Maize Transposable Element Research Articles

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

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", Accl) as the source of transactivating transposase and a nonautonomous "Ds" element, DsA, which is inserted into a chimaeric neomycinphosphotransferase gene used as excision marker. After separate introduction of Acc1 and DsA into Arabidopsis thaliana, progeny analysis of crosses between five different Accl lines and seven different DsA lines shows that: (1) different Accl lines differ greatly in their capacity to transactivate DsA; (2) different DsA lines do not differ significantly with respect to DsA transactivation by one Accl line; (3) reintegration of excised DsA 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 DsA transpositions, allowing the creation of a large number of independent DsA insertions.

Control of excision frequency of maize transposable element Ds in Petunia protoplasts.

The complete coding region of maize transposable element Ac and truncated but active derivatives of it were placed under the control of promoters of different strength and tested for the ability to excise transposable element Ds from a beta-glucuronidase reporter gene in a cotransfection assay in Petunia protoplasts. The highest excision values (5% of the protoplasts able to express the beta-glucuronidase gene in a control experiment) were observed with a truncated version of the Ac coding region under the control of the 2' promoter. The weak Ac promoter is sufficient to give rise to excision values not much lower than those found with much stronger promoters such as the 2' and nos promoters. A decrease in excision frequency was observed when translation of the Ac coding region was hindered by out-of-frame ATG codons in addition to the use of weak promoters. Increasing the level of Ac transposase thus does not seem to be sufficient to raise the level of Ds excision observed in this system and possibly also in maize. Therefore another factor limits the excision of Ds elements. Previously, it was reported that in tobacco cells the deletion of Ds sequence between base pairs 186 and 245 led to a decrease of the Ds excision frequency by the full-length but not by the truncated Ac product. In the Petunia assay system, however, deletion of these sequences decreased the excision rate with both the full length and the truncated Ac coding region. A cDNA construct was found similarly active as the corresponding genomic DNA.

Mechanism of Ds1 excision from the genome of maize streak virus

We have previously shown that the maize transposable element Ds1 introduced into maize plants by agroinfection can be excised from the genome of geminivirus maize streak virus (MSV). Excision depended strictly on the presence of an active Ac element in the plants. In this study, the excision products or "footprints" left in the MSV genome after Ds1 excision were extensively characterized and the effects of flanking sequences on Ds1 excision were analysed. Most types of footprints obtained were comparable to those described for Ds1 excision in the maize genome, and could be explained by the models proposed for excision of plant transposable elements. In two revertants, however, some terminal sequences of the Ds1 element were found to have been left behind at the excision site. The finding of this novel type of Ds1 footprint indicated that gene conversion events occurred during and/or after Ds1 excision from the MSV genome. A partial deletion of one copy of the 8 bp duplications flanking the Ds1 element had no effect on the frequency or on the types of footprints of Ds1 excision from the MSV genome. Thus, the duplicated 8 bp sequences flanking the transposable element are not involved in Ds1 excision. These results, as well as a statistical analysis of the modifications of the bases flanking the Ds1 element after excision, are discussed in terms of excision models.

Pattern of somatic transposition in a high copy Ac tomato line

A transgenic tomato line containing between eight and ten copies per genome of an exceptionally active maize transposable element Ac has previously been described. Southern analyses indicated that these elements are somatically active in these plants. In order to characterize further the pattern of somatic transposition in this line, 24 independent Ac insertion events from a single plant were cloned. In 21 cases, Ac inserted into single copy genomic DNA while in three cases Ac inserted into sequences present at two to four copies per genome; none of the insertions occurred into more highly repetitive DNA. The chromosomal locations of 20 insertion sites were determined by RFLP mapping and a pattern of small dispersed clusters emerged. Thirteen of the 20 insertion sites were linked to at least one other insertion site but these were distributed over nine of the 12 tomato chromosomes. Only one Ac insertion was linked to the T-DNA locus. The structural integrity of these Ac elements was examined and no evidence of deletions or other rearrangements suggestive of Ds elements was found. The implications of these findings with respect to the use of Ac as a transposon tag in heterologous species are discussed.

