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.     

Use of the transposon Ac as a gene-searching engine in the maize genome

We show here that, although genes constitute only a small percentage of the maize genome, it is possible to identify them phenotypically as Ac receptor sites. Simple and efficient Ac transposition assays based on the well-studied endosperm markers bz and wx were used to generate a collection of >1300 independent Ac transposants. The majority of transposed Ac elements are linked to either the bz or the wx donor loci on chromosome 9. A few of the insertions produce obvious visible phenotypes, but most of them do not, suggesting that these populations will be more useful for reverse genetics than for forward transposon mutagenesis. An inverse polymerase chain reaction method was adapted for the isolation of DNA adjacent to the transposed Ac elements (tac sites). Most Ac insertions were into unique DNA. By sequencing tac sites and comparing the sequences to existing databases, insertions were identified in a number of putative maize genes. The expression of most of these genes was confirmed by RNA gel blot analysis. We report here the isolation and characterization of the first 46 tac sites from the two insertion libraries.     

Inactivation of the maize transposable element Activator (Ac) is associated with its DNA modification.

The Activator (Ac) element at the waxy locus (wx-m7 allele) has the ability to undergo changes in its genetic activity and cycles between an active and inactive phase. Comparison of active Ac elements at several loci and the inactive Ac at wx-m7 by Southern blot analysis revealed that the inactive Ac sequence was not susceptible to digestion by the methylation sensitive enzyme PvuII while active elements were susceptible to PvuII digestion. Restriction digest comparisons between the clones of the active and inactive Ac elements were indistinguishable. Further analyses with the enzymes SstII and the methylation sensitive and insensitive isoschizomers EcoRII and BstNI showed the inactive Ac sequence was methylated at these sites, whereas the active Ac was hypomethylated. Although the active Ac at the wx-m7 allele in different genetic backgrounds showed differences in the Ac DNA modification pattern, at least a fraction of genomic DNA contained Ac sequences that were unmethylated at all of the internal sites we assayed. These data may suggest a role for DNA modification in the ability of Ac to transpose from the waxy locus and to destabilize unlinked Ds elements.     

Sexual transmission of transposed activator elements in transgenic tomatoes

The transmission of transposed Ac elements in progeny derived by self-pollination of ten transformed tomato plants has been examined by Southern hybridization analysis. We show that six of these primary transformants have transmitted a transposed Ac to at least one progeny. One of the families was segregating for at least two different insertion events. In five of ten families, progeny were detected that contained a transposed Ac but no donor T-DNA sequences, indicating that a recombination event occurred between the original and new Ac insertion site. Somatic transposition of Ac as late as the R2 generation is evidenced. One family contained an empty donor site fragment but Ac was not detected in either the parent or progeny, indicating Ac was lost in this population early in regeneration. While four of ten families were segregating for aberrant phenotypes, there was no evidence that the mutated gene was linked to a transposed Ac.     

Transposon-mediated single-copy gene delivery leads to increased transgene expression stability in barley

Instability of transgene expression in plants is often associated with complex multicopy patterns of transgene integration at the same locus, as well as position effects due to random integration. Based on maize transposable elements Activator (Ac) and Dissociation (Ds), we developed a method to generate large numbers of transgenic barley (Hordeum vulgare var Golden Promise) plants, each carrying a single transgene copy at different locations. Plants expressing Ac transposase (AcTPase) were crossed with plants containing one or more copies of bar, a selectable herbicide (Basta) resistance gene, located between inverted-repeat Ds ends (Ds-bar). F(1) plants were self-pollinated and the F(2) generation was analyzed to identify plants segregating for transposed Ds-bar elements. Of Ds-bar transpositions, 25% were in unlinked sites that segregated from vector sequences, other Ds-bar copies, and the AcTPase gene, resulting in numerous single-copy Ds-bar plants carrying the transgene at different locations. Transgene expression in F(2) plants with transposed Ds-bar was 100% stable, whereas only 23% of F(2) plants carrying Ds-bar at the original site expressed the transgene product stably. In F(3) and F(4) populations, transgene expression in 81.5% of plants from progeny of F(2) plants with single-copy, transposed Ds-bar remained completely stable. Analysis of the integration site in single-copy plants showed that transposed Ds-bar inserted into single- or low-copy regions of the genome, whereas silenced Ds-bar elements at their original location were inserted into redundant or highly repetitive genomic regions. Methylation of the non-transposed transgene and its promoter, as well as a higher condensation of the chromatin around the original integration site, was associated with plants exhibiting transgene silencing.     

Transposition of the maize transposable element Ac in barley (Hordeum vulgare L.)

