Characterization of the Putative Transposase mRNA of Tag1, Which Is Ubiquitously Expressed in Arabidopsis and Can Be Induced by Agrobacterium-Mediated Transformation With dTag1 DNA

Corresponding author: Nigel M. Crawford, Department of Biology, 0116, University of California at San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0116, ncrawford{at}ucsd.edu (E-mail).

Communicating editor: D. PREUSS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Tag1 is an autonomous transposable element of Arabidopsis thaliana. Tag1 expression was examined in two ecotypes of Arabidopsis (Columbia and No-0) that were transformed with CaMV 35S-Tag1-GUS DNA. These ecotypes contain no endogenous Tag1 elements. A major 2.3-kb and several minor transcripts were detected in all major organs of the plants. The major transcript encoded a putative transposase of 84.2 kD with two nuclear localization signal sequences and a region conserved among transposases of the Ac or hAT family of elements. The abundance of Tag1 transcripts varied among transgenic lines and did not correlate with somatic excision frequency or germinal reversion rates, suggesting that factors other than transcript levels control Tag1 excision activity. In untransformed plants of the Landsberg ecotype, which contain two endogenous Tag1 elements, no Tag1 transcripts were detected. Agrobacterium-mediated transformation of these Landsberg plants with a defective 1.4-kb Tag1 element resulted in the appearance of full-length Tag1 transcripts from the endogenous elements. Transformation with control DNA containing no Tag1 sequences did not activate endogenous Tag1 expression. These results indicate that Agrobacterium-mediated transformation with dTag1 can activate the expression of Tag1.

AN autonomous transposable element of Arabidopsis thaliana, Tag1 undergoes somatic and germinal excision late in shoot development (TSAY et al. 1993 ; FRANK et al. 1997 ; LIU and CRAWFORD 1998 ). Tag1 was first uncovered as an insertion in the fourth intron of a nitrate transporter gene, CHL1, which produced plants resistant to chlorate (TSAY et al. 1993 ). Tag1 is 3.3 kb in length, has 22-bp terminal inverted repeats, and produces an 8-bp direct repeat upon insertion. Analysis of Tag1 genomic sequence has revealed that Tag1 is a member of the Ac or hAT family of elements, which includes Ac and Bg in maize, Tam3 in snapdragon, Hobo in Drosophila, Hermes in housefly, Slide in tobacco, and Restless in the fungus Tolypocladium inflatum (CALVI et al. 1991 ; WARREN et al. 1994 ; ESSERS and KUNZE 1995 ; GRAPPIN et al. 1996 ; KEMPKEN and KUCK 1996 ). All members of this family produce an 8-bp target site duplication and share a signature protein sequence near the C terminus of the transposase.

Although Tag1 is endogenous to the Arabidopsis genome, it is not found in all ecotypes (geographical races) of A. thaliana (TSAY et al. 1993 ; BHATT et al. 1998 ; FRANK et al. 1998 ). For example, two Tag1 elements are present in the Landsberg erecta ecotype but none in Columbia, WS, or certain isolates of No-0. Ecotypes that lack endogenous Tag1 elements have been used for studying Tag1 excision from the marker gene CaMV 35S-GUS (FRANK et al. 1997 ). Such studies have shown that Tag1 somatic excision activity is restricted to late stages of vegetative and reproductive development in the shoot (LIU and CRAWFORD 1998 ). These studies also showed that Tag1 germinal excision activity is affected by Tag1 copy number, genetic dosage, and chromosomal location and can be as high as 25–30% (LIU and CRAWFORD 1998 ). Still unknown are the mechanisms that control Tag1 excision and the identity of the gene product(s) that are needed for Tag1 transposition.