The splicing of transposable elements and its role in intron evolution.

Recent studies have demonstrated that transposable elements in maize and Drosophila are spliced from pre-mRNA. These transposable element introns represent the first examples of recent addition of introns into nuclear genes. The eight reported examples of transposable element splicing include members of the maize Ac/Ds and Spm/dSpm and the Drosophila P and 412 element families. The details of the splicing of these transposable elements and their relevance to models of intron origin are discussed.

Behaviour of the maize transposable element Ac in Arabidopsis thaliana

SummaryThe somatic and germinal activity of the maize transposable element, Ac, has been analysed in progeny of 43 transformants of A. thaliana using a streptomycin resistance assay to monitor Ac excision. The ability to assay somatic activity enabled, for the first time, a detailed analysis of Ac activity in individual A. thaliana seedlings to be made. The effects of T‐DNA copy number, generation, dosage at each locus, flanking sequences and orientation of the element were compared. The most striking observation was the variability in Ac activity in genotypically identical individuals and the poor penetrance of the variegated phenotype. In general, increasing Ac dosage increased both somatic and germinal excision frequencies. The majority of families from individuals selected as inheriting an excision event carried transposed Ac elements re‐integrated in different positions in the genome.

Excision of a transposable element from a viral vector introduced into maize plants by agroinfection

The geminivirus maize streak virus (MSV) was used as a vector to introduce the maize transposable element Dissociation (Ds) and to study its excision in maize plants. MSV carrying Ds1 in its genome was introduced into maize plants by agroinfection. Excision of the Ds1 element from the MSV genome was detected only when functions from the transposable element Activator (Ac) were supplied in trans, either endogenously by the recipient maize plant or by co-transformation with Agrobacterium carrying a genomic Ac clone. The excision of Ds1 could easily be visualized by the appearance of viral symptoms induced by the revertant virus. The junction sequences left on the MSV genome after excision revealed 'footprints' typical of transposition as described for maize. From these results, we conclude that transposition functions in our system and that the use of the MSV replicon provides a rapid and simple tool for the investigation of the excision of transposable elements in maize plants.

Changes in state of the Wx-m5 allele of maize are due to intragenic transposition of Ds.

The molecular basis for the unusual phenotype conditioned by the waxy(Wx)-m5 Ds allele has been elucidated. Unlike most Ds alleles, Wx-m5 is phenotypically wild-type in the absence of Ac. We find that the Wx-m5 gene contains a 2-kb Ds element at -470 relative to the start of Wx transcription, representing the most 5' insertion of any transposable element allele characterized to date in plants. Despite its wild type phenotype, Wx-m5 has reduced levels of Wx enzymatic activity indicating that Ds insertion influences Wx gene expression. In the presence of Ac, Wx-m5 kernels have sectors of null expression on a wild-type background and give rise to stable wx and unstable wx-m germinal derivatives. Seventeen of 20 derivatives examined are wx-m alleles and at least 15 of these appear to result from intragenic transposition of Ds from -470 to new sites within the Wx gene. Three wx-m alleles contain two Ds elements, one at -470 and a second in Wx coding sequences. Surprisingly, only 3 out of 20 derivatives are stable wx mutants and these have sustained gross rearrangements of Wx and flanking sequences. For most other maize transposable element alleles somatic sectors and germinal derivatives usually arise following element excision or deletions of element sequences. In contrast, element insertion following intragenic transposition is apparently responsible for most of the somatic sectors and germinal derivatives of Wx-m5.

The maize transposable Ds1 element is alternatively spliced from exon sequences.

The null wx-ml allele contains a 409-bp Dissociation 1 (Ds1) element in exon 9 of the maize waxy (Wx) gene. In the absence of the autonomous Activator (Ac) element, the Ds1 element cannot transpose, and this allele encodes several Wx transcripts that arise following alternative splicing of Ds1 sequences from Wx pre-mRNA. Splicing involves the utilization of three 5' splice sites and three 3' splice sites. All but one of these splice sites are in Ds1 sequences near the ends of the element. The presence of 5' and 3' splice sites near the Ds1 termini and the element's small size and AT richness are features that distinguish Ds1 elements from all other known Ds elements. It is suggested that these features may enhance the ability of Ds1 to function as a mobile intron.

