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American Society of Plant Biologists Somatic and Germinal Excision Activities of the Arabidopsis Transposon Tag1 Are Controlled by Distinct Regulatory Sequences within Tag1Section of Cell and Developmental Biology, Division of Biology, University of California at San Diego, La Jolla, California 92093-0116 3 To whom correspondence should be addressed. E-mail ncrawford{at}ucsd.edu; fax 858-534-1637
Various sequences within Tag1, the endogenous transposon of Arabidopsis, were examined to determine how Tag1 excision and expression are regulated. The 5' intron for the major 2.3-kb Tag1 transcript was found to be critical for the accumulation of Tag1 transcripts and for high rates of somatic excision. This was true for the autonomous element in cauliflower mosaic virus 35S–Tag1–  -glucuronidase constructs and for a two-component system using the 35S promoter to produce Tag1 transposase and a -glucuronidase::dTag1 marker construct to score for excision. The 3' introns of Tag1, although not needed for high transposase expression in primary transgenic plants, were important for maintaining high levels of somatic excision and accumulation of the major but not the minor Tag1 transcripts in subsequent generations. With both 5' and 3' introns present, exchanging the 5' promoter region of Tag1 with the 35S promoter did not affect the timing of Tag1 excision significantly, but it did disrupt germinal excision. Removal of the 5' intron did not abolish germinal excision activity, however. These results indicate that somatic and germinal excision of Tag1 are differentially controlled, with the 5' promoter region being critical for germinal excision activity and the 5' intron playing an important role for somatic excision, possibly via intron-mediated enhancement.
The regulation of eukaryotic transposable elements involves multiple mechanisms and levels of control (reviewed in Labrador and Corces, 1997  ; Haren et al., 1999). A classic example is germline-specific excision of transposons in Drosophila melanogaster. For P elements, regulation is mediated by differential splicing of the transposase RNA. In the germline, P element RNA is spliced by removing three introns to generate a transcript that encodes the 87-kD transposase (reviewed in Rio, 1990; Engels, 1996). Proper splicing of the third intron of the transposase RNA is inhibited in somatic cells by a 97-kD protein that binds just upstream of the 5' splice site. This binding results in an alternatively spliced mRNA that encodes a 66-kD protein that represses transposition (Misra and Rio, 1990; Lemaitre and Coen, 1991). For comparison, the D. melanogaster hobo element is restricted to the germline by transcriptional regulation of promoter activity (Calvi and Gelbart, 1994).
Regulated transposition also has been described for plant transposons (reviewed in Fedoroff, 1989
Another element that shows developmental control of both somatic and germinal excision is Tag1, an endogenous, autonomous transposon of Arabidopsis (Tsay et al., 1993a To investigate how Tag1 excision is regulated and how transposase expression might affect Tag1 excision, we constructed a two-component system using a CaMV 35S expression vector to produce transposase and a target-defective (dTag1) element in a GUS marker gene to score for excision. We began with a Tag1 cDNA clone for the 2.3-kb major transcript as the source of transposase. These experiments revealed that the 2.3-kb cDNA clone transcribed from the 35S promoter is insufficient to express transposase mRNA or activity. Experiments to determine what was missing in these constructs led to the discovery of two regions of Tag1 that are critical for transposase function: one for somatic excision and Tag1 mRNA accumulation, and the other for germinal excision. The results of these experiments are detailed below.
Requirements for Somatic Excision and Tag1 mRNA Accumulation To make an expression construct encoding a functional Tag1 transposase, we tested a Tag1 cDNA clone corresponding to the 2.3-kb major transcript of Tag1 (Figure 1) . This clone contains an open reading frame (ORF) for a protein of 729 amino acids (Liu and Crawford, 1998b ). Tag1 sequences were inserted between the CaMV 35S promoter and a nopaline synthase 3' terminator sequence in a modified pBI121 plasmid as described in Methods (Figure 1C). DNA constructs were transformed into Arabidopsis plants of the Columbia ecotype, which has no endogenous Tag1 elements (Tsay et al., 1993a; Frank et al., 1998). Primary transgenic plants were then crossed to plants homozygous for a 2.0-kb dTag1 element inserted into a 35S-GUS marker gene (pDTG1; Figure 2A)
(Frank et al., 1997). If the expression construct produces active transposase, the dTag1 element would excise, producing (1) blue sectors in leaves of F1 plants, indicative of somatic excision, and (2) entirely blue-staining plants in the F2 generation, indicative of germinal revertants.
