Short Inverted Repeats Research Articles

The complete chloroplast genome sequence of Knema furfuracea (Myristicaceae)

Knema furfuracea is a member of Myristicaceae. The K. furfuracea chloroplast genome is found to be 154,527 bp in length and has a base composition of A (29.99%), G (19.31%), C (19.92%), and T (30.78%). The genome contained two short inverted repeat (IRa and IRb) regions (48,110 bp) which were separated by a large single copy (LSC) region (86,188 bp) and a small single copy (SSC) region (20,229 bp). The chloroplast genome has 87 protein-coding genes, 27 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. Further, complete chloroplast sequence of K. furfuracea was aligned together with two species of Myristicaceae and five basal angiosperms species which have reported the complete chloroplast sequence. This complete chloroplast genome will provide valuable information for the development of DNA markers for future species resource development and phylogenetic analysis of K. furfuracea.

A User's Guide to the Inverted Terminal Repeats of Adeno-Associated Virus.

Ongoing development of recombinant vectors based on adeno-associated virus (rAAV) is providing an increasingly powerful and widely used toolkit for gene transfer and genome editing applications. While conceptually simple, the system harbors considerable complexity that presents many potential pitfalls for the inexperienced user. The short inverted terminal repeats (ITRs) can prove to be particularly problematic during vector engineering due to inherent instability necessitating diligent quality control measures during vector manufacture. This is especially important from a clinical standpoint when consistent purity and potency are paramount, and all components of the system are rigorously scrutinized by regulatory agencies. Despite the discovery over 30 years ago that the AAV ITRs are the only cis-acting elements of the virus required for vector production, there is a scarcity of reviews specifically focused on these complex elements. This review provides an overview of the ITR with the dual purpose of acting as a user's guide in the application of AAV vector technology and as a roadmap for ongoing vector development and optimization.

Incomer, a DD36E family of Tc1/mariner transposons newly discovered in animals

BackgroundThe Tc1/mariner superfamily might represent the most diverse and widely distributed group of DNA transposons. Several families have been identified; however, exploring the diversity of this superfamily and updating its classification is still ongoing in the life sciences.ResultsHere we identified a new family of Tc1/mariner transposons, named Incomer (IC), which is close to, but distinct from the known family DD34E/Tc1. ICs have a total length of about 1.2 kb, and harbor a single open reading frame encoding a ~ 346 amino acid transposase with a DD36E motif and flanked by short terminal inverted repeats (TIRs) (22–32 base pairs, bp). This family is absent from prokaryotes, and is mainly distributed among vertebrates (141 species of four classes), including Agnatha (one species of jawless fish), Actinopterygii (132 species of ray-finned fish), Amphibia (four species of frogs), and Mammalia (four species of bats), but have a restricted distribution in invertebrates (four species in Insecta and nine in Arachnida). All ICs in bats (Myotis lucifugus, Eptesicus fuscus, Myotis davidii, and Myotis brandtii) are present as truncated copies in these genomes, and most of them are flanked by relatively long TIRs (51–126 bp). High copy numbers of miniature inverted-repeat transposable elements (MITEs) derived from ICs were also identified in bat genomes. Phylogenetic analysis revealed that ICs are more closely related to DD34E/Tc1 than to other families of Tc1/mariner (e.g., DD34D/mariner and DD × D/pogo), and can be classified into four distinct clusters. The host and IC phylogenies and pairwise distance comparisons between RAG1 genes and all consensus sequences of ICs support the idea that multiple episodes of horizontal transfer (HT) of ICs have occurred in vertebrates. In addition, the discovery of intact transposases, perfect TIRs and target site duplications of ICs suggests that this family may still be active in Insecta, Arachnida, frogs, and fish.ConclusionsExploring the diversity of Tc1/mariner transposons and revealing their evolutionary profiles will help provide a better understanding of the evolution of DNA transposons and their impact on genomic evolution. Here, a newly discovered family (DD36E/Incomer) of Tc1/mariner transposons is described in animals. It displays a similar structural organization and close relationship with the known DD34E/Tc1 elements, but has a relatively narrow distribution, indicating that DD36E/IC might have originated from the DD34E/Tc1 family. Our data also support the hypothesis of horizontal transfer of IC in vertebrates, even invading one lineage of mammals (bats). This study expands our understanding of the diversity of Tc1/mariner transposons and updates the classification of this superfamily.

