Characterization of theAc/Ds behaviour in transgenic tomato plants using plasmid rescue

We describe the use of plasmid rescue to facilitate studies on the behaviour ofDs andAc elements in transgenic tomato plants. The rescue ofDs elements relies on the presence of a plasmid origin of replication and a marker gene selective inEscherichia coli within the element. The position within the genome of modifiedDs elements, rescued both before and after transposition, is assigned to the RFLP map of tomato. Alternatively to the rescue ofDs elements equipped with plasmid sequences,Ac elements are rescued by virtue of plasmid sequences flanking the element. In this way, the consequences of the presence of an (active)Ac element on the DNA structure at the original site can be studied in detail. Analysis of a library ofAc elements, rescued from the genome of a primary transformant, shows thatAc elements are, infrequently, involved in the formation of deletions. In one case the deletion refers to a 174 bp genomic DNA sequence immediately flankingAc. In another case, a 1878 bp internalAc sequence is deleted.     

Mapping Ds insertions in barley using a sequence-based approach

A transposon tagging system, based upon maize Ac/Ds elements, was developed in barley (Hordeum vulgare subsp. vulgare). The long-term objective of this project is to identify a set of lines with Ds insertions dispersed throughout the genome as a comprehensive tool for gene discovery and reverse genetics. AcTPase and Ds-bar elements were introduced into immature embryos of Golden Promise by biolistic transformation. Subsequent transposition and segregation of Ds away from AcTPase and the original site of integration resulted in new lines, each containing a stabilized Ds element in a new location. The sequence of the genomic DNA flanking the Ds elements was obtained by inverse PCR and TAIL-PCR. Using a sequence-based mapping strategy, we determined the genome locations of the Ds insertions in 19 independent lines using primarily restriction digest-based assays of PCR-amplified single nucleotide polymorphisms and PCR-based assays of insertions or deletions.The proncipal strategy was to identify and map sequence polymorphisms in the regions corresponding to the flanking DNA using the Oregon Wolfe Barley mapping population. The mapping results obtained by the sequence-based approach were confirmed by RFLP analyses in four of the lines. In addition, cloned DNA sequences corresponding to the flanking DNA were used to assign map locations to Morex-derived genomic BAC library inserts, thus integrating genetic and physical maps of barley. BLAST search results indicate that the majority of the transposed Ds elements are found within predicted or known coding sequences. Transposon tagging in barley using Ac/Ds thus promises to provide a useful tool for studies on the functional genomics of the Triticeae.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by M.-A. GrandbastienThe first three authors contributed equally to this work     

A maize cryptic Ac-homologous sequence derived from an Activator transposable element does not transpose.

Summary Sequences sharing homology to the transposable element Activator (Ac) are prevalent in the maize genome. A cryptic Ac-like DNA, cAc-11, was isolated from the maize inbred line 4Co63 and sequenced. Cryptic Ac-11 has over 90% homology to known Ac sequences and contains an 11 by inverted terminal repeat flanked by an 8 by target site duplication, which are characteristics of Ac and Dissociation (Ds) transposable elements. Unlike the active Ac element, which encodes a transposase, the corresponding sequence in cAc-11 has no significant open reading frame. A 44 by tandem repeat was found at one end of cAc-11, which might be a result of aberrant transposition. The sequence data suggest that cAc-11 may represent a remnant of an Ac or a Ds element. Sequences homologous to cAc-11 can be detected in many maize inbred lines. In contrast to canonical Ac elements, cAc-11 DNA in the maize genome is hypermethylated and does not transpose even in the presence of an active Ac element.     

Ds transposition mediated by transient transposase expression in Heiracium aurantiacum

The feasibility of using transient transposase expression to mobilize Ds elements for gene tagging in Hieracium aurantiacum was evaluated. A T-DNA construct carrying the Ac transposase gene and either a visible marker gene (uidA) or the conditionally-lethal marker gene (codA) was transferred to H. aurantiacum leaf discs (previously transformed with a Ds element) by co-cultivation with Agrobacterium tumefaciens. Shoots were regenerated directly from the co-cultivated leaf discs under selection for antibiotic resistance resulting from Ds excision. Most regenerants carried unique transposition events. Of 84 regenerated plants, twenty one (25%) did not express the marker gene and the DNA coding sequence of the transposase could not be detected in seven (8.3%). Potential advantages of this method over conventional gene-tagging methods are: rapid recovery of individual transposition events; regenerated plants are isogenic; and the transient nature of transposase expression should facilitate the stabilisation of the transposed element.     