To begin elucidating the mechanisms and gene products that control Tag1 transposition, we have characterized the mRNA expression patterns of Tag1 and compared them to somatic and germinal excision rates of the element. We have also isolated and sequenced Tag1 cDNA clones and found that the major Tag1 transcript encodes a putative transposase protein containing the signature sequence common to transposases of the Ac superfamily. We also found that expression of endogenous Tag1 elements is ubiquitous, encompasses all major organs of the plant, and can be activated by Agrobacterium-mediated transformation with dTag1 DNA. The results of these experiments are presented below.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plant material:
DNA constructs were first transformed into Agrobacterium tumefaciens strain C58 AGL-0 (LAZO et al. 1991 ) and then into Arabidopsis plants using vacuum infiltration (BECHTOLD et al. 1993 ). Tissue from organs other than the root was obtained from Arabidopsis plants grown in peat soil and grown under continuous light at 23–25° for 3 wk before harvesting. For root tissue, plants were grown for 10 days in submerged liquid culture as described (LABRIE and CRAWFORD 1994 ).

Molecular cloning and sequence analysis:
A PCR-based strategy was used for cloning Tag1 mRNA. Poly(A)⁺ RNA was isolated from plant leaves of 35S-Tag1-GUS transgenic plants (FRANK et al. 1997 ) using QuickPrep Micro mRNA Purification Kit (Pharmacia Biotech, Piscataway, NJ). cDNA was synthesized using the Ready-To-Go cDNA Synthesis Kit (Pharmacia Biotech). Primers for subsequent reactions are given below and shown in Figure 1A. Primers 1 and 2 were used to generate the middle 1.6-kb fragment of Tag1 cDNA. Primer 3 was used with oligo(dT) (primer 4) to amplify the 3' part of Tag1 cDNA. The 5' end of the Tag1 cDNA was generated by the 5'-RACE (5'-rapid amplification of cDNA end) procedure of FROHMAN et al. 1988 . Poly(A)⁺ RNA was reverse transcribed by first heating 0.5 µg RNA in 38 µl of water at 65° for 5 min, mixed with 10 µl of 5x reaction buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl, 5 mM DTT, 5 mM of each dNTP) and 1 µl (1 µg) of primer 5, 1 µl (200 units) of Superscript II RNaseH^- reverse transcriptase (GIBCO BRL Life Technologies, Gaithersburg, MD) and incubated at 37° for 30 min. cDNA products were gel-purified to remove the excess primer and dissolved in 22 µl water. cDNA was mixed with 6 µl 5x buffer (500 mM potassium cacodylate, pH 7.2, 10 mM CoCl₂, 1 mM DTT) and 1.5 µl 10 mM dCTP, 1.5 µl terminal deoxynucleotidyl transferase (15 units) (GIBCO BRL Life Technologies) and incubated at 37° for 1 hr. One microliter of poly(C)-tailed cDNA was used for PCR reaction with oligo(dG) (primer 7) and primer 6. One PCR band was produced and subsequently cloned. The complete cDNA clone (see Figure 1B) was assembled from the three fragments. The complete Tag1 cDNA sequence was deposited in GenBank under the accession number AF051562. The sequence of the primers used in RT-PCR (reverse transcription-polymerase chain reaction) and 5' RACE procedures are listed below where (+) refers to upper strand and (-) to bottom strand. The first nucleotide of the 5' inverted repeat is designated as position 1 (Figure 1A).

• Primer 1: 5'-GAAACACCATCTTGCTGG-3' (+: 725–742)

  
  

  View larger version (19K): In this window In a new window Download PPT slide   Figure 1. —Diagram of Tag1, its products and DNA constructs. DNA cloning steps are described in text. (A) Position of primers used in cloning procedures is shown. The double line represents the cDNA clone of the Tag1 transcript with poly(C) and poly(G) tails attached at the ends. (B) Diagram of major Tag1 transcript and introns is shown relative to the Tag1 genomic DNA. Vertical bars at each end represent terminal inverted repeats. The numbers 262 and 3141 refer to the beginning and end of the Tag1 transcript. Boxed, hatched line below the diagram refers to the ORF with introns indicated by numbers. Boxes above diagram refer to probes that were a 1.4-kb EcoRI fragment (probe A, nucleotides 1096–2424), a 475-bp fragment (probe B, nucleotides 900–1373), and a 662-bp fragment (probe C, nucleotides 1762–2424). (C) Schematic diagram of the protein encoded by major Tag1 transcript is shown with NLS () and Ac-transposase homology sequences () indicated. (D) Schematic diagram of Tag1 in the CaMV 35S-GUS construct used for transformation. (E) Schematic diagram of the defective dTag1 in the CaMV 35S-GUS construct used for transformation. The dTag1 is missing the internal 1.4-kb EcoRI fragment of Tag1.