The simplest transposase construct tested had the 729–amino acid ORF and no untranslated regions (UTRs) (pCTC5; Figure 1C). Six primary transformants containing pCTC5 were made and crossed to the 35S-dTag1-GUS marker line. At least 20 F1 hybrid seedlings from each cross were examined for GUS gene expression, and none showed any GUS sectors (data not shown). This result indicated that either the 729–amino acid protein was insufficient to produce a functional transposase or it was not expressed. To test this, RNA gel blot analyses were performed on progeny from all six primary transformants, and none showed any detectable Tag1 mRNA (data not shown). Even though the strong and constitutive 35S promoter was used to initiate transcription, no Tag1 mRNA accumulated. To determine if any of the UTRs of the 2.3-kb major transcript were needed for proper expression, several more constructs were made fusing the ORF to the 5' UTR only (pCTC4), the 3' UTR only (pCTC3), or to both the 5' UTR and the 3' UTR (pCTC2) (Figure 1C). These constructs were transformed into Arabidopsis, and four primary transformants for each construct were crossed to line pDTG1 containing the 35S-dTag1-GUS marker. At least 20 F1 hybrid seedlings from each cross were examined for GUS expression, and again none showed GUS sectors (data not shown). RNA gel blot analyses were performed on the progeny of the primary transformants, and none showed any Tag1 mRNA (data not shown). The next sequences tested were the introns and nontranscribed regions of Tag1. Constructs with different combinations of the four introns and the 3' nontranscribed sequences of Tag1 were included along with the complete ORF and the 5' and 3' UTRs (Figure 3) . pCTC1 had only the 5' intron and neither 3' introns nor the 3' nontranscribed region. pCTC10 and pCTC12 had the 3' introns but not the 5' intron, with pCTC10 retaining and pCTC12 omitting the 3' downstream sequences. pCTC9 and pCTC11 had all four introns, with pCTC9 retaining and pCTC11 omitting the 3' downstream sequences. Five primary transformants were generated and analyzed for each construct. Each was crossed to the 35S-dTag1-GUS marker line (pDTG1; Figure 2A), and F1 plants were examined for GUS sectors. Seed were collected from any F1 plants showing activity, and the F2 progeny were examined. In addition, Tag1 mRNA was analyzed in the progeny of each transgenic plant.
Analysis of these new constructs revealed that the 5' intron plays a major role in somatic excision activity and in Tag1 mRNA accumulation. Plants containing constructs with the 3' introns but lacking the 5' intron (pCTC10 and pCTC12) had very weak somatic excision activity in both F1 plants and their F2 progeny (Figure 4A) . Ninety of 130 F1 plants (70%) and 204 of 241 F2 plants (85%) had no activity, and those plants that did have activity had very few sectors (Table 1). RNA analyses of the pCTC10 and pCTC12 transgenic plants showed no Tag1 mRNA signals (Figure 5) , but on longer exposure these plants showed a very low level of Tag1 mRNAs (data not shown). Next, constructs with both 5' and 3' introns (pCTC9 and pCTC11) were tested. Both F1 and F2 plants showed abundant somatic activity (Figure 4B), with a higher percentage of plants (74% for the F1 plants and 51% for the F2 plants) showing sectors than did those lines lacking the 5' intron (Table 1). RNA analyses revealed that a major transcript as well as several smaller transcripts were present at signal intensities similar to the range found for plants containing the Tag1 element itself (Figure 5; cf. data reported by Liu and Crawford [1998b] ). These results show that the 3' nontranscribed region plays no obvious role in these constructs; however, the 5' intron is needed, even in the presence of the 3' introns, for accumulation of the Tag1 transcripts and strong somatic excision activity.