The maize transposable Ds1 element is alternatively spliced from exon sequences.

The null wx-ml allele contains a 409-bp Dissociation 1 (Ds1) element in exon 9 of the maize waxy (Wx) gene. In the absence of the autonomous Activator (Ac) element, the Ds1 element cannot transpose, and this allele encodes several Wx transcripts that arise following alternative splicing of Ds1 sequences from Wx pre-mRNA. Splicing involves the utilization of three 5' splice sites and three 3' splice sites. All but one of these splice sites are in Ds1 sequences near the ends of the element. The presence of 5' and 3' splice sites near the Ds1 termini and the element's small size and AT richness are features that distinguish Ds1 elements from all other known Ds elements. It is suggested that these features may enhance the ability of Ds1 to function as a mobile intron.

The ORFa protein, the putative transposase of maize transposable element Ac, has a basic DNA binding domain.

Ac encodes the 807 amino acid ORFa protein which binds specifically to multiple AAACGG motifs that are subterminally located in both ends of Ac. The wild-type ORFa protein and a number of deletion and amino acid exchange mutants were expressed in Escherichia coli, renatured and used for mobility shift assays. At least 136 amino acids from the N-terminus and 537 C-terminal amino acids may be removed from the ORFa protein without destroying the DNA binding domain, whereas a protein starting at amino acid 189 is DNA binding deficient. Certain basic amino acids between positions 190 and 200 are essential for DNA binding, as their substitution with uncharged amino acids leads to the loss of this function. The DNA binding domain of ORFa protein has an overall basic character, but no obvious sequence homology to any other known DNA binding protein. The homologies to the major open reading frames of transposable elements Tam3 from Antirrhinum majus and Hobo from Drosophila are found between the C-terminal two thirds of the three proteins. The ORFa protein forms discrete complexes with target DNA that appear, depending on the protein concentration, as a 'ladder' of bands on gels, indicating the occupation of target DNA by multiple ORFa protein molecules.

Wide hybridization of Hordeum vulgare × Zea mays

Two barley (Hordeum vulgare L.) varieties ('Klages' and 'Harrington') were crossed with an array of maize (Zea mays L.) stocks carrying active transposons. From 1204 florets pollinated and cultured, 98 embryos were rescued. Forty-one of these embryos germinated to give green plants, 38 of which were haploid (2n = 7) and 3 of which were diploid (2n = 14). The overall efficiency of barley haploid production was 4.7% with 'Klages' and 1.5% with 'Harrington'. No maize DNA introgression was detected cytologically or in DNA hybridizations using maize transposable elements and highly repeated sequences as probes.Key words: barley, maize, haploids, DNA hybridization.

Introduction and transposition of the maize transposable element Ac in rice (Oryza sativa L.).

To develop a transposon tagging system in an important cereal plant, rice (Oryza sativa L.), the maize transposable element Ac (Activator) was introduced into rice protoplasts by electroporation. We employed a phenotypic assay for excision of Ac from the selectable hph gene encoding resistance to hygromycin B. Southern blot analysis of hygromycin B-resistant calli showed that the Ac element can transpose from the introduced hph gene into the rice chromosomes. Sequence analysis of several Ac excision sites in the hph genes revealed sequence alterations characteristic of the excision sites of this plant transposable element. The Ac element appears to be active during development of transgenic rice plants from calli. Moreover, hybridization patterns of different leaves from the same plant indicated that some Ac elements are stable whereas others are able to transpose further during development of leaves. The results indicate that the introduced Ac element can transpose efficiently in transgenic rice plants.