• Primer 2: 5'-GCTCACATCCAGATGAAG-3' (-: 2440–2457)

• Primer 3: 5'-GGGATGTACCGAGCA-3' (+: 1959–1973)

• Primer 4: Oligo(dT)18

• Primer 5: 5'-TGAAGGACCCACATATCC-3' (-: 1149–1166)

• Primer 6: 5'-CCAGCAAGATGGTGTTTC-3' (-: 725–742)

• Primer 7: Oligo(dG)18

PCR reactions were performed at 94°, 1 min; 60°, 2 min; and 72°, 3 min for 35 cycles. All PCR products were cloned into the EcoRV site of pBluescript (SK) vector (Stratagene, La Jolla, CA) and sequenced using dideoxy chain termination methods. Sequence analysis was performed by Wisconsin Sequence Analysis Package "GCG" program (Version 8.0).

Northern hybridization:
Total RNAs were isolated as described (CRAWFORD et al. 1986 ). Twenty micrograms total RNA was separated on 1.2% agarose gels containing 6% formaldehyde and transferred to nylon membranes. Hybridizations were performed at 42° for 24 hr in a solution containing 50% formamide, 5x SSPE, 5x Denhardt's solution, 0.1% SDS, and 100 µg/ml herring sperm DNA. After hybridization, membranes were washed twice with 2x SSPE, 0.5% SDS for 15 min, then twice with 0.1x SSPE, 0.1% SDS. The first three washes were at room temperature, and the final wash was at 42°.

Tag1 excision assay in leaves and germinal activity:
Tag1 excision assays of plant leaves were performed by histochemical staining for GUS (ß-glucuronidase) activity as described (LIU and CRAWFORD 1998 ). Tag1 germinal reversion rate was determined by counting the number of progeny from primary transformants that stained completely blue.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Expression of Tag1 element:
To examine the mRNA transcripts expressed by Tag1, RNA blot hybridization experiments were performed using a 1.4-kb internal EcoRI fragment of Tag1 as probe (probe A in Figure 1B). Total RNA was extracted from leaves of 3-wk-old plants that were transformed with a CaMV 35S-Tag1-GUS construct (Figure 1D; FRANK et al. 1997 ; LIU and CRAWFORD 1998 ). In untransformed control plants of the No-O or Columbia ecotype, which have no endogenous Tag1 elements, no Tag1 transcripts were detected (Figure 2A, lanes 2 and 4). In transformed plants, one major band of ~2.3 kb in size was detected along with two minor bands of 1.0 and 1.2 kb (Figure 2A, lane 3, lanes 5–12).

View larger version (61K): In this window In a new window Download PPT slide   Figure 2. —RNA blot analysis of Tag1 mRNA in various plant lines. RNA blot conditions are given in MATERIALS AND METHODS, and the radiolabeled fragment used for RNA blots was probe A in Figure 1B. (A) RNA blot shows Tag1 mRNAs along with somatic and germinal reversion frequencies for different transgenic lines of No-0 and Columbia ecotypes carrying 35S-Tag1-GUS construct. RNA samples were as follows: lane 1, RNA molecular markers; lane 2, untransformed No-0 plants; lane 3, transformed No-0 plants; lane 4, untransformed Columbia plants; lanes 5–12, transformed Columbia plants. Each transgenic line carries from one to nine copies of Tag1 at a single locus. Somatic excision frequency is given as the average number of GUS sectors per leaf in leaf 3 or 4 observed in five to 10 plants. Germinal reversion rate was determined by scoring completely blue-staining seedlings among the progeny from primary transformants as described (LIU and CRAWFORD 1998 ). "H" means high excision frequency, that is, more than 1000 GUS sectors per leaf. (B) RNA blot shows Tag1 mRNA expression in different Landsberg plant lines. Lane 1, untransformed Landsberg plants; lanes 2–4, Landsberg plants transformed with 35S-Tag1-GUS construct; lanes 5 and 6, Landsberg plants transformed with 35S-dTag1-GUS construct; lanes 7–9, Landsberg plants transformed with plant expression vector pCGN1578 (MCBRIDE and SUMMERFELT 1990 ). The defective Tag1 element (dTag1) used for the Landsberg plants is missing the 1.4-EcoRI fragment of Tag1 (FRANK et al. 1997 ) that was used as probe A for the RNA blots.