Development of Ac and Ds transposon tagging lines for gene isolation in diploid potato

For the development of an efficient transposon tagging strategy it is important to generate populations of plants containing unique independent transposon insertions that will mutate genes of interest. To develop such a transposon system in diploid potato the behavior of the autonomous maize transposable element Ac and the mobile Ds element was studied. A GBSS (Waxy) excision assay developed for Ac was used to monitor excision in somatic starch-forming tissue like tubers and pollen. Excision of Ac results in production of amylose starch that stains blue with iodine. The frequency and patterns of blue staining starch granules on tuber slices enabled the identification of transformants with different Ac activity. After excision the GBSS complementation was usually not complete, probably due to the segment of DNA flanking Ac that is left behind in the GBSS gene. Molecular and phenotypic analyses of 40 primary transformants classified into 4 phenotypic classes revealed reproducible patterns. A very high percentage (32.5%) of the primary transformants clearly showed early excision in the first transformed cell as displayed both by the analysis of the GBSS excision marker gene as well as DNA blot analyses. Genotypes useful for tagging strategies were used for crosses and the frequency of independent germinal transpositions was assessed. In crosses to Ds genotypes, excision of Ds was revealed that correlated to the activity of the Ac genotype. A line displaying Ac amplification to multiple copies conferred a high frequency of independent Ds transpositions. The genotypes described here are useful in somatic insertion mutagenesis aimed at the isolation of tagged mutations in diploid potato.     

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 -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 -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: GEF_1481J = 24 %, GEF_1501D = 25 %, FR_1481J = 42 %, FR_1601D = 46 %.     

Establishment of a gene tagging system in Arabidopsis thaliana based on the maize transposable element Ac

Summary An Ac-derived, two-component transposable element system has been developed and analyzed with respect to its use in Arabidopsis thaliana. This system consists of an immobilized Ac element (Ac clipped wing, Ac_cl) as the source of transactivating transposase and a nonautonomous Ds element, Ds_A, which is inserted into a chimaeric neomycinphosphotransferase gene used as excision marker. After separate introduction of Ac_c1 and Ds_A into Arabidopsis thaliana, progeny analysis of crosses between five different Ac_cl lines and seven different Ds_A lines shows that: (1) different Ac_cl lines differ greatly in their capacity to transactivate Ds_A; (2) different Ds_A lines do not differ significantly with respect to Ds_A transactivation by one Ac_cl line; (3) reintegration of excised Ds_A elements, both at (genetically) linked and unlinked sites, occurs in about 50% of the excision events; and (4) plants with a high rate of somatic excisions can be used as source of new Ds_A transpositions, allowing the creation of a large number of independent Ds_A insertions.     

Transposition of a Ds element from a plasmid into the plant genome in Nicotiana plumbaginifolia protoplast-derived cells

Nicotiana plumbaginifolia haploid protoplasts were co-transformed with two plasmids, one with a NPT-II/Ds element and one with a gene encoding an amino-terminal truncated Ac transposase. It is shown that Ds can efficiently transpose from extrachromosomal DNA to N. plumbaginifolia chromosomes when the Ac transposase gene is present in trans. Ds has been shown to have transposed into the plant genome in a limited number of copies (1.9 copies per genome), for 21/32 transgenic lines tested. The flanking sequences present in the original plasmid are missing in these 21 plants. In only two of 21 plants was part of the transposase construct integrated. By segregation analysis of transgenic progeny, Ds was shown to be present in the heterozygous state in 10 lines even though haploid protoplasts had been originally transformed. This observation could indicate that integration occurred after or during DNA replication that leads to protoplast diploidization.     

Variegation patterns caused by excision of the maize transposable element Dissociation (Ds) are autonomously regulated by allele-specific Activator (Ac) elements and are not due to trans-acting modifier genes

The Ac elements present in the unstable wxm7 and wx-m9 alleles of maize trigger different patterns of Ds excision in trans. To determine whether this differential regulation is a feature of the Ac alleles themselves or is mediated by genetically distinct factors, maize plants heterozygous for the wx-m7 and wx-m9 alleles were crossed to tester strains homozygous for Ds reporter alleles. Kernels showing the variegation pattern characteristic for the Ac elements carried in the wx-m7 and wx-m9 alleles were found to be present in the ratios expected from the genetic constitution of the strains. The aleurone variegation caused by excision of the Ds reporter element and the endosperm variegation caused by excision of Ac from the wx-m7 and wx-m9 alleles themselves segregated with the original wx-m alleles. In addition, stable Wx and wx derivatives of wx-m9 that have lost Ac no longer exert any trans effect on the wx-m7 allele (and vice versa). Therefore it is concluded that the observed variegation patterns are autonomously determined by specific trans effects of the particular Ac element.     

