Germinal Excision Events 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.

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.

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.

Factors affecting the excision frequency of the maize transposable element Ds in Arabidopsis thaliana

A two-element transposon system based on the maize elements Ac and Ds is currently being used for insertional mutagenesis in Arabidopsis. With the aim of making this system as efficient as possible we have continued to analyse several parameters which affect Ds activity in Arabidopsis. The influence of genomic position on Ds excision has been analysed in five lines carrying Ds integrated in different genomic locations. Differences in both somatic and germinal excision were observed between the different lines. The relationship between somatic and germinal excision, the timing of excision events and environmental influences on transposition frequency have been investigated. The effect of varying dosage of the different elements was also analysed. A strong positive dosage effect was observed for the transposase source, but not for the Ds element. Analysis of germinal excision events showed that the majority of them occurred very late in the development of the plant, resulting in the majority of Ds transpositions being independent events.

Effects of gene dosage and sequence modification on the frequency and timing of transposition of the maize element Activator (Ac) in tobacco

The effect of Ac copy number on the frequency and timing of germinal transposition in tobacco was investigated using the streptomycin phosphotransferase gene (SPT) as an excision marker. The activity of one and two copies of the element was compared by selecting heterozygous and homozygous progeny of transformants carrying single SPT::Ac inserts. It was observed that increasing gene copy not only increases the transposition frequency, but also occasionally alters the timing of transposition such that earlier events are obtained. The result is that some homozygous plants generate multiple streptomycin resistant progeny carrying the same transposed Ac (trAc) element. We have also investigated the effect of modification of the sequence in the region around 82 bp downstream of the polyadenylation site and 177 bp from the 3' end of the element on germinal excision frequencies. Alteration of three bases to create a Bgl II site at this location caused a minor decrease in germinal excision events, but insertion of four bases to create a Cla I site caused a 10-fold decrease in the transposition activity of the Ac element.

Germinal and somatic activity of the maize element Activator (Ac) in Arabidopsis.

We have investigated the germinal and somatic activity of the maize Activator (Ac) element in Arabidopsis with the objective of developing an efficient transposon-based system for gene isolation in that plant. Transposition activity was assayed with a chimeric marker that consists of the cauliflower mosaic virus 35S promoter and a bacterial streptomycin phosphotransferase gene (SPT). Somatic activity was detected in seedlings germinated on plates containing streptomycin as green-resistant sectors against a background of white-sensitive cells. Germinal excisions resulted in fully green seedlings. The transposition frequency was extremely low when a single copy of the transposon was present, but appeared to increase with an increase in Ac copy number. Plants that were selected as variegated produced an increased number of green progeny. The methylation state of the Ac elements in lines with either low or high levels of excision was assessed by restriction analysis. No difference was found between these lines, indicating that the degree of methylation did not contribute to the level of Ac activity. Germinal excision events were analyzed molecularly and shown to carry reinserted transposons in about 50% of the cases. In several instances, streptomycin-resistant siblings carried the same transposed Ac element, indicating that excision had occurred prior to meiosis in the parent. We discuss parameters that need to be considered to optimize the use of Ac as a transposon tag in Arabidopsis.

Promoter fusions to the Activator transposase gene cause distinct patterns of Dissociation excision in tobacco cotyledons.

To explore the effects of altering the level of Activator (Ac) transposase (TPase) expression, a series of plasmids was constructed in which heterologous promoters were fused to the TPase gene. Promoters for the cauliflower mosaic virus (CaMV) 35S transcript and the octopine synthase (ocs) and nopaline synthase (nos) genes were tested. These fusions, and constructs expressing TPase from the wild-type Ac promoter, were introduced into tobacco, and their activity was monitored by crossing to a line carrying Dissociation (Ds) in a streptomycin phosphotransferase gene (Ds::SPT). The SPT marker provides a record of somatic excisions of Ds that occur during embryo development. The patterns of somatic variegation that resulted from transactivation by each fusion were distinct and strikingly different from the pattern triggered by the wild-type Ac constructs. Unlike wild-type Ac, which caused transposition throughout embryo development, each fusion gave rise to sectors of discrete size. Sectors triggered by the CaMV 35S fusion were largest, ocs sectors were intermediate, and nos were smallest. These patterns appear to indicate differential timing of the activation of these promoters during embryogeny. Measurement of transcript abundance for each transformant indicated that the CaMV 35S-transformed plants accumulated approximately 1000-fold more TPase mRNA than plants containing wild-type Ac, whereas ocs- and nos-transformed lines accumulated about 100-fold and 20-fold higher levels, respectively. Measurements of germinal excision frequencies driven by the chimeric TPase fusions, however, indicated that increasing transcription does not necessarily result in an increase in germinal excision. These measurements showed that the ocs and nos fusions have very low rates of germinal excision. Only the CaMV 35S fusion transformants were found to have higher rates than the Ac constructs, although significant pod-to-pod variation was observed. Gel blot analysis of DNA from progeny carrying germinal excision events resulting from the CaMV 35S fusion showed that excision is associated with reinsertion and that siblings sometimes carry the same transposition events. These findings suggest that in tobacco there is no direct proportionality between TPase expression and Ac-Ds transposition activity. This possibility has important implications for understanding the regulation of Ac transposition and for designing efficient gene tagging systems.

Molecular analysis of the maize wx-B3 allele indicates that precise excision of the transposable Ac element is rare.

The somatic and germinal behavior of the maize wx-B3 mutation indicates that this Ac allele rarely reverts. Endosperms containing wx-B3 display tiny and infrequent Wx revertant sectors while no significant reversion is detected when wx-B3 pollen is stained with I/KI. Previous studies of other transposable element alleles that revert infrequently have implicated low levels of element excision. Unlike these other alleles, the wx-B3 Ac element is indistinguishable from fully active Ac elements with respect to its structure, and its ability to transpose from the Wx gene or to trans-activate a Ds element. Characterization of somatic and germinal excision events lead us to conclude that excision of the wx-B3 Ac element almost always produces null alleles. Furthermore, the excellent correlation between the position of the wx-B3 mutation on the physical and genetic maps indicates that the Ac insertion is the only lesion of wx-B3. As a result, precise excision of this Ac should restore Wx function. The fact that revertant sectors and pollen grains are rare indicates that precise excision of Ac is also rare. The finding that the wx-B3 reversion frequency is comparable whether wx-B3 is hemizygous or over a wx allele with a wild-type insertion site illustrates a fundamental difference between the excision mechanisms of Ac and Drosophila P elements.

The maize autonomous element Activator (Ac) shows a minimal germinal excision frequency of 0.2%–0.5% in transgenic Arabidopsis thaliana plants

The autonomous mobile element Activator from Zea mays was introduced into Arabidopsis thaliana via Agrobacterium-mediated gene transfer. The use of a chimaeric construct, where the Ac element is located in the leader of the neomycin phosphotransferase (NPT II) gene, enabled the excision of Ac to be monitored by assaying for the reconstitution of NPT II gene activity. Using this approach, the transpositional activity of AC was initially studied in primary transformants. About 50% of the regenerating Ac transformants showed evidence for excision of the element. Reintegration of Ac was confirmed by Southern blot analysis. Transposition events are transmitted to the F1 generation with a minimal frequency of 0.3%. In a few exceptional cases they are detected in a high proportion of the F1 generation. Seedlings from the F2 and F3 generations were assayed for the rate of germinal excisions by scoring for kanamycin resistance. The minimal frequency of germinal excision events amounts to 0.2%–0.5% and hence allows the use of the Ac element for gene tagging purposes in A. thaliana.