The transcripts observed in Figure 2A came from Tag1 elements introduced into plants with the GUS transgene adjacent to the 35S promoter. To determine the transcript pattern produced by endogenous Tag1 elements, plants of the Landsberg erecta ecotype were examined by RNA blot analysis. Plants of this ecotype, hereafter called Landsberg, have two endogenous Tag1 elements (FRANK et al. 1997 ) but show no detectable Tag1 transcripts by RNA blot analysis (Figure 2B, lane 1). After transformation with the 35S-Tag1-GUS construct, the Landsberg plants showed the same Tag1 transcripts observed for transformed No-0 and Columbia plants as described above (Figure 2B, lanes 2–4). However, one cannot distinguish transcripts of the endogenous elements from those of the transgene in these lines; therefore, transgenic lines were generated that contained a defective Tag1 element lacking an internal 1.4-kb EcoRI fragment (Figure 1E; FRANK et al. 1997 ). RNA blots from these transgenic lines probed with the same 1.4-kb EcoRI fragment (probe A in Figure 1B) should display only transcripts from the endogenous elements. RNA from two such transformed Landsberg lines was found to have the same pattern of major and minor transcripts observed in plants carrying the 35S-Tag1-GUS construct (Figure 2B, lanes 5 and 6). No evidence of the probe hybridizing to transcripts from the dTag1 construct was found. We conclude that, when expressed, Tag1 primarily produces a 2.3-kb transcript along with several smaller, minor transcripts in leaves and that an adjacent 35S promoter does not affect the transcript pattern produced by Tag1.

This RNA analysis also indicated that expression of endogenous Tag1 elements is much higher in transgenic as compared with untransformed lines, where expression is undetectable in leaves. Perhaps Agrobacterium-mediated transformation activates Tag1 elements in Landsberg. To test this idea, three Landsberg transgenic lines that had been transformed with control DNA lacking Tag1 (i.e., with the plant expression vector pCGN1578; MCBRIDE and SUMMERFELT 1990 ) were examined. No Tag1 transcripts were detected in the leaves of these lines (Figure 2B, lanes 7–9). Care was taken to examine plants of the same generation (i.e., T₁ progeny from primary transformants) for both the pCGN and Tag1-transformed lines to eliminate propagation effects. These results indicate that transformation with Tag1 containing DNA, not just transformation itself, activates the expression of the endogenous Tag1 elements.

The RNA blots in Figure 2A also show the relative abundance of the Tag1 transcripts in several different lines. Transcript abundance varied from line to line and was compared to the excision activity of Tag1 in each line. Each transgenic line contained an excision marker (35S-Tag1-GUS). Those cells that inherit a 35S-GUS excision allele will stain blue for GUS expression. Somatic excision was assessed by counting the number of GUS sectors in leaf 3 or 4; germinal reversion rate was determined by counting completely blue-staining progeny, as described in LIU and CRAWFORD 1998 . No correlation was found between Tag1 mRNA levels and excision frequency (Figure 2A). Therefore, the Tag1 mRNA levels in the leaves of these lines do not affect the level of Tag1 somatic excision activity in leaves and do not correlate with germinal reversion rates.

