Germinal Excision Research Articles

Development of an activation tagging system for maize.

Activation Tagging, distributing transcriptional enhancers throughout the genome to induce transcription of nearby genes, is a powerful tool for discovering the function of genes in plants. We have developed a transposable element system to distribute a novel activation tagging element throughout the genome of maize. The transposon system is built from the Enhancer/Suppressor (En/Spm) transposon system and uses an engineered seed color marker to show when the transposon excises. Both somatic and germinal excision events can be detected by the seed color. The activation tagging element is in a Spm‐derived non‐autonomous transposon and contains four copies of the Sugarcane Bacilliform Virus‐enhancer (SCBV‐enhancer) and the AAD1 selectable marker. We have demonstrated that the transposon can give rise to germinal excision events that can re‐integrate into non‐linked genomic locations. The transposon has remained active for three generations and events displaying high rates of germinal excision in the T2 generation have been identified. This system can generate large numbers of activation tagged maize lines that can be screened for agriculturally relevant phenotypes.

TED, an Autonomous and Rare Maize Transposon of theMutatorSuperfamily with a High Gametophytic Excision Frequency

Mutator (Mu) elements, one of the most diverse superfamilies of DNA transposons, are found in all eukaryotic kingdoms, but are particularly numerous in plants. Most of the present knowledge on the transposition behavior of this superfamily comes from studies of the maize (Zea mays) Mu elements, whose transposition is mediated by the autonomous Mutator-Don Robertson (MuDR) element. Here, we describe the maize element TED (for Transposon Ellen Dempsey), an autonomous cousin that differs significantly from MuDR. Element excision and reinsertion appear to require both proteins encoded by MuDR, but only the single protein encoded by TED. Germinal excisions, rare with MuDR, are common with TED, but arise in one of the mitotic divisions of the gametophyte, rather than at meiosis. Instead, transposition-deficient elements arise at meiosis, suggesting that the double-strand breaks produced by element excision are repaired differently in mitosis and meiosis. Unlike MuDR, TED is a very low-copy transposon whose number and activity do not undergo dramatic changes upon inbreeding or outcrossing. Like MuDR, TED transposes mostly to unlinked sites and can form circular transposition products. Sequences closer to TED than to MuDR were detected only in the grasses, suggesting a rather recent evolutionary split from a common ancestor.

Transposition behavior of nonautonomous a hAT superfamily transposon nDart in rice (Oryza sativa L.)

Transposable elements (TEs) have a significant impact on the evolution of gene function and genome structures. An endogenous nonautonomous transposable element nDart was discovered in an albino mutant that had an insertion in the Mg-protoporphyrin IX methyltransferase gene in rice. In this study, we elucidated the transposition behavior of nDart, the frequency of nDart transposition and characterized the footprint of nDart. Novel independent nDart insertions in backcrossed progenies were detected by DNA blotting analysis. In addition, germinal excision of nDart occurred at very low frequency compared with that of somatic excision, 0-13.3%, in the nDart1-4(3-2) and nDart1-A loci by a locus-specific PCR strategy. A total of 253 clones from somatic excision at five nDart loci in 10 varieties were determined. nDart rarely caused deletions beyond target site duplication (TSD). The footprint of nDart contained few transversions of nucleotides flanking to both sides of the TSD. The predominant footprint of nDart was an 8-bp addition. Precise excision of nDart was detected at a rate of only 2.2%, which occurred at two loci among the five loci examined. Furthermore, the results in this study revealed that a highly conserved mechanism of transposition is involved between maize Ac/Ds and rice Dart/nDart, which are two-component transposon systems of the hAT superfamily transposons in plant species.

Role of RAD51 in the Repair ofMuDR-Induced Double-Strand Breaks in Maize (Zea maysL.)

Rates of Mu transposon insertions and excisions are both high in late somatic cells of maize. In contrast, although high rates of insertions are observed in germinal cells, germinal excisions are recovered only rarely. Plants doubly homozygous for deletion alleles of rad51A1 and rad51A2 do not encode functional RAD51 protein (RAD51-). Approximately 1% of the gametes from RAD51+ plants that carry the MuDR-insertion allele a1-m5216 include at least partial deletions of MuDR and the a1 gene. The structures of these deletions suggest they arise via the repair of MuDR-induced double-strand breaks via nonhomologous end joining. In RAD51- plants these germinal deletions are recovered at rates that are at least 40-fold higher. These rates are not substantially affected by the presence or absence of an a1-containing homolog. Together, these findings indicate that in RAD51+ germinal cells MuDR-induced double-strand breaks (DSBs) are efficiently repaired via RAD51-directed homologous recombination with the sister chromatid. This suggests that RAD51- plants may offer an efficient means to generate deletion alleles for functional genomic studies. Additionally, the high proportion of Mu-active, RAD51- plants that exhibit severe developmental defects suggest that RAD51 plays a critical role in the repair of MuDR-induced DSBs early in vegetative development.