Detection and abundance of mRNA and protein encoded by transposable element Activator (Ac) in maize

The 3.5 kb long mRNA of the maize transposable element Ac contains an open reading frame (ORFa) which encodes a polypeptide of 807 amino acids, the putative transposase of Ac. The Ac mRNA is a rare transcript: we now estimate the fraction of Ac mRNA in wx-m7::Ac seedlings to be 2-13 x 10(-5) of the polyA RNA. Assuming that maize cells contain similar amounts of polyA RNA as another monocot (0.16 pg/cell), this is equivalent to 1.5-10 transcripts in each cell. A protein with an apparent molecular weight of 112 kDa is detected, by five antisera directed against different segments of ORFa, exclusively in nuclear extracts from Ac-containing maize. This protein is most likely the full-length Ac ORFa protein. We estimate its concentration to be in the range of 3 x 10(-7) of the nuclear proteins, or about 1000 molecules per triploid endosperm cell containing one Ac element.

The Mutator transposable element family of maize.

It has been nearly 15 years since the first report about the Mutator phenomenon in maize (1). The utility of Mutator plants for efficiently generating tagged mutable alleles is probably the primary reason so many maize geneticists have worked with this transposable element family. On the other hand, the elusive nature of the transposase-encoding element for this family and the complexities of the Mu elements themselves and their regulation have become research topics in their own right. The purpose of this review is to summarize the major findings about Mu elements and to highlight the similarity and differences between Mutator and the “classical” transposable elements of maize, Ac and Spm. The structural features of the diverse Mu elements, the assays employed to measure Mutator activity, the role of methylation in element regulation, the timing of Mutator events during the life of the plant, the impact of stress on Mutator activity, and the molecular analysis of Mu-induced mutants will be discussed. In addition, the unresolved problems and implications of recent findings will be presented.

Activation of the maize transposable element Suppressor-mutator (Spm) in tissue culture

Previous experiments have revealed that the maize transposable element Activator (Ac) may become active during tissue culture. The objective of the present study was to determine whether a second transposable element, Suppressor-mutator (Spm), could also be activated in tissue culture and detected in regenerated maize plants. Approximately 500 R1 progeny of 143 regenerated plants (derived from 49 embryo cell lines) were crossed as males onto an Spm-responsive tester stock. Spm activity was observed in two R1 progeny of a single regenerated plant. This plant had been regenerated from Type II (friable embryogenic) callus of an A188 × B73 genetic background after 8 months in culture; the absence of Spm activity in four other plants regenerated from this same callus demonstrates that Spm activity was not present before culturing. Approximately 20 Spm-homologous DNA sequences were detected in each of the inbreds used to initiate the tissue cultures; it is presumed that one of these became active to give rise to Spm activity.

Transposition of maize transposable element Activator in rice

Abstract To explore the possibility of using the maize ( Zea mays L.) transposable element Activator (Ac) as a mutagen and gene tag in rice cells, we have introduced plasmid pKU3, a derivative of an Ac element constructed by Baker, into protoplasts of Oryza sativa L. subsp. japonica by the polyethylene glycol method. Several lines of evidence are presented for the transposition of the Ac element in tranformed calli. (1) In the pKU3 plasmid, the Ac element was inserted in the untranslated leader sequence of the neomycin phosphotransferase II (NPT II) gene. This construction allowed us to follow the excision of Ac by reconstruction of NPT II gene activity. For this, we have obtained 12 Km r calli selected from about 10 6 pKU3 DNA-treated protoplasts. The NPT II enzyme activity could be detected in most of the selected calli. (2) Southern blot analysis confirmed the presence of the Ac element in genomic DNA from transformed Km r calli. (3) A probe comprising leader sequence P1′ of the NPT II gene hybridized to the Eco RI- Hind III digested genomic DNA from transformed Km r calli to produce a 3.0-kb fragment which indicates that the interrupted NPT II gene had been reconstructed. These facts, taken together, show that the maize Ac element was introduced into some transformed rice cells. In some transformed cells, this element had been excised from its original location in the NPT II gene and had moved elsewhere in the transformed rice genome.