Transposition of the maize autonomous element Ac (Activator) was investigated in barley (Hordeum vulgare L.) with the aim of developing a transposon tagging system for the latter. The Ac element was introduced into meristematic tissue of barley by microprojectile bombardment. Transposon activity was then examined in the resulting transgenic plants. Multiple excision events were detected in leaf tissue of all plant lines. The mobile elements generated empty donor sites with small DNA sequence alterations, similar to those found in maize. Reintegration of Ac at independent genomic loci in somatic tissue was demonstrated by isolation of new element-flanking regions by AIMS-PCR (amplification of insertion-mutagenized sites). In addition, transmission of transposed Ac elements to progeny plants was confirmed. The results indicate that the introduced Ac element is able to transpose in barley. This is a first step towards the establishment of a transposon tagging system in this economically important crop.     

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.     

Linked and Unlinked Transposition of a Genetically Marked Dissociation Element in Transgenic Tomato

We have introduced a genetically marked Dissociation transposable element (Ds(neo)) into tomato. In the presence of Ac transposase, Ds(neo) excised from an integrated T-DNA and reinserted at numerous new sites in the tomato genome. The marker genes of Ds(neo) (NPTII) and the T-DNA (HPT) facilitated identification of plants bearing transposon excisions and insertions. To explore the feasibility of gene tagging strategies in tomato using Ds(neo), we examined the genomic distribution of Ds(neo) receptor sites, relative to the location of the donor T-DNA locus. Restriction fragment length polymorphism mapping of transposed Ds(neo) elements was conducted in two tomato families, derived from independent primary transformants each bearing Ds(neo) within a T-DNA at a unique position in the genome. Transposition of Ds(neo) generated clusters of insertions that were positioned on several different tomato chromosomes. Ds(neo) insertions were often located on the same chromosome as the T-DNA donor site. However, no insertion showed tight linkage to the T-DNA. We consider the frequency and distance of Ds(neo) transposition observed in tomato to be well suited for transposon mutagenesis. Our study made use of a novel, stable allele of Ac (Ac3) that we discovered in transgenic tomato. We determined that the Ac3 element bears a deletion of the outermost 5 base pairs of the 5'-terminal inverted repeat. Though incapable of transposition itself, Ac3 retained the ability to mobilize Ds(neo). We conclude that a dual element system, composed of the stable Ac3 trans-activator in combination with Ds(neo), is an effective tool for transposon tagging experiments in tomato.     

Transgenic tomato (Lycopersicon esculentum L.) transformed with a binary vector in Agrobacterium rhizogenes: Non-chimeric origin of callus clone and low copy numbers of integrated vector T-DNA

Summary We transformed tomato (Lycopersicon esculentum L.) by using Agrobacterium rhizogenes containing two independent plasmids: the wild-type Ri-plasmid, and the vector plasmid, pARC8. The T-DNA of the vector plasmid contained a marker gene (Nos/Kan) encoding neomycin phosphotransferase which conferred resistance to kanamycin in transformed plant cells. Transgenic plants (R ₀) with normal phenotype were regenerated from transformed organogenic calli by the punctured cotyledon transformation method. Southern blot analysis of the DNA from these transgenic plants showed that one or two copies of the vector plasmid T-DNA, but none of the Ri-plamid T-DNA, were integrated into the plant genome. Different transgenic plants derived from the same callus clone showed an identical DNA banding pattern, indicating the non-chimeric origin of these plants. We also transformed tomato by using A. tumefaciens strain LBA4404 containing a disarmed Ti-plasmid (pAL4404), and a vector plasmid (pARC8). Transgenic plants derived via A. tumefaciens transformation, like those via A. rhizogenes, contained one to two copies of the integrated vector T-DNA. The kanamycin resistance trait in the progeny (R ₁) of most transgenic plants segregated at a ratio of 3:1, suggesting that the vector T-DNAs were integrated at a single site on a tomato chromosome. In some cases, the expression of the marker gene (Nos/Kan) seemed to be suppressed or lost in the progeny.     

Multiple Divergent ITS1 Copies Were Identified in Single Tomato Genome Using DGGE Analysis

The intra-genomic variation in the internal transcribed spacer (ITS) region has led to misleading conclusions in the evolutionary analysis of plants; understanding this variation is critical for correct evolutionary analysis based on ITS sequences. To reveal the ITS variation in tomato, entire copies of ITS1 sequences within tomato species were separated using denaturing gradient gel electrophoresis (DGGE) and DNA sequence analysis. ITS1 copies varied significantly in sequence composition, but not in sequence length within the same tomato cultivar. DNA sequence similarity of the ITS1 copies was 77–100 %. Moreover, AT and GC contents in ITS1 copies from each tomato cultivar were significantly different, ranging from 50.4 to 64.3 % for GC and from 35.7 to 49.6 % for AT. However, the length variation of ITS1 was insignificant, ranging from 279 to 282 bp. Multiple copies of divergent ITS1 present in the tomato genome indicate that some copies may be paralogues. In conclusion, DGGE technique is a reliable and novel approach to reveal the entire ITS copy variation and the possible evolutionary relationship of tomato.     

