Filler DNA Research Articles

PEI-Mediated Transient Gene Expression in CHO Cells.

We describe a method for polyethyleneimine (PEI)-mediated transient transfection of suspension-adapted Chinese hamster ovary (CHO-DG44) cells for protein expression applicable at scales from 2mL to 2L. The method involves transfection at a high cell density (5×106 cells/mL) by direct addition of plasmid DNA (pDNA) and PEI to the culture and subsequent incubation at 31°C with agitation by orbital shaking. This method requires 0.3mg/L of coding pDNA, 2.7mg/L of nonspecific (filler) DNA, and 15mg/L of PEI. The production phase is performed at 31°C in the presence of 0.25% N,N-dimethylacetamide (DMA). If desired, the method can be modified to avoid use of DMA by increasing the amount of coding DNA. We also provide information on culture vessel options, recommended working volumes, and recommended shaking speeds for transfections at scales from 2mL to 2L.

IKZF1 DELETIONS IN B-ALL: FROM GENETIC BASIS TO DIAGNOSTIC ENHANCEMENT

Because IKZF1 deletions are associated with an increased risk of relapse in patients with B-cell precursor acute lymphoblastic leukemia (B-ALL), the development of methods for rapid and accurate detection of this alteration has a great clinical impact. Although multiplex ligation-dependent probe amplification (MLPA) has been widely used for the evaluation of IKZF1 deletions, multiplex (M)-PCR is a valuable tool for the validation of those IKZF1 deletions. However, this method is still limited to detect recurrent intragenic deletions (Δ2-3, Δ2-7, Δ2-8, Δ4-7, Δ4-8). Therefore, here we evaluate the breakpoint map of IKZF1 deletions with a special focus on non-recurrent deletions to update methodologies for the diagnosis of this genetic alteration. Moreover, we explore the genetic features underlying those IKZF1 deletions to infer its molecular pathogenesis. This study included four international cohorts of pediatric and adult patients with B-ALL. IKZF1 deletions were identified by MLPA (P335 and P202 assays) and M-PCR. Non-recurrent deletions were verified by next-generation sequencing using the Illumina DNA Prep with Enrichment assay. We also revised previously published data regarding IKZF1 deletions in B-ALL for compiling breakpoint sequence information of these deletions. Sequence data from our cohorts and literature were used in further analyses. After identifying the breakpoint clusters, we performed an agnostic motif search using MEME and annotated the presence of filler DNA or microhomologies at the junctions of IKZF1 deletions. A total of 1,472 B-ALL samples were screened, and IKZF1 deletions were found in 16% of these patients. Although non-recurrent deletions were associated with 1% of the total cohort, it represents 8% of all IKZF1 deletions. The analysis of breakpoint sequences at DNA level revealed that non-recurrent IKZF1 deletions comprises miscellaneous alterations, but the majority of them encompases Δ1 and Δ1-3. Considering that IKZF1 exon 1 deletions are more difficult to reliably detect by MLPA due to the high GC content (>50%) of this region, we mapped several exon 1 deletions at DNA level and identified one commonly deleted region which is ideal for designing MLPA probes. Next, we searched for motifs, additional nucleotides/microhomologies associated with IKZF1 deletion sequences. We verified an enrichment of the 5’-CACACAGTG-3’sequence within 21 out of 24 breakpoint clusters of IKZF1 deletion (E-value: 2.9e-008). Additionally, the filler DNA within the junctions of IKZF1 deletions presented a higher GC nucleotide content at these clusters as compared to the deletions outside of them (71% vs. 56%). This study provides the genetic landscape of IKZF1 deletions in B-ALL. Although rare types of deletion may be identified at the routine diagnosis, they should be considered after using adapted MLPA protocols, including additional probes within the common deleted region nearby IKZF1 exon 1. We also verified the occurrence of the heptamer sequence and an enrichment of GC nucleotides exclusively within breakpoint clusters, suggesting that RAG recombination and TdT activity may promote most IKZF1 deletions, although rare types of alterations may be associated with another molecular mechanism of leukemogenesis.