Identification and Molecular Characterisation of mariner-Like Elements in the Tomato Leaf Miner Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae)

The tomato leafminer, Tuta absoluta (Meyrick), (Lepidoptera: Gelechiidae), is a devastating pest of tomato that has recently become a major threat to tomato production in many countries in the Mediterranean Basin, Africa and Asia. Mariner-like elements (MLEs) are class-II transposons with short inverted terminal repeats (ITR) flanking a single open reading frame (ORF) coding for a transposase. In the present study, we have isolated eight full-length MLEs from the tomato leaf miner by PCR amplification, using a single primer designed from the ITR of Hyalophora cecropia L. Mariner signature sequence D,D(34)D, as well as the conserved amino acid motifs, WVPHEL and YSPDLAP(T)D, were identified. However, sequences analysis revealed defective elements with the presence of several kinds of mutations generating stop codons, frameshifts, and non-functional transposase. Based on predicted transposase sequences, T. absoluta MLEs (Tamar1) belongs to the cecropia subfamily and clustered closely with MLEs from other Lepidoptera members suggesting vertical genetic transmission from a common ancestor. The identification of mariner-like elements in this invasive species will be useful to understand the genome dynamics in Lepidoptera.

De Novo Assembly Discovered Novel Structures in Genome of Plastids and Revealed Divergent Inverted Repeats in Mammillaria (Cactaceae, Caryophyllales).

The complete sequence of chloroplast genome (cpDNA) has been documented for single large columnar species of Cactaceae, lacking inverted repeats (IRs). We sequenced cpDNA for seven species of the short-globose cacti of Mammillaria and de novo assembly revealed three novel structures in land plants. These structures have a large single copy (LSC) that is 2.5 to 10 times larger than the small single copy (SSC), and two IRs that contain strong differences in length and gene composition. Structure 1 is distinguished by short IRs of <1 kb composed by rpl23-trnI-CAU-ycf2; with a total length of 110,189 bp and 113 genes. In structure 2, each IR is approximately 7.2 kb and is composed of 11 genes and one Intergenic Spacer-(psbK-trnQ)-trnQ-UUG-rps16-trnK-UUU-matK-trnK-UUU-psbA-trnH-GUG-rpl2-rpl23-trnI-CAU-ycf2; with a total size of 116,175 bp and 120 genes. Structure 3 has divergent IRs of approximately 14.1 kb, where IRA is composed of 20 genes: psbA-trnH-GUG-rpl23-trnI-CAU-ycf2-ndhB-rps7-rps12-trnV-GAC-rrn16-ycf68-trnI-GAU-trnA-AGC-rrn23-rrn4.5-rrn5-trnR-ACG-trnN-GUU-ndhF-rpl32; and IRB is identical to the IRA, but lacks rpl23. This structure has 131 genes and, by pseudogenization, it is shown to have the shortest cpDNA, of just 107,343 bp. Our findings show that Mammillaria bears an unusual structural diversity of cpDNA, which supports the elucidation of the evolutionary processes involved in cacti lineages.

Breakpoint junction features of seven DMD deletion mutations

Duchenne muscular dystrophy is an inherited muscle wasting disease with severe symptoms and onset in early childhood. Duchenne muscular dystrophy is caused by loss-of-function mutations, most commonly deletions, within the DMD gene. Characterizing the junction points of large genomic deletions facilitates a more detailed model of the origins of these mutations and allows for a greater understanding of phenotypic variations associated with particular genotypes, potentially providing insights into the deletion mechanism. Here, we report sequencing of breakpoint junctions for seven patients with intragenic, whole-exon DMD deletions. Of the seven junction sequences identified, we found one instance of a “clean” break, three instances of microhomology (2–5 bp) at the junction site, and three complex rearrangements involving local sequences. Bioinformatics analysis of the upstream and downstream breakpoint regions revealed a possible role of short inverted repeats in the initiation of some of these deletion events.