The low level of activity of Arabidopsis thaliana Tag1 transposon correlates with the absence of two minor transcripts in Medicago truncatula

Tag1 is an element from the hAT (hobo, Ac, Tam) family of DNA transposable elements present in Arabidopsis thaliana ecotype Landsberg. We have compared its behavior in the A. thaliana ecotype Columbia and in the legume model plant Medicago truncatula. As previously described Tag1 transposed efficiently in A. thaliana ecotype Columbia. In contrast, only somatic excision, but no germinal excision or transposition events could be detected in M. truncatula, indicating that Tag1 is poorly active in this plant. These differences in activity correlated with the absence of two minor Tag1 transcripts in M. truncatula transgenic lines but not with the structure of the T-DNA loci or the methylation of the element. These results indicate that Tag1 is not suitable for insertion mutagenesis in M. truncatula.

Genetic and molecular characterization of Candystripe1 transposition events in sorghum

In sorghum, the Candystripe1 (Cs1) transposable element causes a variegated pericarp phenotype due to its excision activity from the yl (yellow seed1) locus. The Y1 is a transcription regulator which is required for the biosynthesis of red 3-deoxyflavonoid pigments. Somatic variability in the transposition behavior of Cs1 was observed via biochemical analysis of 3-deoxyflavonoids in the leaf tissues of the Y1-cs alleles. Using somatic excisions of Cs1 as a tool, we establish that the Cs1 is active in young seedlings and the y1 locus is also functional in these tissues. Several somatic and germinal excision events were characterized and sequence analysis of independent events predominantly showed 2-bp footprints. Further, with the goal of ur.derstanding the properties of Cs1 that would facilitate the development of a transposon tagging system in sorghum, germinal excisions of Cs1 from y1 were used as a marker. Transposition of Cs1 was followed by characterization of putative insertion events. Genetic linkage between mutant phenotypes and the cosegregating restriction fragments of Cs1 provided additional evidence that Cs1 is an active transposable element in sorghum.

Surgical Treatment of In-Growing Toenails Performed by Senior House Officers: Are They Good Enough?

Although the surgical treatment of in-growing toenails is a common procedure, the success of ingrown toenail surgery is extremely variable and recurrences frequently impair the quality of lift of those who have this condition. In most hospitals this procedure is commonly performed by junior surgical trainees who may have little experience. We proposed to find out if the success of this procedure is operator-dependent by comparing the results of standard of toenail surgery performed by basic surgical trainees (BSTs) in our hospitals with already published data. A retrospective analysis of nail bed ablation surgery performed by BSTs under local anaesthesia over a 15-month period in a district general hospital was conducted. 106 phenol ablations and 46 germinal matrix excisions were prformed. Symptomatic recurrence rates 12 months following the procedure were 5.7% for phenol ablation and 4.3% for germinal matrix excisions. Our results are comparable to published data, and we conclude that toenail ablation surgery can be just as successfully performed by junior surgeons after relatively little training in the procedure.

Tissue-culture enhanced transposition of the maize transposable element Dissociation in Brassica oleracea var. 'Italica'

To investigate the potential of heterologous transposons as a gene tagging system in broccoli ( Brassica oleracea var. Italica), we have introduced a Ds-based two-element transposon system. Ds has been cloned into a 35S-SPT excision-marker system, with transposition being driven by an independent 35S-transposase gene construct ( Tpase). In three successive selfed generations of plants there was no evidence of germinal-excision events. To overcome this apparent inability to produce B. oleracea plants with germinal excisions, we performed a novel tissue-culture technique to select for fully green shoots from seed with somatic-excision events. The results showed a very high efficiency of regeneration of fully green plants (up to 65%) and molecular analysis indicated that the plants genetically were like plants that contain a germinal-excision event. Further molecular analysis of these plants showed that 69% exhibited reinsertion of Ds back into the plant genome. Sequencing of donor-site footprints after Ds excision, revealed that there is an indication of more-severe deletions and rearrangements when higher concentrations of streptomycin are used in the tissue-culture selection process. Adapted versions of this regeneration technique have a high potential for providing germinal excision-like events in heterologous plants species which show low transposon activity. Alternatively, there is the potential to increase the proportion of 'germinal' plants in earlier generations of more-active plant species.

