Transposable elements can be used to study cell lineages in transgenic plants.

The beta-glucuronidase reporter gene has been used to develop a sensitive assay for the excision of transposable elements introduced into transgenic plants. The reporter gene, inactivated by the insertion of the maize transposable element Activator (Ac) into the 5'-untranslated leader, was introduced into the genome of tobacco by Agrobacterium-mediated transformation. Reactivation of the beta-glucuronidase gene was detected in transgenic plants using a fluorometric or histochemical assay. Reactivation of the reporter gene was dependent on the presence of the transposase of Ac, and resulted from the excision of the Ac element. This assay, together with the improved methods for visualization, will provide a valuable and rapid method for studying the basic mechanism of transposition in plants and for developing modified transposable element systems suitable for gene tagging in transgenic plants.     

A comparative study of Tam3 and Ac transposition in transgenic tobacco and petunia plants

Transposition of the Anthirrinum majus Tam3 element and the Zea mays Ac element has been monitored in petunia and tobacco plants. Plant vectors were constructed with the transposable elements cloned into the leader sequence of a marker gene. Agrobacterium tumefaciens-mediated leaf disc transformation was used to introduce the transposable element constructs into plant cells. In transgenic plants, excision of the transposable element restores gene expression and results in a clearly distinguishable phenotype. Based on restored expression of the hygromycin phosphotransferase II (HPTII) gene, we established that Tam3 excises in 30% of the transformed petunia plants and in 60% of the transformed tobacco plants. Ac excises from the HPTII gene with comparable frequencies (30%) in both plant species. When the -glucuronidase (GUS) gene was used to detect transposition of Tam3, a significantly lower excision frequency (13%) was found in both plant species. It could be shown that deletion of parts of the transposable elements Tam3 and Ac, removing either one of the terminal inverted repeats (TIR) or part of the presumptive transposase coding region, abolished the excision from the marker genes. This demonstrates that excision of the transposable element Tam3 in heterologous plant species, as documented for the autonomous element Ac, also depends on both properties. Southern blot hybridization shows the expected excision pattern and the reintegration of Tam3 and Ac elements into the genome of tobacco plants.     

The transposon Tip100 from the common morning glory is an autonomous element that can transpose in tobacco plants

The mutable flaked or a (flaked) (a(f)) line of the common morning glory (Ipomoea purpurea) displays white flowers with colored flakes, and the a(f) mutation is caused by the insertion of a transposable element named Tip100 into the CHS-D gene for anthocyanin biosynthesis. The 3.9-kb Tip100 element belongs to the Ac/Ds family and contains an ORF encoding a polypeptide of 808 amino acids. The frequency and timing of flower variegation vary in different a(f) lines, and a genetic element termed Modulator has been postulated to affect the variegation pattern. Since the pattern of flower variegation is determined by the frequency and timing of excision of Tip100 from the CHS-D gene, we wished to determine whether Tip100 is an autonomous element that is itself capable of transposition in a heterologous host. To do this, we introduced the element into the genome of tobacco plants by Agrobacterium-mediated transformation. The intact Tip100 element was able to excise from its original position in the chromosome and reinsert into new sites in the tobacco genome, whereas an internal deletion derivative was not. Based on these results, we conclude that Tip100 is an autonomous element. We also discuss the nature of the putative Modulator element affecting flower and leaf variegation in various mutable lines of the morning glory.     

Cell-autonomous behavior of the rolC gene of Agrobacterium rhizogenes during leaf development: a visual assay for transposon excision in transgenic plants.

We describe a genetic switch based on the Ac transposable element of maize and the rolC gene of Agrobacterium rhizogenes, a dominant gene, which has pleiotropic effects on plant growth and morphology. Moreover, rolC gene expression under the control of the 35S cauliflower mosaic virus promoter decreases chlorophyll content in transgenic tobacco plants. Chlorophyll is a visible cell-autonomous marker, and it is shown here that the reduction in chlorophyll content caused by the rolC gene product allows us to monitor, in palisade or spongy mesophyll cells, Ac excision events resulting in rolC gene expression as pale-green sectors and spots. Our results indicate that the rolC gene product behaves in a cell-autonomous manner during leaf development, at least as far as chlorophyll accumulation is concerned. In addition, the rolC gene can be useful to evaluate visually if and when a transposable element is active. Most important, we propose the use of a transposable element as a tool to activate expression of morphogenetic genes in a clonal population of cells. This could be particularly useful when studying genes affecting growth and development whose constitutive expression can severely impair regeneration of transgenic plants.     