We checked to make sure that the 5' intron was needed for somatic excision and was not playing some other role in generating instabilities in gene expression that would produce blue sectors without excision of Tag1. Polymerase chain reaction (PCR) analysis was performed using primers that hybridized to the 35S promoter and the GUS sequence surrounding the Tag1 element. Genomic DNA was prepared from the leaves of several pCTC9 and pCTC11 lines showing many blue sectors. Amplification of the DNA by PCR showed DNA products corresponding to the excision of Tag1. Genomic DNA also was prepared from leaves of plants from pCTC10 and pCTC12 that showed very few to no blue sectors. One round of PCR (30 cycles) produced no detectable excision bands. Using the amplified DNA as a template for a second round of PCR (30 cycles), an excision band for pCTC12 but not for pCTC10 was produced. Thus, the appearance of blue sectors, which requires the 5' intron, is caused by excision of Tag1. We next examined the role of the 3' introns. Because the pCTC10 and pCTC12 constructs with the 3' introns alone showed so little somatic activity, we expected that the 3' introns would be dispensable if the 5' intron were present. Transgenic plants containing the pCTC1 construct, which incorporate only the 5' intron, were analyzed (Figure 3). RNA blots of the progeny of three pCTC1 transgenic plants showed strong Tag1 mRNA levels for the major transcript (Figures 5 and 6) . Excision activity in F1 plants containing the 35S-dTag1-GUS marker was present but definitely lower than that found for the pCTC9 and pCTC11 lines (Table 1). When the F2 progeny were examined, the pCTC1 lines showed even lower levels of somatic activity (Table 1).
The diminution of activity in the F2 plants suggested that there was a progressive loss of transposase activity or expression from generation to generation. RNA analyses were performed to determine the level of Tag1 mRNA in both the T2 transgenic lines and the F2 progeny (Figure 6). Progeny from three of the original transgenic lines (pCTC1-5, pCTC1-10, and pCTC1-12) showed high levels of mRNA; the other two lines had low (1 to 8) to very low (1 to 11) levels of Tag1 mRNA (Figure 6, pCTC1-T2). In contrast, the mRNA profiles for the F2 plants were very different from those of the original transgenic lines. The F2 plants had little or no major transcript but high levels of the smaller transcripts. For comparison, we examined the Tag1 mRNA profiles of the F2 plants from the highly active lines pCTC9-1, pCTC9-4, pCTC11-1, and pCTC11-4 containing all four introns. For these four lines, high levels of the major transcript were observed (Figure 6). These results indicate that the 5' intron is important for transposase mRNA accumulation and activity in the context of the 35S promoter but that the 5' intron is not sufficient for maintenance of transposase expression. The additional components needed for maintenance are the 3' introns.
The constructs used previously to express the Tag1 transposase all relied on the 35S promoter to initiate transcription of the transposase coding region. In this context, the 5' intron is critical for the expression of functional transposase. To determine if this is true for the Tag1 element itself, a deletion of the 5' intron was made in an intact Tag1 element. This Tag1 element was inserted into the pBI121 35S-GUS vector to produce pTG
One complication from deleting the 5' intron is that it could remove a cis-acting sequence needed for excision. To test this, a line containing the pTG  5 construct in the 35S-GUS marker was crossed to plants containing an intact element in a 35S-Tag1-SPT construct (Liu et al., 2001). F1 plants showed 50 to 300 sectors per leaf (medium level of activity; data not shown), demonstrating that the Tag1 deletion derivative missing the 5' intron still is capable of excision if provided transposase.
Requirements for Germinal Excision
To determine what was missing, we compared sequences between an intact Tag1 element, which shows germinal excision, and the pCTC11 construct, which supports high somatic excision but no germinal excision. The only difference between these constructs is the 5' promoter region. Tag1 has its own promoter, whereas the pCTC11 constructs have the 35S promoter. This comparison indicates that it is the Tag1 promoter region that is necessary for germinal excision activity. We then examined the necessity of the 5' intron for germinal excision activity. Germinal revertants were counted among T2 progeny containing the pTG
Timing of Excision
Molecular Cloning of Several Tag1 Minor Transcripts As reported previously (Liu and Crawford, 1998b ) and as can been seen in the RNA blots from the various pCTC lines (Figures 5 and 6), minor RNAs accumulate in plants containing Tag1 or the 35S-transposase constructs. To determine the nature of these RNAs, we analyzed them in more detail. First, RNA gel blot analysis was performed. An RNA blot using a 1.4-kb EcoRI fragment from the middle of Tag1 (Figure 7A
, probe A) hybridized to both major and minor transcripts (Figure 8)
, as reported previously (Liu and Crawford, 1998b). When the same RNA blot was rehybridized with probe B (Figure 7A), the major transcript showed a strong signal as expected, but no distinct minor transcripts were apparent, only a light smear (Figure 8).