Characterization of T-Circles and Their Formation Reveal Similarities to Agrobacterium T-DNA Integration Patterns.

Agrobacterium transfers T-DNA to plants where it may integrate into the genome. Non-homologous end-joining (NHEJ) has been invoked as the mechanism of T-DNA integration, but the role of various NHEJ proteins remains controversial. Genetic evidence for the role of NHEJ in T-DNA integration has yielded conflicting results. We propose to investigate the formation of T-circles as a proxy for understanding T-DNA integration. T-circles are circular double-strand T-DNA molecules, joined at their left (LB) and right (RB) border regions, formed in plants. We characterized LB-RB junction regions from hundreds of T-circles formed in Nicotiana benthamiana or Arabidopsis thaliana. These junctions resembled T-DNA/plant DNA junctions found in integrated T-DNA: Among complex T-circles composed of multiple T-DNA molecules, RB-RB/LB-LB junctions predominated over RB-LB junctions; deletions at the LB were more frequent and extensive than those at the RB; microhomology was frequently used at junction sites; and filler DNA, from the plant genome or various Agrobacterium replicons, was often present between the borders. Ku80 was not required for efficient T-circle formation, and a VirD2 ω mutation affected T-circle formation and T-DNA integration similarly. We suggest that investigating the formation of T-circles may serve as a surrogate for understanding T-DNA integration.

Analysis of T-DNA integration events in transgenic rice

Agrobacterium-mediated plant transformation has been widely used for introducing transgene(s) into a plant genome and plant breeding. However, our understanding of T-DNA integration into rice genome remains limited relative to that in the model dicot Arabidopsis. To better elucidate the T-DNA integration into the rice genome, we investigated extensively the T-DNA ends and their flanking rice genomic sequences from two transgenic rice plants carrying Cowpea Trypsin Inhibitor (CpTI)-derived gene Signal-CpTI-KDEL (SCK) and Bacillus thuringiensis (BT) gene, respectively, by TAIL-PCR method. Analysis of the junction sequences between the T-DNA ends and rice genome DNA indicated that there were three joining patterns of microhomology, filler DNA sequences, and exact joining, and both the T-DNA ends tend to adopt identical manner to join the rice genome. After T-DNA integration, there were several variations of rice genomic sequences, including small deletions at the integration sites, superfluous DNA inserted between T-DNA and genome, and translocation of genomic DNA in the flanking regions. The translocation block could be from a noncontiguous region in the same chromosome or different chromosomes at the integration sites, and the originating position of the translocated block resulted in comparable deletion based on a cut/paste mechanism rather than a replication mechanism. Our study may lead to a better understand of T-DNA integration mechanism and facilitate functional genomic studies and further crop improvement.

Plant DNA Repair and Agrobacterium T-DNA Integration.

Agrobacterium species transfer DNA (T−DNA) to plant cells where it may integrate into plant chromosomes. The process of integration is thought to involve invasion and ligation of T-DNA, or its copying, into nicks or breaks in the host genome. Integrated T−DNA often contains, at its junctions with plant DNA, deletions of T−DNA or plant DNA, filler DNA, and/or microhomology between T-DNA and plant DNA pre-integration sites. T−DNA integration is also often associated with major plant genome rearrangements, including inversions and translocations. These characteristics are similar to those often found after repair of DNA breaks, and thus DNA repair mechanisms have frequently been invoked to explain the mechanism of T−DNA integration. However, the involvement of specific plant DNA repair proteins and Agrobacterium proteins in integration remains controversial, with numerous contradictory results reported in the literature. In this review I discuss this literature and comment on many of these studies. I conclude that either multiple known DNA repair pathways can be used for integration, or that some yet unknown pathway must exist to facilitate T−DNA integration into the plant genome.