Frequency and distance of transposition of a modifiedDissociation element in transgenic tobacco

Effective transposon tagging with theAc/Ds system in heterologous plant species relies on the accomplishment of a potentially high transposon-induced mutation frequency. The primary parameters that determine the mutation frequency include the transposition frequency and the transposition distance. In addition, the development of a generally applicable transposon tagging strategy requires predictable transposition behaviour. We systematically analysedDs transposition frequencies andDs transposition distances in tobacco. An artificialDs element was engineered with reporter genes that allowed transposon excision and integration to be monitored visually. To analyse the variability ofDs transposition between different tobacco lines, eight single copy T-DNA transformants were selected. Fortrans-activation of theDs elements, differentAc lines were used carrying an unmodifiedAc ⁺ element, an immobilizedsAc element and a stableAc element under the control of a heterologous chalcone synthas (chsA) promoter. With allAc elements, eachDs line showed characteristic and heritable variegation patterns at the seedling level. SimilarDs line-specificity was observed for the frequency by whichDs transpositions were germinally transmitted, as well as for the distances of theDs transpositions. ThesAc element induced transposition ofDs late in plant development, resulting in low germinal transposition frequencies (0.37%) and high incidences of independent transposition (83%). The majority of theseDs elements (58%) transposed to genetically closed linked sites (10 cM).     

A genetic analysis of mutations recovered from tomato following Agrobacterium-mediated transformation with the maize transposahle elements Activator and Dissociation

Summary The maize autonomous transposable element, Activator (Ac), and the nonautonomous element Dissociation (Ds), were introduced into the tomato cultivars VF36 and VFNT Cherry by Agrobacterium-mediated transformation. Progeny families from 145 primary transformants were scored at the seedling stage for phenotypically variant individuals. When VF36 was transformed, 20% of families had progeny with aberrant phenotypes. The mutation frequency in VFNT Cherry transformants was lower; in this cultivar 7% of the transformants had progeny segregating for seedling mutations. The majority of the mutations showed monogenic inheritance in the R₁ population, suggesting that the mutations occurred early in the transformation/regeneration process. One mutation, however, was recovered at low frequency in the R₁, suggesting a late mutagenesis event. When tomato was transformed with either the Ac or Ds elements, no differences in mutation frequencies were observed. Since Ac is transpositionally active in tomato transformants while Ds is not, these numbers indicate that the mutation frequency inherent to the transformation process is higher than the mutational activity of Ac. These results demonstrate that efficient gene tagging using heterologous transposable elements will require screening for transposon-induced mutations in later generations.     

Cloning of the Bz2 locus of Zea mays using the transposable element Ds as a gene tag

Summary The Bz2 locus of Zea mays has been cloned, utilizing the presence of the transposable element Dissociation (Ds) at the locus as a gene tag. The Ds element inserted in the bz2-m allele was identified among many members of the Ac/Ds family in a Southern blot analysis of a population segregating for bz2-m and Bz2. After cloning a DNA fragment from the bz2-m allele, sequences flanking the Ds insertion were shown to be Bz2-specific and were used to isolate a homologous fragment from a wild-type Bz2 line. The Ds insertion in the bz2-m allele was found to be a Ds2 element identical to the Ds insertion in adh1-2F11.     

Evolution of Ac and Dsl elements in select grasses (Poaceae)

We present data on evolution of the Ac/Ds family of transposable elements in select grasses (Poaceae). An Ac-like element was cloned from a DNA library of the grass Pennisetum glaucum (pearl millet) and 2387 bp of it have been sequenced. When the pearl millet Ac-like sequence is aligned with the corresponding region of the maize Ac sequence, it is found that all sequences corresponding to intron II in maize Ac are absent in pearl millet Ac. Kimura's evolutionary distance between maize and pearl millet Ac sequences is estimated to be 0.429±0.020 nucleotide substitutions per site. This value is not significantly different from the average number of synonymous substitutions for coding regions of the Adh1 gene between maize and pearl millet, which is 0.395±0.051 nucleotide substitutions per site. If we can assume Ac and Adh1 divergence times are equivalent between maize and pearl millet, then the above calculations suggest Ac-like sequences have probably not been strongly constrained by natural selection. The level of DNA sequence divergence between maize and pearl millet Ac sequences, the estimated date when maize and pearl millet diverged (25–40 million years ago), coupled with their reproductive isolation/lack of current genetic exchange, all support the theory that Ac-like sequences have not been recently introduced into pearl millet from maize. Instead, Ac-like sequences were probably present in the progenitor of maize and pearl millet, and have thus existed in the grasses for at least 25 million years. Ac-like sequences may be widely distributed among the grasses. We also present the first 2 Dsl controlling element sequences from teosinte species: Zea luxurians and Zea perennis. A total of 10 Dsl elements had previously been sequenced from maize and a distant maize relative, Tripsacum. When a maximum likelihood network of genetic relationships is constructed for all 12 sequenced Dsl elements, the 2 teosinte Dsl elements are as distant from most maize Dsl elements and from each other, as the maize Dsl elements are from one another. Our new teosinte sequence data support the previous conclusion that Dsl elements have been accumulating mutations independently since maize and Tripsacum diverged. We present a scenario for the origin of Dsl elements.     

