Molecular analysis of rice plants harboring an Ac/Ds transposable element-mediated gene trapping system

In rice, limited efforts have been made to identify genes by the use of insertional mutagens, especially heterologous transposons such as the maize Ac/Ds. We constructed Ac and gene trap Ds vectors and introduced them into the rice genome by Agrobacterium-mediated transformation. In this report, rice plants that contained single and simple insertions of T-DNA were analysed in order to evaluate the gene-tagging efficiency. The 3' end of Ds was examined for putative splicing donor sites. As observed in maize, three splice donor sites were identified at the 3' end of the Ds in rice. Nearly 80% of Ds elements were excised from the original T-DNA sites, when Ac cDNA was expressed under a CaMV 35S promoter. Repetitive ratoon culturing was performed to induce new transpositions of Ds in new plants derived from cuttings. About 30% of the plants carried at least one Ds which underwent secondary transposition in the later cultures. Eight per cent of transposed Ds elements expressed GUS in various tissues of rice panicles. With cloned DNA adjacent to Ds, the genomic complexities of the insertion sites were examined by Southern hybridization. Half of the Ds insertion sites showed simple hybridization patterns which could be easily utilized to locate the Ds. Our data demonstrate that the Ac/Ds-mediated gene trap system could prove an excellent tool for the analysis of functions of genes in rice. We discuss genetic strategies that could be employed in a large scale mutagenesis using a heterologous Ac/Ds family in rice.     

Plant transposable elements and their application in genetics and biotechnology

Data concerning plant transposable elements and their contribution to plant genome evolution are reviewed. Much attention is focused on utilization of transgenic plants as heterologous hosts of transposons for investigation of transposition mechanisms and gene cloning. Probable ways of the use of plant transposons as genetic tools in biotechnology are discussed.     

McClintock's controlling elements: the full story

The chapter describes the early part of Barbara McClintock's work on DNA transposons in maize, in which she uncovered the Ac-Ds family of mobile "controlling elements". As a basis to understanding the work there is a preamble on the reproductive biology of maize, which explains the particular qualities of the maize plant as an experimental organism for genetics. An account is then given of the cytology of the system that was used to generate intact chromosomes having "sticky" (broken) ends, which is the starting point of the story. Cytogenetic aspects of the chromatid and chromosome breakage-fusion-bridge cycles, deriving from breakage, are then described; this leads on to a discussion about the way in which variegation phenotypes of the maize kernels can be "read" in terms of chromosome breakage. The "genetic earthquake" event of 1944, triggered by introducing broken chromosomes into a zygote from both parents, is then described; and the sequence of events leading to the discovery of Ds and Ac is traced. Finding mobility of Ds from one chromosomal location to another was pure serendipity, and the transposition showed itself while experiments were being undertaken to accurately map Ds. A similar chance observation revealed transposition of Ac as well, and then the relationship between the two elements was elucidated in terms of their autonomous and non-autonomous nature. The chapter concludes with a brief reference to the molecular cloning of Ac and Ds.     

Transposon Ac/Ds-induced chromosomal rearrangements at the rice OsRLG5 locus

Previous studies have shown that pairs of closely-linked Ac/Ds transposable elements can induce various chromosomal rearrangements in plant genomes. To study chromosomal rearrangements in rice, we isolated a line (OsRLG5-161) that contains two inversely-oriented Ds insertions in OsRLG5 (Oryza sativa Receptor like kinase Gene 5). Among approximately 300 plants regenerated from OsRLG5-161 heterozygous seeds, 107 contained rearrangements including deletions, duplications and inversions of various sizes. Most rearrangements were induced by previously identified alternative transposition mechanism. Furthermore, we also detected a new class of rearrangements that contain juxtaposed inversions and deletions on the same chromosome. We propose that these novel alleles were generated by a previously unreported type of alternative transposition reactions involving the 5' and 3' termini of two inversely-oriented Ds elements located on the same chromatid. Finally, 11% of rearrangements contained inversions resulting from homologous recombination between the two inverted Ds elements in OsRLG5-161. The high frequency inheritance and great variety of rearrangements obtained suggests that the rice regeneration system results in a burst of transposition activity and a relaxation of the controls which normally limit the transposition competence of individual Ds termini. Together, these results demonstrate a greatly enlarged potential of the Ac/Ds system for plant chromosome engineering.     