Complete Chloroplast Genome of Pinus densiflora Siebold &amp; Zucc. and Comparative Analysis with Five Pine Trees

Pinus densiflora (Korean red pine) is widely distributed in East Asia and considered one of the most important species in Korea. In this study, the complete chloroplast genome of P. densiflora was sequenced by combining the advantages of Oxford Nanopore MinION and Illumina MiSeq. The sequenced genome was then compared with that of a previously published conifer plastome. The chloroplast genome was found to be circular and comprised of a quadripartite structure, including 113 genes encoding 73 proteins, 36 tRNAs and 4 rRNAs. It had short inverted repeat regions and lacked ndh gene family genes, which is consistent with other Pinaceae species. The gene content of P. densiflora was found to be most similar to that of P. sylvestris. The newly attempted sequencing method could be considered an alternative method for obtaining accurate genetic information, and the chloroplast genome sequence of P. densiflora revealed in this study can be used in the phylogenetic analysis of Pinus species.

The complete chloroplast genome sequence of Horsfieldia amygdalina (Myristicaceae)

Horsfieldia amygdalina is a member of Myristicaceae. The H. amygdalina chloroplast genome is found to be 155,683 bp in length and has a base composition of A (29.99%), G (19.32%), C (19.92%), and T (30.77%). The genome contained two short inverted repeat (IRa and IRb) regions (37,754 bp) which were separated by a large single copy (LSC) region (86,931 bp) and a small single copy (SSC) region (30,998 bp). The genome encodes 121 unique genes, including 86 protein-coding genes, 27 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. Further, complete chloroplast sequence of H. amygdalina was aligned together with Horsfieldia pandurifolia, Myristica yunnanensis and other Magnoliales and basal angiosperms species which have reported the complete chloroplast sequence. This complete chloroplast genome will provide valuable information for the development of DNA markers for future species resource development and phylogenetic analysis of H. amygdalina.

The complete chloroplast genome sequence of Horsfieldia kingii (Myristicaceae)

Horsfieldia kingii is a member of Myristicaceae. The H. kingii chloroplast genome is found to be 155,655 bp in length and has a base composition of A (30.03%), G (19.52%), C (19.72%), and T (30.73%). The genome contained two short inverted repeat (IRa and IRb) regions (48,052 bp) which were separated by a large single copy (LSC) region (86,912 bp) and a small single copy (SSC) region (20,691 bp). The genome encodes 123 unique genes, including 85 protein-coding genes, 27 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. Further, complete chloroplast sequence of H. kingii was aligned together with other 2 species of Myristicaceae and other 5 basal angiosperms species which have reported the complete chloroplast sequence. This complete chloroplast genome will provide valuable information for the development of DNA markers for future species resource development and phylogenetic analysis of H. kingii.

Characteristic and phylogenetic analyses of chloroplast genome for an endangered species Manglietia lucida (Magnoliaceae)

Manglietia lucida is a threatened horticultural plant in Yunnan province of China. Now, the complete chloroplast (cp) genome of M. lucida was assembled. The cp genome of M. lucida was 160,134 bp in length and contained two short inverted repeat regions (26,595 bp), which were separated by a small single copy region (18,825 bp) and a large single copy region (88,119 bp). The cp genome encodes 108 unique genes, including 74 protein-coding genes, 30 transfer RNA genes and 4 ribosomal RNA genes. The topology of the phylogenetic tree showed that M. lucida was not formed a monophyletic clade but clustered together with genus Magnolia.

The complete chloroplast genome sequence of Keteleeria hainanensis (Pinaceae)

Keteleeria hainanensis is an endemic species with extremely narrow distribution. In this study, the complete genome of K._hainanensis was sequenced and analyzed. The genome size is 117,366 bp and it contains two short inverted repeat regions of 1,272 bp, which was separated by a large single-copy (LSC) region of 74,819 bp and a small single-copy (SSC) region of 40,003 bp. The GC content of this genome was 38.57%. The chloroplast genome contained 103 unique genes, including 74 protein-coding gene, 25 tRNA genes, and 4 rRNA gene. Phylogenetic analysis base on 11 chloroplast genomes indicated that K. hainanensis is closely related to K. davidiana.