Double Ds elements are involved in specific chromosome breakage

Summary The structure of the unstable Ds-induced sh-m5933 allele of the maize sucrose synthase gene was analysed and a double Ds structure found in opposite orientation on both sides of a 30 kb insert interrupting the sucrose synthase gene. The double Ds structures bordering the insert are identical over a distance of approximately 3 kb. These double Ds structures and the DNA segments beyond them are in opposite orientation and identical over a distance of approx. 5.3 kb. A hypothesis for how such a symmetrical structure could be formed is proposed. When one complete Ds element was excised from one of the double Ds structures a half Ds element was left behind. This half Ds element was found in one revertant strain which displayed an altered pattern of chromosome breakage compared to revertant strains which had not undergone Ds excision. Nine new maize strains which showed a similarly altered chromosome breakage pattern were isolated. In all nine cases we observed an indistinguishable deletion in the genomic DNA. These excisions are likely to be the result of similar excision events to that described above. We conclude that double Ds structures are responsible for Ds-induced chromosome breakage.     

Characterization of an unusual <Emphasis Type="Italic">Ds</Emphasis> transposable element in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>: Insertion of an abortive circular transposition intermediate

The maize Ac/Dstransposable elements, which belong to the hAT transposon superfamily, are widely used as insertional mutagens in numerous plant species. Molecular studies suggest that Ac/Ds elements transpose in a conservative non-replicative fashion; however the molecular mechanism of transposition remains unclear. We describe here the identification of an unusual Ds element, Ds-mmd1, in a transgenic Arabidopsis line. Ds-mmd1 is rearranged relative to the original Ds element, such that the original 5 and 3 ends are internal and previously internal sequences are the new 5 and 3 termini of Ds-mmd1. Short duplications of plant genomic DNA and Ds sequences are present at the Ds-mmd1 junctions, suggesting that a circular Dsmolecule was part of the events that created the Ds-mmd1 element. In addition, a revertant analysis on mmd1 plants demonstrated that Ds-mmd1 can be eliminated from the genome in an Ac-dependent process.     

Developing a transposon tagging system to isolate rust-resistance genes from flax

Summary A line of flax, homozygous for four genes controlling resistance to flax rust, was transformed with T-DNA vectors carrying the maize transposable elements Ac and Ds to assess whether transposition frequency would be high enough to allow transposon tagging of the resistance genes. Transposition was much less frequent in flax than in Solanaceous hosts such as tobacco, tomato and potato. Transposition frequency in callus tissue, but not in plants, was increased by modifications to the transposase gene of Ac. Transactivation of the excision of a Ds element was achieved by expressing a cDNA copy of the Ac transposase gene from the Agrobacterium T-DNA 2 promoter. Progeny of three plants transformed with Ac and 15 plants transformed with Ds and the transposase gene, were examined for transposition occurring in the absence of selection. Transposition was observed in the descendants of only one plant which contained at least nine copies of Ac. Newly transposed Ac elements were observed in 25–30% of the progeny of some members of this family and one active Ac element was located 28.8 (SE=6.3) map units from the L ⁶ rust-resistance gene. This family will be potentially useful in our resistance gene tagging program.     

Molecular evolutionary characterization of an Activator (Ac)-like transposable element sequence from pearl millet (Pennisetum glaucum) (Poaceae)