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.     

A transgenic system for generation of transposon Ac/Ds-induced chromosome rearrangements in rice

The maize Activator (Ac)/Dissociation (Ds) transposable element system has been used in a variety of plants for insertional mutagenesis. Ac/Ds elements can also generate genome rearrangements via alternative transposition reactions which involve the termini of closely linked transposons. Here, we introduced a transgene containing reverse-oriented Ac/Ds termini together with an Ac transposase gene into rice (Oryza sativa ssp. japonica cv. Nipponbare). Among the transgenic progeny, we identified and characterized 25 independent genome rearrangements at three different chromosomal loci. The rearrangements include chromosomal deletions and inversions and one translocation. Most of the deletions occurred within the T-DNA region, but two cases showed the loss of 72 kilobase pairs (kb) and 79 kb of rice genomic DNA flanking the transgene. In addition to deletions, we obtained chromosomal inversions ranging in size from less than 10 kb (within the transgene DNA) to over 1 million base pairs (Mb). For 11 inversions, we cloned and sequenced both inversion breakpoints; in all 11 cases, the inversion junctions contained the typical 8 base pairs (bp) Ac/Ds target site duplications, confirming their origin as transposition products. Together, our results indicate that alternative Ac/Ds transposition can be an efficient tool for functional genomics and chromosomal manipulation in rice.     

Characterization and mapping of Ds—GUS-T-DNA lines for targeted insertional mutagenesis

The transposition patterns of the Ds —GUS transposon T-DNA in 23 independent single-copy lines have been characterized and the map positions of 10 of them on three of the five Arabidopsis chromosomes are reported. Using overexpressed Activator ( Ac ) elements as a transposase source, it was found that the primary determinant of transposition frequency is the insertion site of the Ac -T-DNA. Neither the structure of the transposon T-DNA nor, in most cases, its insertion site have a significant effect on transposition frequency. Both the frequency and timing of transposition are influenced by the parent through which the transposon and transposase T-DNAs are transmitted. Overall, nearly 75% of plants in which excision has occurred bear a reinserted element and very short-range transpositions predominate, underlining the advantage of using mapped transposons for insertional mutagenesis.     

State II dissociation element formation following activator excision in maize

Active Activator (Ac) elements undergo mutations to become nonautonomous Dissociation (Ds) elements at a low frequency. To understand the mechanism of Ds formation, we have developed high-throughput genetic and molecular screens to identify these rare Ds derivatives generated from any Ac insertion in the maize genome. Using these methods we have identified 15 new Ds elements derived from Ac insertions at eight different loci. Approximately half of the Ds elements contain filler DNA inserted at the deletion junction that is derived from sequences within or adjacent to Ac. In contrast to previous reports, several of these Ds elements lack direct repeats flanking the deletion junctions and filler DNA in the donor Ac. To accommodate our findings and those of others, we propose a model of slip mispairing during error-prone repair synthesis to explain the formation of state II Ds elements in maize. We discuss the use of these lines and molecular techniques developed here to capture somatic Ds transposition events in two-component Ac/Ds tagging programs in maize.     

Circularized Ac/Ds Transposons: Formation, Structure and Fate

The maize Ac/Ds transposable elements are thought to transpose via a cut-and-paste mechanism, but the intermediates formed during transposition are still unknown. In this work we present evidence that circular Ac molecules are formed in plants containing actively transposing elements. In these circles, transposon ends are joined head-to-head. The sequence at the ends' junction is variable, containing small deletions or insertions. Circles containing deleted Ac ends are probably unable to successfully reintegrate. To test the ability of circles with intact transposon ends to integrate into the genome, an artificial Ds circle was constructed by cloning the joined ends of Ac into a plasmid carrying a plant selectable marker. When such a circular Ds was introduced into tobacco protoplasts in the presence of Ac-transposase, no efficient transposase-mediated integration was observed. Although a circular transposition intermediate cannot be ruled out, the findings of circles with deleted transposon ends and the absence of transposase-mediated integration of the circular Ds suggest that some of the joined-ends-carrying elements are not transposition intermediates, but rather abortive excision products. The formation of Ac circles might account for the previously described phenomenon of Ac-loss. The origin of Ac circles and the implications for models of Ac transposition are discussed.     