Trans-activation of the maize transposable element, Ds, in Brassica napus.

A two-component transposable element system consisting of a stabilized Activator ( Ac(st)) and a chimeric Dissociation ( Ds) element has been introduced into the genome of Brassica napus. Analyses performed on F2 progeny derived from crosses between Ac(st)- and Ds-bearing parents confirm that Ac transposase catalyzes the somatic excision of the Ds element in both embryonic and non-embryonic tissues of this important crop species. The data further reveal that the vast majority of plants containing both Ac(st) and Ds exhibit Ds excision. However, the level of excision is low and germinal Ds excision events are not observed. We estimate that germinal excision of Ds occurs at a frequency of < 0.2%. RT-PCR analysis of the Ac(st) transcript in somatically active seedlings reveals that introns III and IV are highly misprocessed. The pattern of transcript processing is very similar to that observed in germinally inactive but somatically active Arabidopsis seedlings. We suggest that Ds excision activity in B. napus is highly dependent on the efficiency of Ac(st) transcript processing.

Somatic and germinal excision activities of the Arabidopsis transposon Tag1 are controlled by distinct regulatory sequences within Tag1.

Various sequences within Tag1, the endogenous transposon of Arabidopsis, were examined to determine how Tag1 excision and expression are regulated. The 5' intron for the major 2.3-kb Tag1 transcript was found to be critical for the accumulation of Tag1 transcripts and for high rates of somatic excision. This was true for the autonomous element in cauliflower mosaic virus 35S-Tag1-beta-glucuronidase constructs and for a two-component system using the 35S promoter to produce Tag1 transposase and a beta-glucuronidase::dTag1 marker construct to score for excision. The 3' introns of Tag1, although not needed for high transposase expression in primary transgenic plants, were important for maintaining high levels of somatic excision and accumulation of the major but not the minor Tag1 transcripts in subsequent generations. With both 5' and 3' introns present, exchanging the 5' promoter region of Tag1 with the 35S promoter did not affect the timing of Tag1 excision significantly, but it did disrupt germinal excision. Removal of the 5' intron did not abolish germinal excision activity, however. These results indicate that somatic and germinal excision of Tag1 are differentially controlled, with the 5' promoter region being critical for germinal excision activity and the 5' intron playing an important role for somatic excision, possibly via intron-mediated enhancement.

Somatic and Germinal Excision Activities of the Arabidopsis Transposon Tag1 Are Controlled by Distinct Regulatory Sequences within Tag1

Somatic and Germinal Excision Activities of the Arabidopsis Transposon Tag1 Are Controlled by Distinct Regulatory Sequences within Tag1

Somatic and Germinal Excision Activities of the Arabidopsis Transposon Tag1 Are Controlled by Distinct Regulatory Sequences within Tag1

Somatic and Germinal Excision Activities of the Arabidopsis Transposon Tag1 Are Controlled by Distinct Regulatory Sequences within Tag1

A novel transposon tagging element for obtaining gain-of-function mutants based on a self-stabilizing Ac derivative.

A novel tagging system AcREH, designed for obtaining gain-of-function mutations, was prepared on the basis of a self-stabilizing Ac transposon derivative. The transposable element, DsAT, was constructed in a way that it can activate transcription of neighboring genes by two 35S promoters and/or by four tandem repeats of the enhancer fragment of this promoter. DsAT revealed somatic excision in the first generation of the tobacco transformants. The element exhibited germinal excision to the next generation, as demonstrated by PCR and Southern hybridization analysis. In spite of the structure of the element, which may inhibit the expression of the transposase gene, the frequency of germinal excision was comparable to or higher than those so far reported, suggesting the applicability of the element for gene tagging.