Characterization of a pollen-specific cDNA from sunflower encoding a zinc finger protein.

The maize transposable element Activator (Ac) carries subterminal CpG-rich sequences which are essential for the transposition of the element. It has previously been shown that the methylation of certain sequences contained in this region can alter their ability to interact with the Ac-encoded protein. The novel hypothesis that the methylation of subterminal Ac sequences is required for transposition was tested. Approximately 150 bp of the 5' subterminal region of the Ac element was examined for the presence of 5-methylcytosines by the ligation-mediated polymerase chain reaction (LMPCR)-aided genomic sequencing method. The methylation status of 22 and 39 cytosines on either strand of the DNA were analysed in each of five different transgenic tobacco cultures carrying transposable Ac sequences. Ten micrograms of tobacco DNA were used for each base-specific cleavage reaction before amplification by LMPCR. All but one of the cytosines were unmethylated. Only a minor fraction of the Ac molecules was methylated at one cytosine residue. It is concluded that DNA methylation at the tested Ac sequences is not required for the transposability of Ac or Ds elements in tobacco cells.     

The transposition pattern of the Ac element in tobacco cultured cells.

We investigated physical distances and directions of transposition of the maize transposable element Ac in tobacco cultured cells. We introduced a T-DNA construct that carried a non-autonomous derivative of Ac (designated dAc-I-RS) that included sites for cleavage by restriction endonuclease MluI. Another cleavage site was also introduced into the T-DNA region outside of the dAc-I-RS transposable element. The tobacco cultured cell line BY-2 was transformed with the T-DNA and several transformed lines that had a single copy of the T-DNA at a different chromosomal location were isolated. These lines were co-cultured with Agrobacterium tumefaciens cells that carried a cDNA for the Ac transposase gene under the control of various promoters. Sublines of cultured cells in which dAc-I-RS had been transposed, were isolated. The genomic DNAs of these sublines were isolated and digested with MluI. Sizes of DNA segments generated by digestion were determined by pulse-field gel electrophoresis. Our results showed that 20 to 70% of transposition events had occurred within several hundreds kilo-base pairs (kb) on the same chromosome. These results demonstrate that the Ac-Ds element preferentially transposed to regions near the original site in a tobacco chromosome. In addition, the present results are an example of asymmetric transposition as demonstrated by the distance of transposition on the chromosome.     

Survey of transposable elements from rice genomic sequences

Oryza sativa L. (domesticated rice) is a monocotyledonous plant, and its 430 Mb genome has been targeted for complete sequencing. We performed a high-resolution computer-based survey for transposable elements on 910 Kb of rice genomic DNA sequences. Both class I and II transposable elements were present, contributing 19.9% of the sequences surveyed. Class II elements greatly outnumbered class I elements (166 versus 22), although class I elements made up a greater percentage (12.2% versus 6.6%) of nucleotides surveyed. Several Mutator-like elements (MULEs) were identified, including rice elements that harbor truncated host cellular genes. MITEs (miniature inverted-repeat transposable elements) account for 71.6% of the mined transposable elements and are clearly the predominant type of transposable element in the sequences examined. Moreover, a putative Stowaway transposase has been identified based on shared sequence similarity with the mined MITEs and previously identified plant mariner-like elements (MLEs). Members of a group of novel rice elements resembling the structurally unusual members of the Basho family in Arabidopsis suggest a wide distribution of these transposons among plants. Our survey provides a preview of transposable element diversity and abundance in rice, and allows for comparison with genomes of other plant species.     

Sequence similarity of putative transposases links the maize Mutator autonomous element and a group of bacterial insertion sequences.