Rapid, large-scale generation of Ds transposant lines and analysis of the Ds insertion sites in rice

Rapid, large-scale generation of a Ds transposant population was achieved using a regeneration procedure involving tissue culture of seed-derived calli carrying Ac and inactive Ds elements. In the F(2) progeny from genetic crosses between the same Ds and Ac starter lines, most of the crosses produced an independent germinal transposition frequency of 10-20%. Also, many Ds elements underwent immobilization even though Ac was expressed. By comparison, in a callus-derived regenerated population, over 70% of plants carried independent Ds insertions, indicating transposition early in callus formation. In the remaining population, the majority of plants carried only Ac. Most of the new Ds insertions were stably transmitted to a subsequent generation. An exceptionally high proportion of independent transposants in the regenerated population means that selection markers for transposed Ds and continual monitoring of Ac/Ds activities may not necessarily be required. By analyzing 1297 Ds-flanking DNA sequences, a genetic map of 1072 Ds insertion sites was developed. The map showed that Ds elements were transposed onto all of the rice chromosomes, with preference not only near donor sites (36%) but also on certain physically unlinked arms. Populations from both genetic crossing and tissue culture showed the same distribution patterns of Ds insertion sites. The information of these mapped Ds insertion sites was deposited in GenBank. Among them, 55% of Ds elements were on predicted open-reading frame (ORF) regions. Thus, we propose an optimal strategy for the rapid generation of a large population of Ds transposants in rice.     

Comparative analysis of non-random DNA repair following Ac transposon excision in maize and Arabidopsis

Ac/Ds transposable elements often leave short DNA rearrangements, or 'footprints,' at the sites where they excise. Previous studies at the maize waxy ( wx ) gene suggest that the DNA repair that forms transposon footprints is not random. Each excision site consistently displays a different, predominant repair product suggesting flanking DNA may influence footprint formation. We have expanded these studies to show that predominant end-joining products also form in association with Ac/Ds excision in Arabidopsis and that chromosomal location of the Ac -containing construct does not appear to influence this repair. The predominant repair product is identical in both maize and Arabidopsis for Ac elements with the same adjacent DNA sequences. However, a broader range of minor footprint types is observed in Arabidopsis , including footprints that are rare in maize, suggesting potential differences in the host proteins involved in either transposition, repair or both. The data also suggest that the sequences influencing footprint formation are within 39 bp 5' and 18 bp 3' of the transposon. These studies demonstrate that transgenic Ac/Ds -containing plants will be useful tools in dissecting plant DNA repair processes.     

Phylogenetic and functional characterization of the hAT transposon superfamily

Transposons are found in virtually all organisms and play fundamental roles in genome evolution. They can also acquire new functions in the host organism and some have been developed as incisive genetic tools for transformation and mutagenesis. The hAT transposon superfamily contains members from the plant and animal kingdoms, some of which are active when introduced into new host organisms. We have identified two new active hAT transposons, AeBuster1, from the mosquito Aedes aegypti and TcBuster from the red flour beetle Tribolium castaneum. Activity of both transposons is illustrated by excision and transposition assays performed in Drosophila melanogaster and Ae. aegypti and by in vitro strand transfer assays. These two active insect transposons are more closely related to the Buster sequences identified in humans than they are to the previously identified active hAT transposons, Ac, Tam3, Tol2, hobo, and Hermes. We therefore reexamined the structural and functional relationships of hAT and hAT-like transposase sequences extracted from genome databases and found that the hAT superfamily is divided into at least two families. This division is supported by a difference in target-site selections generated by active transposons of each family. We name these families the Ac and Buster families after the first identified transposon or transposon-like sequence in each. We find that the recently discovered SPIN transposons of mammals are located within the family of Buster elements.     