The plastomes of Astrocaryum aculeatum G. Mey. and A. murumuru Mart. show a flip-flop recombination between two short inverted repeats.

The plastomes of Astrocaryum murumuru and A. aculeatum revealed a lineage-specific structural feature originated by flip-flop recombination, non-synonymous substitutions in conserved genes and several molecular markers. Astrocaryum murumuru Mart. and A. aculeatum G.Mey. are two palm species of Amazon forest that are economically important as source of food, oil and raw material for several applications. Genetic studies aiming to establish strategies for conservation and domestication of both species are still in the beginning given that the exploitation is mostly by extractive activity. The identification and characterization of molecular markers are essential to assess the genetic diversity of natural populations of both species. Therefore, we sequenced and characterized in detail the plastome of both species. We compared both species and identified 32 polymorphic SSR loci, 150 SNPs, 46 indels and eight hotspots of nucleotide diversity. Additionally, we reported a specific RNA editing site found in the ccsA gene, which is exclusive to A. murumuru. Moreover, the structural analysis in the plastomes of both species revealed a 4.6-kb inversion encompassing a set of genes involved in chlororespiration and plastid translation. This 4.6-kb inversion is a lineage-specific structural feature of the genus Astrocaryum originated by flip-flop recombination between two short inverted repeats. Furthermore, our phylogenetic analysis using whole plastomes of 39 Arecaceae species placed the Astrocaryum species sister to Acrocomia within the tribe Cocoseae. Finally, our data indicated substantial changes in the plastome structure and sequence of both species of the genus Astrocaryum, bringing new molecular markers, several structural and evolving features, which can be applied in several areas such as genetic, evolution, breeding, phylogeny and conservation strategies for both species.

Cruciform DNA structure formed at short inverted repeats: A source of genetic instability in vivo

Genomic instability is a major driving force for many human diseases including cancer. Certain mutation “hotspot” regions of the human genome are susceptible to cancer‐associated instability. These regions often co‐localize with repetitive DNA sequences that can adopt alternative structures (i.e. non‐B DNA). One such structure is cruciform DNA which forms at inverted repeat sequences. Short inverted repeats (&lt;100bp) are abundant in the human genome and our laboratory has shown that they are substantially enriched at translocation breakpoints in human cancer genomes. Also, they are mutagenic and stimulate the formation of DNA double strand breaks (DSBs) leading to deletions in mammalian and yeast cells. The DSBs can be caused by fork stalling (i.e. a replication‐dependent mechanism) or by cleavage of cruciform structures by the repair enzyme XPF (i.e. a replication‐independent mechanism). Together, these studies have provided a mechanistic insight into the role of short inverted repeats in disease etiology. To further delineate the biological significance of these sequences, we have developed a transgenic mouse model containing human inverted repeats. We hypothesize that short inverted repeats capable of forming cruciform structures increase genetic instability in vivo. To test this, we have introduced a recoverable mutation‐reporter shuttle vector, carrying either a GC‐rich short inverted repeat (GC mice) or B‐DNA‐forming sequence (control mice) into the mouse genome. Both male and female GC‐containing mice (n=7) and control mice (n=9) were aged for 2 years and the shuttle‐vector was subsequently recovered from the genomic DNA of spleen tissue. Transformation into competent cells was performed to obtain blue (wild‐type) and white (mutant) colonies. Mutation frequencies were calculated and mutation spectra were obtained by direct DNA sequencing. Preliminary data reveals that GC rich cruciform‐forming sequences induce a significantly higher mutation frequency than the control B‐DNA‐forming sequences. These preliminary results suggest that cruciform‐forming sequences contribute to genetic instability in vivo. The contribution of this study to cancer etiology is significant, as the results provide insights into the molecular changes induced by cruciform structures.Support or Funding InformationNIH/NCI support to KMV (CA193124 and CA093729)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

De novo origination of MIRNAs through generation of short inverted repeats in target genes