Abstract 2097: Cell-free DNA methylome profiling by MBD-seq with ultra-low input

Abstract Methylation signatures in cell-free DNA (cfDNA) have shown great sensitivity and specificity in the characterization of tumor status and classification of tumor types, as well as the response to therapy and recurrence. Currently, most cfDNA methylation studies are based on bisulfite conversion. However, bisulfite treatment may result in the substantial degradation of up to 96% of DNA. This reduction critically impacts the assay's sensitivity, especially for low yield material such as cfDNA. Recently, enrichment-based cfMeDIP-seq that uses cfDNA as starting material is beginning to show potential. Here, we report another enrichment-based ultra-low input cfDNA methylation profiling method using methyl-CpG binding proteins capture, termed cfMBD-seq. We optimized the conditions of cfMBD capture by adjusting the amount of MethylCap protein and beads along with using exogenous λ DNA as filler DNA. Our data showed that cfMBD-seq performed equally to the standard MBD-seq (1000 ng input) even when using 1 ng DNA as the input. cfMBD-seq performed better than a previously published low input MBD-seq protocol without using filler DNA in the enrichment of methylated DNA, with 59.63% vs. 19.77% reads mapped to CpG islands and 94.74% vs. 85.66% reads mapped to combined CpG islands/shores/shelves. cfMBD-seq also demonstrated higher sequencing data quality when compared to cfMeDIP-seq, with more reads that passed filter and less duplicate rate (79.80% vs. 60.91% sequenced reads used for analyses). We showed that cfMBD-seq outperformed cfMeDIP-seq in the enrichment of fragments with higher CpG density (29.59 vs. 23.33, number of CpG per fragments where coverage reaches peak), CpG islands for example (59.63% vs. 38.89% reads mapped on CpG islands). cfMBD-seq also successfully recapitulates methylation profiles from reduced representation bisulfite sequencing and whole-genome bisulfite sequencing. Overall, cfMBD-seq is a method of choice for interrogating epigenetic regulation of gene expression (methylation changes on CpG islands). On the other hand, cfMeDIP-seq would be preferable in investigating transcriptional regulation of non-coding RNAs (methylation changes on gene bodies and CpG shores). Our study demonstrates the potential benefits of using cfMBD-seq to profile the methylome of cfDNA with ultra-low DNA input. Current results provide justification for further validation using case and control plasma samples from different malignancies to perform differential methylation analyses. Another potential for cfMBD-seq is the use in other methylome-wide investigations that are limited by DNA yield. This new bisulfite-free ultra-low input methylation profiling technology has a great potential in non-invasive and cost-effective cancer detection and classification. Citation Format: Jinyong Huang, Alex C. Soupir, Liang Wang. Cell-free DNA methylome profiling by MBD-seq with ultra-low input [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2097.

Cell-free DNA methylome profiling by MBD-seq with ultra-low input

ABSTRACT Methylation signatures in cell-free DNA (cfDNA) have shown great sensitivity and specificity in the characterization of tumour status and classification of tumour types, as well as the response to therapy and recurrence. Currently, most cfDNA methylation studies are based on bisulphite conversion, especially targeted bisulphite sequencing, while enrichment-based methods such as cfMeDIP-seq are beginning to show potential. Here, we report an enrichment-based ultra-low input cfDNA methylation profiling method using methyl-CpG binding proteins capture, termed cfMBD-seq. We optimized the conditions for cfMBD capture by adjusting the amount of MethylCap protein along with using methylated filler DNA. Our data show high correlation between low input cfMBD-seq and standard MBD-seq (>1000 ng input). When compared to cfMEDIP-seq, cfMBD-seq demonstrates higher sequencing data quality with more sequenced reads passed filter and less duplicate rate. cfMBD-seq also outperforms cfMeDIP-seq in the enrichment of CpG islands. This new bisulphite-free ultra-low input methylation profiling technology has great potential in non-invasive and cost-effective cancer detection and classification.

Optimization of a transient antibody expression platform towards high titer and efficiency.