The Mutator transposable element system of maize is the most active transposable element system characterized in higher plants. While Mutator has been used to generate and tag thousands of new maize mutants, the mechanism and regulation of its transposition are poorly understood. The Mutator autonomous element, MuDR, encodes two proteins: MURA and MURB. We have detected an amino acid sequence motif shared by MURA and the putative transposases of a group of bacterial insertion sequences. Based on this similarity we believe that MURA is the transposase of the Mutator system. In addition we have detected two rice cDNAs in genbank with extensive similarity to MURA. This sequence similarity suggests that a Mutator-like element is present in rice. We believe that Mutator, a group of bacterial insertion sequences, and an uncharacterized rice transposon represent members of a family of transposable elements.     

Amplification of genomic sequences flanking transposable elements in host and heterologous plants: a tool for transposon tagging and genome characterization.

The isolation of sequences flanking integrated transposable elements is an important step in gene tagging strategies. We have demonstrated that sequences flanking transposons integrated into complex genomes can be simply and rapidly obtained using the polymerase chain reaction. Amplification of such sequences was established in a model system, a transgenic tobacco plant carrying a single Ac element, and successfully applied to the cloning of a specific Spm element from a maize line carrying multiple Spm hybridizing sequences. The described utilization of methylation sensitive restriction enzymes (including those with degenerate recognition sequences) in the generation of templates for amplification will simplify the cloning and mapping of genomic sequences adjacent to transposable elements.     

Excision of the Drosophila transposable element mariner: identification and characterization of the Mos factor.

Genetic and molecular evidence presented in this paper demonstrate that the Mos factor for inherited mosaicism is a special copy of the transposable element mariner. Mosaicism observed in the presence of the Mos (Mosaic) factor results from a high frequency of excision of the mariner element from an insertion site near the white-eye gene in Drosophila mauritiana. The Mos factor promotes the excision of mariner elements from genomic insertion sites other than the site in wpch, and it also promotes its own loss from the genome. Putative transpositions of Mos to new genomic sites have also been observed. A copy of mariner present at a particular site in a Mos strain has been shown to be missing in derived strains in which the Mos factor has been lost, and in strains with putative transpositions. We propose that this copy of mariner is identical to the Mos factor.     

The En/Spm transposable element of Zea mays contains splice sites at the termini generating a novel intron from a dSpm element in the A2 gene

The A2 locus of Zea mays, identified as one of the genes affecting anthocyanin biosynthesis, was cloned using the transposable elements rcy and dSpm as gene tags. The A2 gene encodes a putative protein of 395 amino acids and is devoid of introns. Two a2-m1 alleles, containing dSpm insertions of different sizes, were characterized. The dSpm element from the original state allele has perfect termini and undergoes frequent transposition. The element from the class II state allele is no longer competent to transpose. It has retained the 13 bp terminal inverted repeat but has lost all subterminal sites at the 5' end, which are recognized by tnpA protein, the most abundant product of the En/Spm transposable element system. The relatively high A2 gene expression of one a2-m1 allele is due to removal of almost all dSpm sequences by splicing. The slightly altered A2 enzyme is still functional as shown by complementation of an a2 mutant with the corresponding cDNA. The 5' and 3' splice sites are constituted by the termini of the dSpm element; it therefore represents a novel intron of the A2 gene.     

Identification of Transposable Element Markers for a Rust (Puccinia arachidis Speg.) Resistance Gene in Cultivated Peanut

Peanut rust (Puccinia arachidis Speg.) affects pod yield and quality up to an extent of 10–50%. Efforts have been made on transferring the rust resistance gene to cultivated peanut species through interspecific hybridization. But, in most of the cases, it failed due to linkage drag of undesirable plant and pod features. Identification of tightly linked molecular markers will help to identify the desirable recombinants more efficiently. A recombinant inbred line population comprising 164 lines was developed from a cross between a rust‐resistant parent VG 9514 and a rust susceptible parent TAG 24. Using a modified bulk segregant analysis, 243 transposable element (TE) primer pairs were screened for putative linkage with rust resistance. Of the 243, 40 TE primer pairs were found polymorphic between parents and two transposable element markers, and TE 360 and TE 498 were found associated with rust resistance gene. Based on genetic mapping, TE 360 was found linked to the rust resistance gene at 4.5 cM distance. Identification of such markers could be applied for marker‐assisted selection of rust resistance plants in peanut.