ABSTRACTMIRNA (MIR) gene origin and early evolutionary processes, such as hairpin precursor sequence origination, promoter activity acquirement and the sequence of these two processes, are fundamental and fascinating subjects. Three models, including inverted gene duplication, spontaneous evolution and transposon transposition, have been proposed for de novo origination of hairpin precursor sequence. However, these models still open to discussion. In addition, de novo origination of MIR gene promoters has not been well investigated. Here, I systematically investigated the origin of evolutionarily young polyphenol oxidase gene (PPO)-targeting MIRs, including MIR1444, MIR058 and MIR12112, and a genomic region termed AasPPO-as-hp, which contained a hairpin-forming sequence. I found that MIR058 precursors and the hairpin-forming sequence of AasPPO-as-hp originated in an ancient PPO gene through forming short inverted repeats. Palindromic-like sequences and imperfect inverted repeats in the ancient PPO gene contributed to initiate the generation of short inverted repeats probably by causing errors during DNA duplication. Analysis of MIR058 and AasPPO-as-hp promoters showed that they originated in the 3ʹ-flanking region of the ancient PPO gene. Promoter activities were gained by insertion of a CAAT-box and multiple-copper-response element (CuRE)-containing miniature inverted-repeat transposable element (MITE) in the upstream of AT-rich TATA-box-like sequence. Gain of promoter activities occurred before hairpin-forming sequence origination. Sequence comparison of MIR1444, MIR058 and MIR12112 promoters showed frequent birth and death of CuREs, indicating copper could be vital for the origination and evolution of PPO-targeting MIRs. Based on the evidence obtained, a novel model for plant MIR origination and evolution is proposed.

The complete chloroplast genome sequence of Horsfieldia pandurifolia (Myristicaceae)

Horsfieldia pandurifolia is a member of Myristicaceae. The H. pandurifolia chloroplast genome is found to be 155,695 bp in length and has a base composition of A (30.01%), G (19.31%), C (19.90%), and T (30.78%). The genome contained two short inverted repeats (IRa and IRb) regions (48,062 bp) which were separated by a large single copy (LSC) region (92,561 bp) and a small single copy (SSC) region (15,072 bp). The genome encodes 121 unique genes, including 86 protein-coding genes, 27 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. Further, complete chloroplast sequence of H. pandurifolia was aligned together with five other species which have reported the complete chloroplast sequence. This complete chloroplast genome will provide valuable information for the development of DNA markers for future species resource development and phylogenetic analysis of H. pandurifolia.

The complete chloroplast genome sequence of Myristica yunnanensis (Myristicaceae)

Myristica yunnanensis is a member of Myristicaceae. The M. yunnanensis chloroplast genome is found to be 155,923 bp in length and has a base composition of A (30.04%), G (19.28%), C (19.91%), and T (30.77%). The genome contained two short inverted repeat (IRa and IRb) regions (48,004 bp) which were separated by a large single-copy (LSC) region (87,088 bp) and a small single-copy (SSC) region (20,731 bp). The genome encodes 120 unique genes, including 85 protein-coding genes, 27 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. Further, complete chloroplast sequence of M. yunnanensis was aligned together with Horsfieldia pandurifolia and other Magnoliales and basal angiosperms species which have reported the complete chloroplast sequence. This complete chloroplast genome will provide valuable information for the development of DNA markers for future species resource development and phylogenetic analysis of M. yunnanensis.

The complete chloroplast genome of Malania oleifera (Olacaceae), an endangered species in China

Malania oleifera is a rare and threatened medicinal plant in China. In this study, the complete chloroplast genome (cp) of M. oleifera was sequenced, assembled, and analyzed. The circular complete cp genome of M. oleifera was 158,163 bp in length and contained a large single copy region (LSC, 87,212 bp), a small single copy region (SSC, 439 bp), and 2 short inverted repeat (IRa and IRb) regions (35,256 bp). The cp genome encodes 97 unique genes, including 62 protein-coding genes, 30 transfer RNA genes, 4 ribosomal RNA genes, and a pseudogene. Phylogenetic analysis of M. oleifera showed that M. oleifera was the sister group with Champereia manillana from Opiliaceae and Osyris alba from Santalaceae.