These RNA blot results suggest that the minor transcripts are generated by premature termination or by alternative splicing. To test these possibilities, cDNA was prepared from poly(A)+ RNA isolated from leaves containing active Tag1 elements. The cDNA was used as a template for PCR with the following primers: an oligonucleotide corresponding to the transcription start site of the major transcript and oligo(dT). Two PCR products with approximate lengths corresponding to minor transcripts (1.2 and 1.0 kb) were isolated. Sequencing of these two cDNAs revealed that they have the same sequence as the 5' end of the major transcript and then terminate in a poly(A) sequence at residues Ala-1474 [CCCAACTA-poly(A)] for the 1.0-kb cDNA and Thr-1707 [ACACGTT-poly(A)] for the 1.2-kb cDNA (Figure 7B). This result is similar to what has been described for the Bacillus thuringiensis cryIA gene in tobacco, in which multiple polyadenylation sites lead to small transcripts that are terminated prematurely (Diehn et al., 1998 ). It is interesting that both Tag1 transcripts have an in-frame stop codon that is generated by adding adenines to the TA or T nucleotides at the end of the RNAs. Thus, some of the minor Tag1 transcripts arise from premature polyadenylation. On the basis of these analyses, we surmised that the minor transcripts are unlikely to encode a complete functional transposase or to be sufficient for Tag1 excision in Arabidopsis plants. One test of this hypothesis comes from our results with the pCTC1 lines, which contained the 35S promoter and a 5' intron but no 3' introns. The F2 plants from the cross pCTC1 x 35S-dTag1-GUS showed weak or no signals for the major transcript but high levels of minor transcripts (Figure 6, pCTC1-F2 lines). The somatic excision activity of these F2 lines was low or very low (Table 1). This finding suggests that the major transcript is necessary for transposase activity. We were not able to test this directly with an expression construct that produced only the major transcript, but we could test for transposase activity from the small transcripts. Both the 1.2- and 1.0-kb cDNA clones were inserted between the 35S promoter and the nopaline synthase terminator to make pCTC7 and pCTC8, respectively (Figure 9) . Both constructs were transformed into Arabidopsis of the Columbia ecotype. Four primary transformants from each construct were crossed to the pDTG1 35S-dTag1-GUS line. Twenty F1 hybrid seedlings were examined for each construct, and none showed GUS sectors (data not shown). Further crosses were performed to combine both pCTC7 and pCTC8 constructs into one transgenic line, which was then crossed to the pDTG1 line. GUS staining of the F1 hybrids showed no sectors (data not shown). These results support the hypothesis that the minor transcripts are not sufficient for transposase function; however, we cannot exclude the possibility that they may be part of the functional transposase.
The major finding described here is that there are distinct sequence requirements for somatic and germinal excision of Tag1 in 35S-GUS constructs. Germinal excision is dependent on the Tag1 5' promoter region being present, and somatic excision is dependent on the 5' intron, as is accumulation of Tag1 mRNA. Additional findings are that the maintenance of somatic excision depends on the 3' introns and that the timing of excision is not affected significantly by replacing the Tag1 5' promoter region with the 35S promoter. These results provide important insights into the regulation of Tag1 excision.