Distribution of Unlinked Transpositions of a Ds Element from a T-DNA Locus on Tomato Chromosome 4

In maize, receptor sites for unlinked transpositions of Activator (Ac) elements are not distributed randomly. To test whether the same is true in tomato, the receptor sites for a Dissociation (Ds) element derived from Ac, were mapped for 26 transpositions unlinked to a donor T-DNA locus on chromosome 4. Four independent transposed Dss mapped to sites on chromosome 4 genetically unlinked to the donor T-DNA, consistent with a preference for transposition to unlinked sites on the same chromosome as opposed to sites on other chromosomes. There was little preference among the nondonor chromosomes, except perhaps for chromosome 2, which carried seven transposed Dss, but these could not be proven to be independent. However, these data, when combined with those from other studies in tomato examining the distribution of transposed Acs or Dss among nondonor chromosomes, suggest there may be absolute preferences for transposition irrespective of the chromosomal location of the donor site. If true, transposition to nondonor chromosomes in tomato would differ from that in maize, where the preference seems to be determined by the spatial arrangement of chromosomes in the interphase nucleus. The tomato lines carrying Ds elements at known locations are available for targeted transposon tagging experiments.     

Ac as a tool for the functional genomics of rice

To examine whether the maize autonomous transposable element Ac can be used for the functional analysis of the rice genome, we used Southern blot analysis to analyze the behaviour of Ac in 559 rice plants of four transgenic families through three successive generations. All families showed highly active transposition of Ac, and 103 plants (18.4%) contained newly transposed Ac insertions. In nine of the 12 independent transpositions analyzed, their germinal transmission was detected. Partial sequencing of 99 Ac-flanking sequences revealed that 21 clones exhibited significant similarities with protein-coding genes in databases and four of them matched rice cDNA sequences. These results indicate preferential Ac transposition into protein-coding rice genes. To examine the feasibility of PCR-based screening of gene knockouts in rice Ac plants, we prepared bulked genomic DNA from the leaves of approximately 6000 rice Ac plants and pooled the DNA according to a three-dimensional matrix. Of 14 randomly selected genes, two gene knockouts were identified, and one encoding a rice cytochrome P450 (CYP86) gene was shown to be stably inherited to the progeny. Together, these results suggest that Ac can be efficiently used for the functional analysis of the rice genome.     

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.     

Molecular analysis of single copy and repetitive genes on chromosome 2 in intergeneric tomato somatic hybrid plants

Restriction fragment polymorphisms were used to identify and quantify the nuclear contributions from each parent to somatic hybrid plants between tomato (Lycopersicon esculentum Mill.) cv. Sub-Arctic Maxi and Solanum lycopersicoides Dun. Three single-copy clones, 2–13, 2–17, and 3–288, and a clone for the 45s ribosomal RNA, pHA2, all mapped to chromosome 2 of tomato, were used in analysis of 47 somatic hybrids. The amount of hybridizing probe for each parental band was quantified by densitometry of the autoradiograph film. Analyses with the three single-copy clones indicated that there were more than two S. lycopersicoides copies in most somatic hybrid plants. For at least one somatic hybrid there was a loss of one tomato copy. No evidence was found for more than two copies donated from tomato or loss of a copy from S. lycopersicoides. Most of the observed variation in copy number of the single-copy clones was consistent with chromosomal changes occurring in the suspension cells from which S. lycopersicoides parental protoplasts were derived.The number of copies of rDNA derived from each parent varied independently of the number of copies of single-copy clones from each parent. Changes in the copy number of rDNA occurred in both tomato and S. lycopersicoides genomes.     

Transformation of Brassica napus by using the aadA gene as selectable marker and inheritance studies of the marker genes

An Agrobacterium tumefaciens -mediated transformation system for Brassica napus has been improved. We investigated several marker genes for transformation of Brassica napus , and the aadA gene, which confers resistance to streptomycin and spectinomycin, was found to be the most suitable. Forty-three out of 193 putative transformants in the T₁ generation were investigated by Southern blot analysis. Transformants containing a range of 1 to 10 integrated T-DNA copies per genome were found. Total DNA from 35 plants showed hybridisation to both the aadA and the nptll marker gene probes, from 5 plants only to one marker gene probe and from 3 plants DNA did not hybridise to any of the gene probes. Furthermore, more complex integration patterns such as direct repeated copies of the T-DNA, both as tandem and inverted copies, were observed. Inheritance of the marker genes in the T₂ generation was studied in 37 of the plants. This revealed that 22% of the plants that contained both marker genes, segregated as one single locus (3:1) for both genes, while 46% of the plants gave a segregation pattern corresponding to one T-DNA locus for at least one of the marker genes. Moreover, these inheritance patterns appeared to be more or less independent of the number of genes seen in the Southern blot analysis of the T, generation. In this study we show that the introduced marker genes are inherited by the T; generation in a less predictable way than was earlier reported for B. napus .     