Germinal Excision and Reinsertion Frequencies of the Mobile Element Ds Transposed from Two Unlinked T-DNA Loci in Tomato

Acceptor sites of unlinked transposed Ds element from two T-DNA loci in tomato were mapped. Experimental data obtained from TC1 progeny testing were employed for estimation of germinal excision frequency (GEF) of Ds element and frequency of its reinsertion (FR). The donor T-DNAs 1481J and 1601D, containing a 35S:NPT transformation marker, a 35S:BAR or nos:BAR excision marker conferring phosphinothricine resistance and a Ds element in the 5' untranslated leader of the nos (or 35S): BAR gene, were located on chromosome 7 and 8, respectively. Ds transposition was induced by 105121 T-DNA carrying stabilized Ac (sAc) which provides a source of transposase and 2':GUS marker conferring β-glucuronidase activity. Tomato plants harbouring the Ds in 1481J or 1601D locus and sAc were crossed and F1D, were crossed individually as seed parents to wild-type plants to generate TC1 progenies. TC1 seed was germinated on phosphinothricine (Basta)-containing medium, and individual seedlings carrying a transposed Ds and lacking sAc were identified by PCR (to detect the Ds) on phosphinothricine resistant individuals that lacked β-glucuronidase activity. From segregation ratio in TC1 the germinal excision and reinsertion frequencies of the Ds element were estimated for individual F1 plants. A total of 14560 TC1 seedlings of 1481J and 16195 TC1 seedlings of 1601D was analyzed. We observed high variation between individual plants as regards both GEF and FR despite of donor locus (1481J or 1601D), however, the average germinal excision frequencies as well as average frequencies of reinsertion were very similar for both donor loci: GEF1481J = 24 %, GEF1501D = 25 %, FR1481J = 42 %, FR1601D = 46 %.

Frequency and pattern of transposition of the maize transposable element Ds in transgenic rice plants.

Two kinds of T-DNA constructs, I-RS/dAc-I-RS and Hm(R)Ds, carrying a non-autonomous transposable element of Ac of maize were introduced into rice plants by Agrobacterium-mediated gene transfer. Six transgenic rice plants identified as containing a single copy of the element were crossed with two transgenic rice plants carrying a gene for Ac transposase under the control of the cauliflower mosaic virus 35S promoter. In F2 progenies, excision of the element was detected by PCR analysis and re-integration of the element was investigated by Southern blot analysis. The frequency of the excision of the element was found to vary from 0 to 70% depending on the crossing combination. The frequency of the number of individual transposition events out of the total number of F2 plants with germinal excision was 44% in one crossing combination and 38% in the other. In the most efficient case, 10 plants with independent transposition were obtained out of the 49 F2 plants tested. Linkage analysis of the empty donor site and the transposed Ds-insertion site in F3 plants demonstrated that one of five Ds-insertion sites was not linked to the empty donor site. The transgenic rice obtained in this study can be used for functional genomics of rice.

Psl: a novel Spm-like transposable element from Petunia hybrida.

The identification of a spontaneous mutable Hf1 allele in Petunia hybrida provided an opportunity to isolate and characterize a novel transposable element. This 9.9 kb element has features in common with members of the Spm family, such as homologous terminal inverted repeats and a 3 bp target site direct duplication within the Hf1 gene. The element is named Petunia Spm-like (Psl). The footprints left by excising elements have been isolated from several germinal revertants and sequence analysis shows similarities to those left by other Spm family members. Southern analysis shows that the transposon is present at low copy number in the genome of different inbred lines and species of Petunia. The germinal excision frequency of Psl was 21-33% in outcross populations. The element appears to be very mobile somatically in the inbred line V26, with 38% of plants from an inbred population showing new Psl-hybridizing bands by Southern analysis. The high somatic and germinal excision frequency demonstrated by Psl suggests that this element may have utility for gene tagging in petunia.

Insertions of a novel class of transposable elements with a strong target site preference at the r locus of maize.

The r locus of maize regulates anthocyanin synthesis in various tissues of maize through the production of helix-loop-helix DNA binding proteins capable of inducing expression of structural genes in the anthocyanin biosynthetic pathway. The complex r variant, R-r: standard (R.r), undergoes frequent mutation through a variety of mechanisms including displaced synapsis and crossing over, and intrachromosomal recombination. Here we report a new mechanism for mutation at the R-r complex: insertion of a novel family of transposable elements. Because the elements were first identified in the R-p gene of the R-r complex, they have been named P instability Factor (PIF). Two different PIF elements were cloned and found to have identical sequences at their termini but divergent internal sequences. In addition, the PIF elements showed a marked specificity of insertion sites. Six out of seven PIF-containing derivatives examined had an element inserted at an identical location. Two different members of the PIF element family were identified at this position. The seventh PIF-containing derivative examined had the element inserted at a distinct position within r. Even at this location, however, the element inserted into a conserved target sequence. The timing of PIF excision is unusual. Germinal excision rates can range up to several percent of progeny. Yet somatic sectors are rare, even in lines exhibiting high germinal reversion rates.