The reduction in somatic excision caused by deletion of the 5' intron can best be explained by a severe reduction in Tag1 mRNA accumulation. Quantification of transcript levels (by scanning autoradiography of the RNA blots) revealed that levels of Tag1 mRNA for the pCTC10 lines (which lack the 5' intron but contain the 3' introns) were 10 to 1% of those found in the pCTC11 lines (which contain all introns). In this context with the 35S promoter, the role of the 5' intron is likely to be post-transcriptional or at least after the initiation of transcription. If the intron were a transcriptional regulator, as in the case of the second intron of the Arabidopsis AGAMOUS gene (Sieburth and Meyerowitz, 1997
The requirements for somatic excision of Tag1 stand in contrast to the requirements for germinal excision, which include the Tag1 5' promoter region. For transposase expression constructs containing only the 35S promoter and no Tag1 5' promoter sequences, almost no germinal excision was observed, even for lines that had multiple transgenes. This is in contrast to what we observed with the intact Tag1 element in the 35S-Tag1-GUS constructs, in which the higher the number of transgenes, the greater the average germinal excision (Liu and Crawford, 1998a
A study of 35S promoter activity in Arabidopsis flowers using a 35S-APETALA3 construct showed that these constructs lead to the accumulation of APETALA3 RNA in all four whorls in buds as late as stage 6 (Jack et al., 1994 Our findings provide a way to design a transposon that is specific to the germinal lineages in Arabidopsis. By simply deleting the 5' intron, the somatic excision of Tag1 is essentially eliminated, yet fully active germinal excision is retained. Such a germline-specific element might be useful for insertional mutagenesis in cases in which somatic revertant sectors are lethal, such as when systemic toxins are applied to select for resistant mutants. As mentioned above, an intriguing result from our work is that the use of the 35S promoter to produce transposase mRNA did not qualitatively change the overall somatic excision behavior of Tag1 as measured using 35S-GUS constructs. The frequency of excision increased and the production of large sectors (a measure of excision timing) decreased with the pCTC9 and pCTC11 35S-transposase constructs compared with intact Tag1 in the 35S-Tag1-GUS constructs. However, overall excision behavior (the range of excision frequencies and a few early excision events) was similar. It is clear from these results that internal Tag1 sequences affect transposase production in our 35S-transposase constructs. What we do not know is how much the 35S promoter affects the expression and somatic excision of the intact Tag1 element in the 35S-Tag1-GUS constructs. Further experiments are needed to address this point.
Plasmid Construction To express the Tag1 cDNA sequences in Arabidopsis thaliana, the pBI121 vector (Jefferson, 1989 ) was used, which has a cauliflower mosaic virus 35S–-glucuronidase–nopaline synthase (35S-GUS-NOS) expression cassette with a kanamycin resistance gene as a selectable marker for plant transformation. pBI121 was modified by removing the GUS gene by digesting with SmaI and SacI, filling in with Klenow, and religating. The resultant plasmid was designated pCN1. Tag1 sequences were inserted into the XbaI and BamHI sites between the 35S promoter and the nopaline synthase 3' terminator sequences of pCN1.
Constructs pCTC2 to pCTC5 (Figure 1) were made using a cDNA clone of the major 2.3-kb Tag1 transcript (from plasmid pTC20 [Liu and Crawford, 1998b
pCTC1 (Figure 3) was made by replacing the 0.6-kb 5' XbaI-EcoRI fragment of pTC20 with a 0.8-kb PCR product covering from nucleotide 262 (transcriptional start site) to the first EcoRI site of Tag1 DNA using a Tag1 genomic clone in the pBT1 plasmid (Liu and Crawford, 1998b pCTC9 and pCTC10 (Figure 3) were made by replacing the 1.1-kb 5' XbaI-EcoRI fragment of Tag1 in pBT1 with (1) the same 0.8-kb PCR product used for pCTC1 described above (containing the 5' intron) to make pCTC9 or (2) the 0.6-kb 5' XbaI-EcoRI fragment from pTC20 (lacking the 5' intron) to make pCTC10. The two DNAs were inserted into pCN1. pCTC11 and pCTC12 (Figure 3) were made as follows. The Tag1 plasmid pBT1 was digested with SmaI and ClaI, filled in by Klenow enzyme, and religated. The resulting plasmid was then partially digested with EcoRI and XhoI. A 2.0-kb fragment was used to replace the EcoRI-XhoI fragment of plasmid pTC12 (the precursor of pCTC1). The resulting insert was excised with XbaI and BamHI and inserted into pCN1 to make pCTC11 containing the 5' intron. The same 2.0-kb fragment from pBTI also was used to replace the EcoRI-XhoI fragment of pTC20. The resulting insert was excised with XbaI and BamHI and inserted into pCN1 to make pCTC12 lacking the 5' intron.
The Tag1 clone lacking the 5' intron (pTG
Molecular Cloning of Small Tag1 Transcripts
Plant Transformation
RNA Gel Blot Hybridization
Phenotypic Assays for Tag1 Excision Frequencies
This work was supported by Grant MCB-9808215 from the National Science Foundation.
1 These authors contributed equally to this article. 
2 Current address: Dow AgroSciences LLC, 9330 Zionsville Rd., Indianapolis, IN 46268. Received January 19, 2001; accepted May 18, 2001.
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Abstract
INTRODUCTION
1.2 and 
