mediated by IS26 This genetic structure could mediate the transposition of the virulence-encoding fragment into various plasmid backbones and chromosomes through formation of a circular intermediate. Therefore, findings in this work provide important insights into the transmission mechanisms of mobile virulence profiles in K. pneumoniae strains and lay the foundation for devising effective intervention approaches aimed at preventing the dissemination of these virulence-encoding elements.

Tn6603, a Carrier of Tn5053 Family Transposons, Occurs in the Chromosome and in a Genomic Island of Pseudomonas aeruginosa Clinical Strains.

Transposons of the Pseudomonas aeruginosa accessory gene pool contribute to phenotype and to genome plasticity. We studied local P. aeruginosa strains to ascertain the encroachment of mer-type res site hunter transposons into clinical settings and their associations with other functional modules. Five different Tn5053 family transposons were detected, all chromosomal. Some were solitary elements; one was in res of Tn1013#, a relative of a reported carrier of int-type res site hunters (class 1 integrons), but most were in res of Tn6603, a new Tn501-related transposon of unknown phenotype. Most of the Tn6603::Tn elements, and some Tn6603 and Tn6603::Tn elements found in GenBank sequences, were at identical sites in an hypothetical gene of P. aeruginosa genomic island PAGI-5v. The island in clonally differing strains was at either of two tRNALys loci, suggesting lateral transfer to these sites. This observation is consistent with the membership of the prototype PAGI-5 island to the ICE family of mobile genetic elements. Additionally, the res site hunters in the nested transposons occupied different positions in the Tn6603 carrier. This suggested independent insertion events on five occasions at least. Tn5053 family members that were mer-/tni-defective were found in Tn6603- and Tn501-like carriers in GenBank sequences of non-clinical Pseudomonas spp. The transposition events in these cases presumably utilized tni functions in trans, as can occur with class 1 integrons. We suggest that in the clinical context, P. aeruginosa strains that carry Tn6603 alone or in PAGI-5v can serve to disseminate functional res site hunters; these in turn can provide the requisite trans-acting tni functions to assist in the dissemination of class 1 integrons, and hence of their associated antibiotic resistance determinants.

Ectopic Expression of a Maize Gene Is Induced by Composite Insertions Generated Through Alternative Transposition.

Transposable elements (TEs) are DNA sequences that can mobilize and proliferate throughout eukaryotic genomes. Previous studies have shown that in plant genomes, TEs can influence gene expression in various ways, such as inserting in introns or exons to alter transcript structure and content, and providing novel promoters and regulatory elements to generate new regulatory patterns. Furthermore, TEs can also regulate gene expression at the epigenetic level by modifying chromatin structure, changing DNA methylation status, and generating small RNAs. In this study, we demonstrated that Ac/fractured Ac (fAc) TEs are able to induce ectopic gene expression by duplicating and shuffling enhancer elements. Ac/fAc elements belong to the hAT family of class II TEs. They can undergo standard transposition events, which involve the two termini of a single transposon, or alternative transposition events that involve the termini of two different nearby elements. Our previous studies have shown that alternative transposition can generate various genome rearrangements such as deletions, duplications, inversions, translocations, and composite insertions (CIs). We identified >50 independent cases of CIs generated by Ac/fAc alternative transposition and analyzed 10 of them in detail. We show that these CIs induced ectopic expression of the maize pericarp color 2 (p2) gene, which encodes a Myb-related protein. All the CIs analyzed contain sequences including a transcriptional enhancer derived from the nearby p1 gene, suggesting that the CI-induced activation of p2 is affected by mobilization of the p1 enhancer. This is further supported by analysis of a mutant in which the CI is excised and p2 expression is lost. These results show that alternative transposition events are not only able to induce genome rearrangements, but also generate CIs that can control gene expression.