Transposition of the maize activator element in transgenic rice plants.

Transposition of the maize Activator (Ac) element was observed in transgenic rice. After protoplast transformation, Ac excision from an interrupted hygromycin phosphotransferase gene was monitored by appearance of the hygromycin-resistant colonies. The frequency of Ac excision, based on the biological assay was up to 19%. Southern hybridization analysis indicated that at least one copy per genome of the hygromycin-resistance gene was reconstituted after Ac excision and that the transposed Ac element was reintegrated into the rice genome. Analysis of DNA sequences at 14 empty donor sites indicated that the Ac element was excised in rice in a similar manner as maize. The excision of an Ac mutant in which a 1.3 kbp Tn903 fragment was inserted at a unique BamHI site so as to disrupt binding of the putative transposase was not detected by DNA analysis. These results demonstrated that the maize Ac element might be used as an effective heterologous transposon for mutagenesis and gene tagging in rice, an important food crops.     

Ac Transposition from a T-DNA Can Generate Linked and Unlinked Clusters of Insertions in the Tomato Genome

We have investigated the distribution of transposed Acs in the tomato genome. Our approach has been to clone the regions flanking the T-DNAs and transposed Acs from two transgenic lines of tomato and place these sequences on the tomato restriction fragment length polymorphism (RFLP) map. The distribution of transposed Acs around the T-DNA and at locations unlinked to the T-DNA indicates that Ac transposes to linked and unlinked sites in tomato as it does in maize. The structure and terminal sequence of these cloned elements shows that Ac remains intact after transposition. We discuss these results and their bearing on gene tagging strategies using Ac and Ds.     

Frequency and pattern of transposition of the maize transposable element Ds in transgenic rice plants

Two kinds of T-DNA constructs, I-RS/dAc-I-RS and Hm(R)Ds, carrying a non-autonomous transposable element of Ac of maize were introduced into rice plants by Agrobacterium-mediated gene transfer. Six transgenic rice plants identified as containing a single copy of the element were crossed with two transgenic rice plants carrying a gene for Ac transposase under the control of the cauliflower mosaic virus 35S promoter. In F2 progenies, excision of the element was detected by PCR analysis and re-integration of the element was investigated by Southern blot analysis. The frequency of the excision of the element was found to vary from 0 to 70% depending on the crossing combination. The frequency of the number of individual transposition events out of the total number of F2 plants with germinal excision was 44% in one crossing combination and 38% in the other. In the most efficient case, 10 plants with independent transposition were obtained out of the 49 F2 plants tested. Linkage analysis of the empty donor site and the transposed Ds-insertion site in F3 plants demonstrated that one of five Ds-insertion sites was not linked to the empty donor site. The transgenic rice obtained in this study can be used for functional genomics of rice.     

Ac-Induced Instability at the Xanthophyllic Locus of Tomato

To detect genomic instability caused by Ac elements in transgenic tomatoes, we used the incompletely dominant mutation Xanthophyllic-1 (Xa-1) as a whole plant marker gene. Xa-1 is located on chromosome 10 and in the heterozygote state causes leaves to be yellow. Transgenic Ac-containing tomato plants which differed in the location and number of their Ac elements were crossed to Xa-1 tester lines and F(1) progeny were scored for aberrant somatic sectoring. Of 800 test and control F(1) progeny screened, only four plants had aberrantly high levels of somatic sectors. Three of the plants had twin sectors consisting of green tissue adjacent to white tissue, and the other had twin sectors comprised of green tissue adjacent to tissue more yellow than the heterozygote background. Sectoring was inherited and the two sectoring phenotypes mapped to opposite homologs of chromosome 10; the green/yellow sectoring phenotype mapped in coupling to Xa-1 while the green/white sectoring phenotype mapped in repulsion. The two sectoring phenotypes cosegregated with different single, non-rearranged Acs, and loss of these Acs from the genome corresponded to the loss of sectoring. Sectoring was still observed after transposition of the Ac to a new site which indicated that sectoring was not limited to a single locus. In both sectored lines, meiotic recombination of the sectoring Ac to the opposite homolog caused the phenotype to switch between the green/yellow and the green/white phenotypes. Thus the two different sectoring phenotypes arose from the same Ac-induced mechanism; the phenotype depended on which chromosome 10 homolog the Ac was on. We believe that the twin sectors resulted from chromosome breakage mediated by a single intact, transposition-competent Ac element.