Transient gene expression (TGE) using mammalian cells is an extensively used technology for the production of antibodies and recombinant proteins and has been widely adopted by both academic and industrial labs. Chinese Hamster Ovary (CHO) cells have become one of the major workhorses for TGE of recombinant antibodies due to their attractive features: post-translational modifications, adaptation to high cell densities, and use of serum-free media. In this study, we describe the optimization of parameters for TGE for antibodies from CHO cells. Through a matrix evaluation of multiple factors including inoculum, transfection conditions, amount and type of DNA used, and post-transfection culture conditions, we arrived at an uniquely optimized process with higher titer and reduced costs and time, thus increasing the overall efficiency of early antibody material supply. We further investigated the amount of coding DNA used in TGE and the influence of kinetics and size of the transfection complex on the in vitro efficiency of the transfection. We present here the first report of an optimized TGE platform using Filler DNA in an early drug discovery setting for the screening and production of therapeutic mAbs.

Strategies to produce T-DNA free CRISPRed fruit trees via Agrobacterium tumefaciens stable gene transfer

Genome editing via CRISPR/Cas9 is a powerful technology, which has been widely applied to improve traits in cereals, vegetables and even fruit trees. For the delivery of CRISPR/Cas9 components into dicotyledonous plants, Agrobacterium tumefaciens mediated gene transfer is still the prevalent method, although editing is often accompanied by the integration of the bacterial T-DNA into the host genome. We assessed two approaches in order to achieve T-DNA excision from the plant genome, minimizing the extent of foreign DNA left behind. The first is based on the Flp/FRT system and the second on Cas9 and synthetic cleavage target sites (CTS) close to T-DNA borders, which are recognized by the sgRNA. Several grapevine and apple lines, transformed with a panel of CRISPR/SpCas9 binary vectors, were regenerated and characterized for T-DNA copy number and for the rate of targeted editing. As detected by an optimized NGS-based sequencing method, trimming at T-DNA borders occurred in 100% of the lines, impairing in most cases the excision. Another observation was the leakage activity of Cas9 which produced pierced and therefore non-functional CTS. Deletions of genomic DNA and presence of filler DNA were also noticed at the junctions between T-DNA and genomic DNA. This study proved that many factors must be considered for designing efficient binary vectors capable of minimizing the presence of exogenous DNA in CRISPRed fruit trees.

Development of a high cell density transient CHO platform yielding mAb titers greater than 2 g/L in only 7 days.

We developed a simple transient Chinese Hamster Ovary expression platform. Titers for a random panel of 20 clinical monoclonal antibodies (mAbs) ranged from 0.6 to 2.7 g/L after 7 days. Two factors were the key in obtaining these high titers. First, we utilized an extremely high starting cell density (20 million cells/ml), and then arrested further cell growth by employing mild hypothermic conditions (32°C). Second, we performed a 6-variable Design of Experiments to find optimal concentrations of plasmid DNA (coding DNA), boost DNA (DNA encoding the XBP1S transcription factor), transfection reagent (polyethylenimine [PEI]), and nutrient feed amounts. High coding DNA concentrations (12.5 mg/L) were found to be optimal. We therefore diluted expensive coding DNA with inexpensive inert filler DNA (herring sperm DNA). Reducing the coding DNA concentration by 70% from 12.5 to 3.75 mg/L did not meaningfully reduce mAb titers. Titers for the same panel of 20 clinical mAbs ranged from 0.7 to 2.2 g/L after reducing the coding DNA concentration to 3.75 mg/L. Finally, we found that titer and product quality attributes were similar for a clinical mAb (rituximab) expressed at very different scales (volumes ranging from 3 ml to 2 L).

Effects of Arabidopsis Ku80 deletion on the integration of the left border of T-DNA into plant chromosomal DNA via Agrobacterium tumefaciens.