A transposon tagging strategy with Ac on plant cell level in heterologous plant species

The maize transposable element Ac can have a ‘late’ excision time during leaf development in certain transgenic tobacco plants. This was visualized with an assay based on Ac-excision restoring GUS-expression. Leaves of the described plants contain over 10 3 small blue spots, each of these spots representing an independent excision event. Leaves showing this ‘late’ excision phenomenon may be used for transposon tagging experiments at plant cell level. Plants which display ‘late’ Ac-excision do not detectably express GUS during the preceding callus phase, thus allowing transformants to be preselected for a ‘late’ timing of excision. To examine the applicability of this phenomenon a phenotypic selection assay for excision of Ac was used. Transformed calli containing Ac within the hygromycin resistance gene were regenerated and protoplasts isolated from leaves of regenerated plants were selected on hygromycin. Up to 0.8% of these protoplasts displayed hygromycin resistance. The hygromycin resistant derivatives analyzed were shown to represent independent transposition events. Ac-insertions which can be generated in this way may be used for transposon tagging experiments at cell level.

Insertion of Mu1 elements in the first intron of the Adh1-S gene of maize results in novel RNA processing events.

Maize transposable elements, when inserted in or near genes, alter expression by several transcriptional and post-transcriptional mechanisms. Three independent, unstable insertions of the transposable element Mutator (Mu) into the first intron of the Alcohol dehydrogenase-1 (Adh1) gene have been shown to decrease expression [Strommer et al. (1982). Nature 300, 542-544]. We have developed an approach to elucidate the underlying molecular mechanisms responsible for the mutant phenotypes. Mu1 elements were inserted into Adh1-S intron 1 in vitro to create plasmid facsimiles of the mutant alleles. The Mu1 element was also inserted at novel positions within intron 1 to create new mutations. The Mu1/intron constructions were placed between the Adh1-S promoter/exon 1 segment and a reporter gene (firefly luciferase or beta-glucuronidase), and these chimeric gene constructs were tested in transient assays in maize protoplasts. When compared with the appropriate control, the Mu1 insertions decreased reporter gene expression to levels approximating the alcohol dehydrogenase enzyme activities observed for the Adh1-S mutants in vivo. The Mu1 insertions also showed a polarity effect with luciferase expression increasing as the insertions were placed nearer the 3' splice junction. In addition, Mu1 insertions within a different intron, actin intron 3, also significantly reduced luciferase expression, indicating that Mu1 insertions within introns are likely to diminish expression in many genes. The presence of the Mu1 sequences was correlated with decreased levels of steady-state luciferase transcript. Deletion analysis of the Mu1 element and RNase mapping indicate that the transposable element contains RNA processing signals in its central region that are largely responsible for the decrease in expression.

Excision of a Ds-like maize transposable element (AcΔ) in a transient assay in Petunia is enhanced by a truncated coding region of the transposable element Ac

The excision of a Ds-like transposable element (Ac delta) is mediated in trans by the transposable element Ac or its derivatives in Petunia protoplasts cotransfected with two plasmid DNAs. Excision restores the activity of the beta-glucuronidase (GUS) gene that is otherwise shut off by the presence of Ac delta in its leader sequence. A transient expression assay (histochemical test) is used to detect the beta-glucuronidase activity at the protoplast to detect the beta-glucuronidase activity at the protoplast level. The number of blue-stained protoplasts is a measure of the excision frequency. With Ac delta alone a near-zero background of GUS activity is detected, which is weakly enhanced by the presence, in trans, of either the wild-type Ac or the coding region (ORFa) transcribed from the 2' promoter of Agrobacterium tumefaciens TR-DNA. A strong enhancement is observed when a truncated Ac coding region, also under the control of the 2' promoter, is supplied in trans. The truncated version has ATG10 at codon 103 in frame with ORFa and is preceded by 7 out-of-frame ATGs. The assay is quick and well suited for detection of excision frequencies above the value obtained with the wild-type Ac. The presence of empty donor sites following excision can be demonstrated by PCR amplification and direct sequencing of the appropriate DNA fragment.