We present data on the evolution of the Ac/Ds family of transposable elements in select grasses (Poaceae). A defective Ac-like element was cloned from a DNA library of the grass Pennisetum glaucum (pearl millet) and its entire 4531 bp sequence has been determined. When the pearl millet Ac-like sequence is aligned with the maize Ac sequence, it is found that there is approximately 70% DNA similarity in the central region spanning most of maize Ac exon II and all of exon III. In addition, there are two smaller regions of similarity at the Ac terminii. Besides these three major structural similarities, Pennisetum Ac has two large regions, one 5 and one 3, that show little similarity to Zea Ac. Furthermore, most of the sequences corresponding to intron II in maize Ac are absent in pearl millet Ac. Kimura's evolutionary distance between the central region of maize and pearl millet Ac sequences is estimated to be 0.429±0.020 nucleotide substitutions per site. This value is not significantly different from the average number of synonymous substitutions for coding regions of the Adh1 gene between maize and pearl millet, which is 0.395±0.051 nucleotide substitutions per site. If we assume Ac and Adh1 divergence times are equivalent between maize and pearl millet, then the above calculations suggest Ac-like sequences have probably not been strongly constrained by natural selection. Conserved DNA and amino acid sequence motifs are also examined. The level of DNA sequence divergence between maize and pearl millet Ac sequences, the estimated date when maize and pearl millet diverged (25–40 million years ago), coupled with their reproductive isolation/lack of current genetic exchange, all support the theory that Ac-like sequences have not been recently introduced into pearl millet from maize. Instead, Ac-like sequences were probably present in the progenitor of maize and pearl millet and have thus existed in the grasses for at least 25 million years.     

Transposition of the maize Ds element from a viral vector to the rice genome

The geminivirus miscanthus streak virus (MiSV) was used as a gene vector to study the transposition of the maize Ds element in rice protoplasts. Efficient excision of the Ds from the MISV vector was observed only when the MiSV vector was allowed to replicate and the plasmid expressing the transposase gene encoded by Ac was co-transfected. Under the same condition, the Ds carrying a hygromycin phosphotransferase gene (Ds::HPT) was also efficiently excised. Hygromycin-resistant calli were obtained by culturing these transfected protoplasts in order to examine the transposition of the excised Ds::HPT into the rice genome. In five out of 16 calli examined, the Ds::HPT, but not the vector sequence, was integrated into the rice genome and 8 bp target site duplication typical of Ac/Ds transposition was generated. These results show that the Ds::HPT inserted in the MISV vector transposed directly into the rice genome. This demonstrates the direct transposition of a cloned plant transposable element into the plant genome. Implications of these finding are discussed.     

A Ds-mutation of the Adh1 gene in Zea mays L.

Summary An unstable spontaneous mutation in the maize Adh1 gene, coding for alcohol dehydrogenase, was selected by allyl alcohol poisoning of wild type Adh1 pollen from a maize line carrying Ds at the Bz2 locus and one copy of Ac in an unknown position. The mutant has a null phenotype. No wild type pollen grains were detected in strains devoid of Ac, but in the presence of Ac, wild type pollen grains were detected with a frequency of between 10^-4 and 10^-3. In addition, events have been identified in the aleurone in which reversions of both bz2-m and the unstable adh1 mutation occurred in the same patch of tissue, presumably in response to an alteration of Ac. By these criteria, the Adh1 mutant is caused by Ds. DNA blotting experiments have shown the presence of a 1.3 kb insertion in the Adh1 gene. All or part of this Ds insertion is transcribed, and is detected as an insertion within the ADH1-mRNA. The longer mRNA hybridizes to an authentic Ds probe.This Ds element differs in size from other known Ds insertions.     

Ac/Ds transposon mutagenesis in Arabidopsis thaliana: mutant spectrum and frequency of Ds insertion mutants

Using a two-component Ac/Ds system consisting of a stabilized Ac element (Ac_c1) and a non-autonomous element (Ds_A), 650 families of plants carrying independent germinal Ds_A excisions/transpositions were isolated. Progenies of 559 of these Ac_c1/Ds_A families, together with 43 families of plants selected for excision/transposition of wild-type (wt)Ac, were subjected to a broad screening program for mutants exhibiting visible alterations. This resulted in the identification of 48 mutants showing a wide variety of mutant phenotypes, including embryo lethality (24 mutants), chlorophyll defects (5 mutants), defective seedlings (2 mutants), reduced fertility (5 mutants), reduced size (3 mutants), altered leaf morphology (2 mutants), dark green, unexpanded rosette leaves (3 mutants), and aberrant flower or shoot morphology (4 mutants). To test whether these mutants were due to transposon insertions, a series of Southern blot experiments was performed on 28 families, comparing in each case several mutant plants with others showing the wild-type phenotype. A preliminary analysis revealed in 4 of the 28 families analyzed a common, novel Ds_A fragment in all mutant plants, which was present only in heterozygous plants with wt phenotype, as expected for Ds_A insertion mutations. These four mutants included two showing embryo lethality, one with dark green, unexpanded rosette leaves and stunted inflorescences, and one with curly growth of stems, leaves and siliques. Further evidence for Ds_A insertion mutations was obtained for one embryo lethal mutant and for the stunted mutant, while in case of the second embryo lethal mutant, the Ds_A insertion could be separated from the mutant locus by genetic recombination.