Development of a simple transient assay for Ac/Ds activity in cells of intact barley tissue

The development of a barley ( Hordeurn vulgare L.) transformation system made it possible to consider the use of maize Activator/Dissociation ( Ac/Ds ) transposable elements for gene tagging in transgenic barley plants. However, barley transformation is time-consuming, and therefore a simple transient assay for Ac/Ds activity in intact barley tissues was developed to test the components of a proposed gene tagging system, prior to their stable introduction into plants. In this assay, barley scutellar tissue is co-transformed with constructs containing the maize Ac transposase gene and an Escherichia coli uid A reporter gene ( Gus ), the expression of which is interrupted by a maize Ds element. In transformed barley scutellar cells, Ac transposase-mediated excision of the Ds element generates a functional Gus gene, leading to histochemically detectable GUS activity. Characterization of the excision products showed that they had a pattern of nucleotide deletions and/or transversions similar to that found in maize and other heterologous plant systems. In addition, although contrary to the situation observed in heterologous dicot systems, efficient Ds excision in barley, a heterologous monocot system, appears to be inversely associated with Ac copy number, a finding similar to the Ac dosage effects observed in maize. The transient assay was used to demonstrate functional transposase activity in barley callus lines stably transformed with an Ac transposase gene.     

Regulation of activator/dissociation transposition by replication and DNA methylation

In maize the transposable elements Activator/Dissociation (Ac/Ds) transpose shortly after replication from one of the two resulting chromatids ("chromatid selectivity"). A model has been suggested that explains this phenomenon as a consequence of different affinity for Ac transposase binding to holo-, hemi-, and unmethylated transposon ends. Here we demonstrate that in petunia cells a holomethylated Ds is unable to excise from a nonreplicating vector and that replication restores excision. A Ds element hemi-methylated on one DNA strand transposes in the absence of replication, whereas hemi-methylation of the complementary strand causes a >6.3-fold inhibition of Ds excision. Consistently in the active hemi-methylated state, the Ds ends have a high binding affinity for the transposase, whereas binding to inactive ends is strongly reduced. These results provide strong evidence for the above-mentioned model. Moreover, in the absence of DNA methylation, replication enhances Ds transposition in petunia protoplasts >8-fold and promotes formation of a predominant excision footprint. Accordingly, replication also has a methylation-independent regulatory effect on transposition.     

Molecular evidence that chromosome breakage by Ds elements is caused by aberrant transposition.

The transposable Dissociation (Ds) element of maize was first discovered as a site of high-frequency chromosome breakage. Because both Ds-mediated breakage and transposition require the presence of the Activator (Ac) element, it has been suggested that chromosome breakage may be the outcome of an aberrant transposition event. This idea is consistent with the finding that only complex structures containing multiple Ds or Ac and Ds elements have been correlated with chromosome breakage. In this report, we describe two chromosome-breaking maize alleles that contain pairs of closely linked but separate Ds elements inserted at the Waxy locus. A polymerase chain reaction assay was utilized to isolate intermediates in the breakage process. The DNA sequence of these intermediates reveals deletions and base pair changes consistent with transposon footprints that may represent the junctions between fused sister chromatids. These results provide direct molecular evidence that chromosome breakage is the result of aberrant transposition events.     

Tam3 produces a suppressible allele of the DAG locus of Antirrhinum majus similar to Mu-suppressible alleles of maize

Tam3 from Antirrhinum majus belongs to the Ac/Ds family of transposable elements. An allele of the DAG locus of Antirrhinum ( dag ::Tam3), which is required for chloroplast development and leaf palisade differentiation, has been generated by Tam3 insertion into the untranslated leader sequence of the gene. This allele gives rise to a cold-sensitive phenotype, where mutant tissue containing wild-type revertant somatic sectors is observed in the leaves of plants grown at 15°C, while leaves of plants grown at 25°C appear near wild-type. The temperature sensitivity of dag ::Tam3 results from expression of the DAG locus responding to the activity of the transposable element, the transposition of which is very sensitive to growing temperature. Genetic suppression of Tam3 transposition, using the STABILISER locus, also results in suppression of the dag mutant phenotype. dag ::Tam3 represents a Tam3-suppressible allele similar to those described for Mu transposons in maize. Suppression of the dag mutant phenotype in response to element inactivation appears to result from use of an alternative promoter at the 3' end of the Tam3 element. The production of suppressible alleles by an Ac-like element is discussed in relation to the mutagenic potential of plant transposons in producing complex genetic diversity.