Increased Ac excision (iae): Arabidopsis thaliana mutations affecting Ac transposition

The maize transposable element Ac is highly active in the heterologous hosts tobacco and tomato, but shows very much reduced levels of activity in Arabidopsis. A mutagenesis experiment was undertaken with the aim of identifying Arabidopsis host factors responsible for the observed low levels of Ac activity. Seed from a line carrying a single copy of the Ac element inserted into the streptomycin phosphotransferase (SPT) reporter fusion, and which displayed typically low levels of Ac activity, were mutagenized using gamma rays. Nineteen mutants displaying high levels of somatic Ac activity, as judged by their highly variegated phenotypes, were isolated after screening the M2 generation on streptomycin-containing medium. The mutations fall into two complementation groups, iae1 and iae2, are unlinked to the SPT::Ac locus and segregate in a Mendelian fashion. The iae1 mutation is recessive and the iae2 mutation is semi-dominant. The iae1 and iae2 mutants show 550- and 70-fold increases, respectively, in the average number of Ac excision sectors per cotyledon. The IAE1 locus maps to chromosome 2, whereas the SPT::Ac reporter maps to chromosome 3. A molecular study of Ac activity in the iae1 mutant confirmed the very high levels of Ac excision predicted using the phenotypic assay, but revealed only low levels of Ac re-insertion. Analyses of germinal transposition in the iae1 mutant demonstrated an average germinal excision frequency of 3% and a frequency of independent Ac re-insertions following germinal excision of 22%. The iae mutants represents a possible means of improving the efficiency of Ac/Ds transposon tagging systems in Arabidopsis, and will enable the dissection of host involvement in Ac transposition and the mechanisms employed for controlling transposable element activity.

The maize transposable element Ac is mobile in the legume Lotus japonicus.

To evaluate the prospects for transposon mutagenesis in the autogamous diploid legume Lotus japonicus, the behaviour of the maize transposable element Ac was analysed in the progeny of 38 independent transgenic plants. The conditions for monitoring donor site excision using histochemical localization of beta-glucuronidase activity or the alternative spectinomycin resistance assay were established, and used to follow Ac mobility through two generations. Somatic excision was monitored as variegated cotyledons in the T2 generation and germinal excision events were scored in segregating T3 families as complete beta-glucuronidase-mediated staining of cotyledons or as a fully green spectinomycin-resistant phenotype. Using these assays an average germinal excision frequency of 12% was estimated in the T3 offspring from variegated plants. The fidelity of the excision assays was ascertained by comparing the frequency of germinal excision to the frequency of Ac reinsertion at new positions of the genome. Transposition of Ac in 42% of the plants and detection of the characteristic Ac insertion/excision footprints suggests that insertion mutagenesis with the autonomous maize Activator element is feasible in Lotus japonicus. Parameters influencing Ac behaviour, such as dosage, position effects and modification of the element itself, were also investigated comparing homozygous and hemizygous plants from the same family and by analysing different transformants.

Germinal transpositions of the maize element Dissociation from T-DNA loci in tomato.

We have analyzed the pattern of germinal transpositions of artificial Dissociation (Ds) transposons in tomato. T-DNA constructs carrying Ds were transformed into tomato, and the elements were trans-activated by crossing to lines transformed with a stabilized Activator (sAc) that expressed the transposase gene. The sAc T-DNA carried a GUS gene to monitor its segregation. The Ds elements were inserted in a marker gene so that excision from the T-DNA could be monitored. The Ds elements also carried a genetic marker that was intended to be used for reinsertion selection of the elements after excision. Unfortunately, this gene was irreversibly inactivated on crossing to sAc. Germinal excision frequencies of Ds averaged 15-40%, but there was large variation between and within plants. Southern hybridization analysis of stable transposed Ds elements indicated that although unique transpositions predominate, early transposition events can lead to large clonal sectors in the germline of developing plants and to sibling offspring carrying the same transposition event. Multiple germinal transpositions from three different loci were examined for uniqueness, and 15 different transpositions were identified from each of three T-DNA loci that carried a single independent Ds. These were mapped relative to the donor T-DNA loci, and for each locus 70-80% of the transposed elements were closely linked to the donor site.