T-DNA integration into plant chromosomal DNA via Agrobacterium tumefaciens can be achieved by exploiting the double-strand break repair system of the host's DNA. However, the detailed mechanism of T-DNA integration remains unclear. Here, a sequence analysis of the junction sequences of T-DNA and chromosomal DNA was performed to assess the mechanism of T-DNA integration. T-DNA was introduced into Arabidopsis wild-type and NHEJ-deficient ku80 mutant plants using the floral dip method; the junctions of the left border (LB) of T-DNA were subsequently analyzed by adapter PCR. The most frequent junction of the LB of T-DNA with chromosomal DNA was of the filler DNA type in both lines. The lengths of direct or inverted repeat sequences within or around the filler DNA sequence were greater in the ku80 mutant. In addition, the frequency of T-DNA integration near a transcription start site was significantly higher in the ku80 mutant. Our observations suggest that the presence of the Ku80 protein affects the location of the integration of T-DNA and the pattern of formation of repeat sequences within or around the filler DNA during LB integration into chromosomal DNA.

Preparation of cfMeDIP-seq libraries for methylome profiling of plasma cell-free DNA

Circulating cell-free DNA (cfDNA) comprises small DNA fragments derived from normal and tumor tissue that are released into the bloodstream. Recently, methylation profiling of cfDNA as a liquid biopsy tool has been gaining prominence due to the presence of tissue-specific markers in cfDNA. We have previously reported cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq) as a sensitive, low-input, cost-efficient and bisulfite-free approach to profiling DNA methylomes of plasma cfDNA. cfMeDIP-seq is an extension of a previously published MeDIP-seq protocol and is adapted to allow for methylome profiling of samples with low input (ranging from 1 to 10 ng) of DNA, which is enabled by the addition of 'filler DNA' before immunoprecipitation. This protocol is not limited to plasma cfDNA; it can also be applied to other samples that are naturally sheared and at low availability (e.g., urinary cfDNA and cerebrospinal fluid cfDNA), and is potentially applicable to other applications beyond cancer detection, including prenatal diagnostics, cardiology and monitoring of immune response. The protocol presented here should enable any standard molecular laboratory to generate cfMeDIP-seq libraries from plasma cfDNA in ~3-4 d.

Characterization of a novel Helitron family in insect genomes: insights into classification, evolution and horizontal transfer

BackgroundHelitrons play an important role in shaping eukaryotic genomes due to their ability to transfer horizontally between distantly related species and capture gene fragments during the transposition. However, the mechanisms of horizontal transfer (HT) and the process of gene fragment capturing of Helitrons still remain to be further clarified.ResultsHere, we characterized a novel Helitron family discontinuously distributed in 27 out of 256 insect genomes. The most prominent characteristic of Hel1 family is its high sequence similarity among species of different insect orders. Related elements were also identified in two spiders, representing the first report of spider Helitrons. All these elements were classified into 2 families, 9 subfamilies and 35 exemplars based on our new classification criteria. Autonomous partners of Helitron were reconstructed in the genomes of three insects and one spider. Integration pattern analysis showed that majority of Hel1A elements in Papilio xuthus and Pieris rapae inserted into introns. Consistent with filler DNA model, stepwise sequence acquisition was observed in Sfru_Hel1Aa, Sfru_Hel1Ab and Sfru_Hel1Ac in Spodoptera frugiperda. Remarkably, the evidence that Prap_Hel1Aa in a Lepdidoptera insect, Pieris rapae, was derived from Cves_Hel1Aa in a parasitoid wasp, Cotesia vestalis, suggested the role of nonregular host-parasite interactions in HT of Helitrons.ConclusionsWe proposed a modified classification criteria of Helitrons based on the important role of the 5′-end of Helitrons in transposition, and provided evidence for stepwise sequence acquisition and recurrent HT of a novel Helitron family. Our findings of the nonregular host-parasite interactions may be more conducive to the HT of transposons.

PEI-Mediated Transient Gene Expression in CHO Cells.