Complete chloroplast genome sequence of the Mongolian oak, Quercus mongolica (Fagaceae)

Quercus mongolica is a major tree species in East Asian hardwood forests. In this study, we assembled a complete chloroplast (cp) genome of Q. mongolica based on next-generation sequencing data. The cp genome was 161,295 bp, with a large single-copy region (90,583 bp), a small single-copy region (19,060 bp), and two short inverted repeat regions (25,826bp). The genome contains 121 genes, including 85 protein-coding genes, eight rRNA genes, and 29 tRNA genes. Phylogenetic analyses based on cp genome sequences revealed that Q. mongolica was grouped with species from section Quercus.

Single-cell copy number variant detection reveals the dynamics and diversity of adaptation.

Copy number variants (CNVs) are a pervasive source of genetic variation and evolutionary potential, but the dynamics and diversity of CNVs within evolving populations remain unclear. Long-term evolution experiments in chemostats provide an ideal system for studying the molecular processes underlying CNV formation and the temporal dynamics with which they are generated, selected, and maintained. Here, we developed a fluorescent CNV reporter to detect de novo gene amplifications and deletions in individual cells. We used the CNV reporter in Saccharomyces cerevisiae to study CNV formation at the GAP1 locus, which encodes the general amino acid permease, in different nutrient-limited chemostat conditions. We find that under strong selection, GAP1 CNVs are repeatedly generated and selected during the early stages of adaptive evolution, resulting in predictable dynamics. Molecular characterization of CNV-containing lineages shows that the CNV reporter detects different classes of CNVs, including aneuploidies, nonreciprocal translocations, tandem duplications, and complex CNVs. Despite GAP1’s proximity to repeat sequences that facilitate intrachromosomal recombination, breakpoint analysis revealed that short inverted repeat sequences mediate formation of at least 50% of GAP1 CNVs. Inverted repeat sequences are also found at breakpoints at the DUR3 locus, where CNVs are selected in urea-limited chemostats. Analysis of 28 CNV breakpoints indicates that inverted repeats are typically 8 nucleotides in length and separated by 40 bases. The features of these CNVs are consistent with origin-dependent inverted-repeat amplification (ODIRA), suggesting that replication-based mechanisms of CNV formation may be a common source of gene amplification. We combined the CNV reporter with barcode lineage tracking and found that 102–104 independent CNV-containing lineages initially compete within populations, resulting in extreme clonal interference. However, only a small number (18–21) of CNV lineages ever constitute more than 1% of the CNV subpopulation, and as selection progresses, the diversity of CNV lineages declines. Our study introduces a novel means of studying CNVs in heterogeneous cell populations and provides insight into their dynamics, diversity, and formation mechanisms in the context of adaptive evolution.

A strong structural correlation between short inverted repeat sequences and the polyadenylation signal in yeast and nucleosome exclusion by these inverted repeats

DNA sequences that read the same from 5′ to 3′ in either strand are called inverted repeat sequences or simply IRs. They are found throughout a wide variety of genomes, from prokaryotes to eukaryotes. Despite extensive research, their in vivo functions, if any, remain unclear. Using Saccharomyces cerevisiae, we performed genome-wide analyses for the distribution, occurrence frequency, sequence characteristics and relevance to chromatin structure, for the IRs that reportedly have a cruciform-forming potential. Here, we provide the first comprehensive map of these IRs in the S. cerevisiae genome. The statistically significant enrichment of the IRs was found in the close vicinity of the DNA positions corresponding to polyadenylation [poly(A)] sites and ~ 30 to ~ 60 bp downstream of start codon-coding sites (referred to as ‘start codons’). In the former, ApT- or TpA-rich IRs and A-tract- or T-tract-rich IRs are enriched, while in the latter, different IRs are enriched. Furthermore, we found a strong structural correlation between the former IRs and the poly(A) signal. In the chromatin formed on the gene end regions, the majority of the IRs causes low nucleosome occupancy. The IRs in the region ~ 30 to ~ 60 bp downstream of start codons are located in the + 1 nucleosomes. In contrast, fewer IRs are present in the adjacent region downstream of start codons. The current study suggests that the IRs play similar roles in Escherichia coli and S. cerevisiae to regulate or complete transcription at the RNA level.