We describe a method for polyethyleneimine (PEI) mediated transient transfection of suspension-adapted Chinese hamster ovary (CHO-DG44) cells for protein expression applicable at scales from 2mL to 2L. The method involves transfection at a high cell density (5×106 cells/mL) by direct addition of plasmid DNA (pDNA) and PEI to the culture and subsequent incubation at 31°C with agitation by orbital shaking. This method requires 0.3mg/L of coding pDNA, 2.7mg/L of nonspecific (filler) DNA and 15mg/L of PEI. The production phase is performed at 31°C in the presence of 0.25% N,N-dimethylacetamide (DMA). We also provide information on culture vessel options, recommended working volumes, and recommended shaking speeds for transfections at scales from 2mL to 2L.

Large-Scale Transient Transfection of Chinese Hamster Ovary Cells in Suspension.

We describe a one-liter transfection of suspension-adapted Chinese hamster ovary (CHO-DG44) cells using polyethyleneimine (PEI) for DNA delivery. The method involves transfection at a high cell density (5 × 106 cells/mL) by direct addition of plasmid DNA (pDNA) and PEI to the culture and subsequent incubation at 31 °C with agitation by orbital shaking. We also describe an alternative method in which 90% of the pDNA is replaced by nonspecific (filler) DNA, and the production phase is performed at 31 °C in the presence of 0.25% N, N-dimethylacetamide (DMA).

T-DNA integration in plants results from polymerase-θ-mediated DNA repair.

Agrobacterium tumefaciens is a pathogenic bacterium, which transforms plants by transferring a discrete segment of its DNA, the T-DNA, to plant cells. The T-DNA then integrates into the plant genome. T-DNA biotechnology is widely exploited in the genetic engineering of model plants and crops. However, the molecular mechanism underlying T-DNA integration remains unknown1. Here we demonstrate that in Arabidopsis thaliana T-DNA integration critically depends on polymerase theta (Pol θ). We find that TEBICHI/POLQ mutant plants (which have mutated Pol θ), although susceptible to Agrobacterium infection, are resistant to T-DNA integration. Characterization of >10,000 T-DNA-plant genome junctions reveals a distinct signature of Pol θ action and also indicates that 3' end capture at genomic breaks is the prevalent mechanism of T-DNA integration. The primer-template switching ability of Pol θ can explain the molecular patchwork known as filler DNA that is frequently observed at sites of integration. T-DNA integration signatures in other plant species closely resemble those of Arabidopsis, suggesting that Pol-θ-mediated integration is evolutionarily conserved. Thus, Pol θ provides the mechanism for T-DNA random integration into the plant genome, demonstrating a potential to disrupt random integration so as to improve the quality and biosafety of plant transgenesis.

Enhanced plasmid DNA utilization in transiently transfected CHO-DG44 cells in the presence of polar solvents.

Although the protein yields from transient gene expression (TGE) with Chinese hamster ovary (CHO) cells have recently improved, the amount of plasmid DNA (pDNA) needed for transfection remains relatively high. We describe a strategy to reduce the pDNA amount by transfecting CHO-DG44 cells with 0.06 μg pDNA/10(6) cells (10% of the optimal amount) in the presence of nonspecific (filler) DNA and various polar solvents including dimethylsufoxide, dimethyl formamide, acetonitrile, dimethyl acetamide (DMA), and hexamethyl phosphoramide (HMP). All of the polar solvents with the exception of HMP increased the production of a recombinant antibody in comparison to the untreated control transfection. In the presence of 0.25% DMA, the antibody yield in a 7-day batch culture was 500 mg/L. This was fourfold higher than the yield from the untreated control transfection. Mechanistic studies revealed that the polar solvents did not affect polyethylenimine-mediated pDNA delivery into cells or nuclei. The steady-state transgene mRNA level was elevated in the presence of each of the polar solvents tested, while the transgene mRNA half-life remained the same. These results indicated that the polar solvents enhanced transgene transcription. When screening a panel of recombinant antibodies and Fc-fusion proteins for production in the presence of the polar solvents, the highest increase in yield was observed following DMA addition for 11 of the 12 proteins. These results are expected to enhance the applicability of high-yielding TGE processes with CHO-DG44 cells by decreasing the amount of pDNA required for transfection.

Identification of human-specific AluS elements through comparative genomics

Mobile elements are responsible for ~45% of the human genome. Among them is the Alu element, accounting for 10% of the human genome (>1.1million copies). Several studies of Alu elements have reported that they are frequently involved in human genetic diseases and genomic rearrangements. In this study, we investigated the AluS subfamily, which is a relatively old Alu subfamily and has the highest copy number in primate genomes. Previously, a set of 263 human-specific AluS insertions was identified in the human genome. To validate these, we compared each of the human-specific AluS loci with its pre-insertion site in other primate genomes, including chimpanzee, gorilla, and orangutan. We obtained 24 putative human-specific AluS candidates via the in silico analysis and manual inspection, and then tried to verify them using PCR amplification and DNA sequencing. Through the PCR product sequencing, we were able to detect two instances of near-parallel Alu insertions in nearby sites that led to computational false negatives. Finally, we computationally and experimentally verified 23 human-specific AluS elements. We reported three alternative Alu insertion events, which are accompanied by filler DNA and/or Alu retrotransposition mediated-deletion. Bisulfite sequencing was carried out to examine DNA methylation levels of human-specific AluS elements. The results showed that fixed AluS elements are hypermethylated compared with polymorphic elements, indicating a possible relation between DNA methylation and Alu fixation in the human genome.

The Spectrum and Frequency of Self-Inflicted and Host Gene Mutations Produced by the Transposon Ac in Maize

The autonomous transposon Activator (Ac) is a powerful mutagen. Ac-induced mutations range from small footprints of host sequences to large rearrangements of transposon or host sequences. These mutations arise by different repair mechanisms of the double-strand break produced by Ac excision: footprints by nonhomologous end joining and rearrangements by various mechanisms, including DNA replication repair. Footprints greatly outnumber other mutations, masking them because they usually share a nonfunctional phenotype. To determine the spectrum and frequencies of host and self-mutations generated by Ac, we used an allele harboring Ac in the 5' untranslated region bronze (bz). In this system, simple excisions produce purple revertants, whereas deletions of host or transposon sequences produce stable bronze (bz-s) mutants. Internal and terminal deletions of Ac predominated among the 72 bz-s derivatives. Most internal deletions (52 of 54) behaved as nonautonomous Dissociation (Ds) elements. All nine terminal deletions or fractured Ac (fAc) elements had rearrangements of adjacent host sequences. Most Ds and fAc deletion junctions displayed microhomologies and contained filler DNA from nearby sequences, suggesting an origin by DNA repair synthesis followed by microhomology-mediated end joining. All mutations occurred more frequently in pollen, where one in 200 grains carried new Ds or fAc elements.

Inter‐species sequence comparison of Brachypodium reveals how transposon activity corrodes genome colinearity

Intergenic sequences evolve rapidly in plant genomes through a process known as genomic turnover. To investigate the influence of DNA transposons on genomic turnover, we compared 1 Mbp of orthologous genomic sequences from Brachypodium distachyon and Brachypodium sylvaticum. We found that B. distachyon and B. sylvaticum diverged approximately 1.7-2.0 million years ago. Of a total of 219 genes identified on the analyzed sequences, 211 were colinear. However, only 24 transposable elements of a total of 451 were orthologous (i.e. inserted in the common ancestor). We characterized in detail 59 insertions and 60 excisions of DNA transposons in one or other species, which altered 17% of the intergenic space. The DNA transposon excision sites showed complex and highly diagnostic sequence motifs for double-strand break (DSB) repair. DNA transposon excisions can lead to extensive deletions of hundreds of base pairs of flanking sequence if the DSB is repaired by 'single-strand annealing', or insertions of up to several hundred base pairs of 'filler DNA' if the DSB is repaired by 'synthesis-dependent strand annealing'. In some cases, DSBs were repaired by a combination of both methods. We present a model for the evolution of intergenic sequences in which repair of DSBs upon DNA transposon excision is a major factor in the rapid turnover and erosion of intergenic sequences.