Next, Tag1 expression studies were expanded to include other organs of the plant: root, leaf, stem, fully opened flower, young flower bud, and silique. RNA blot analysis was performed using transgenic lines carrying 35S-Tag1-GUS or 35S-dTag1-GUS constructs. Fairly uniform Tag1 expression was found throughout the plant (Figure 3). This finding was true both for Tag1 elements introduced as transgenes in the 35S-Tag1-GUS lines (Figure 3A) and for endogenous elements in the 35S-dTag1-GUS lines (Figure 3B). This non-organ-specific expression pattern correlates with our finding of Tag1 excision in all plant organs (LIU and CRAWFORD 1998 ).

View larger version (57K): In this window In a new window Download PPT slide   Figure 3. —RNA blot analysis of Tag1 mRNA expression in various plant organs. Hybridization conditions are given in Figure 2 legend. Blots were hybridized with probe A shown in Figure 1B. (A) RNA samples from a Columbia plant line (see Figure 2A, lane 6) transformed with 35S-Tag1-GUS construct. (B) RNA samples from a Landsberg plant line (see Figure 2B, lane 6) transformed with 35S-dTag1-GUS construct.

Cloning and sequence analysis of the major Tag1 transcript:
The major Tag1 transcript was cloned using RT-PCR and 5' RACE as described in MATERIALS AND METHODS. Sequence analysis showed that four introns are removed to produce the final transcript (Figure 1B and Figure 4). All four introns have GT/AG border sequences and contain AT-rich sequences (75–89% AT). All PCR products analyzed had the same 5' end starting at position 262, but the 3' end varied to produce spliced products of 2.3–2.4 kb in length (see Figure 4). When we compared the cDNA sequence to that published for the Tag1 genomic clone (accession number L12220; TSAY et al. 1993 ), several discrepancies were found. The original sequence has an extra A, T, T, C, and A at positions 1033, 1630, 1721, 1722, and 3044, respectively, and lacks a T at position 1108. We resequenced the Tag1 element and found that the cDNA sequence was correct. The correct Tag1 nucleotide sequence is 3295 bp in length and was provided to GenBank.

View larger version (118K): In this window In a new window Download PPT slide   Figure 4. —Tag1 nucleotide and protein sequence. Terminal inverted repeat sequences at each end are in boldface. Tag1 cDNA sequence is in uppercase. Untranscribed regions and intron sequences are in lowercase. Transcription start site is indicated by an arrow. Two putative TATA sequences are boxed. Stop codon is indicated by an asterisk. Four different transcription termination sites are indicated by open circles. Putative polyadenylation signal sequence ATTAAA is double underlined. Two nuclear localization signal sequences are underlined. Protein sequences that show high homology to other transposases are shaded.

Translation of the Tag1 cDNA reveals a single open reading frame (ORF) that encodes a 729-amino-acid protein with a calculated molecular mass of 84.2 kD and pI of 6.74. Two putative nuclear localization signal (NLS) sequences are located at amino acids 47–51, and 127–144 (Figure 1C and Figure 4). The first NLS sequence consists of five basic amino acids, which is a SV40-like NLS, and the second has a combination of two regions of basic amino acids separated by a space of about 10 residues, which is a bipartite NLS (HICKS et al. 1995 ). The predicted Tag1 protein also contains the conserved transposase sequence found among members of the Ac or hAT family (Figure 1C and Figure 4). The conserved region is also shown in Figure 5, which includes the sequence of the protein encoded by the Tag1 cDNA. Previous comparisons (WARREN et al. 1994 ; ESSERS and KUNZE 1995 ) used the Tag1 genomic sequence, which has an extra 4–5 amino acids at the 5' end of the conserved regions that are not present in the cDNA sequence due to splicing.

View larger version (26K): In this window In a new window Download PPT slide   Figure 5. —Conserved transposase sequences in Ac or hAT family. Amino acid residues that are identical in at least four sequences are boxed.

Analysis of subterminal regions of Tag1 element:
It has been shown that multiple repetitive sequence motifs in the transposon's subterminal region play an important role for the element's transposition and serve as binding sites for the transposase (reviewed in SAEDLER and GIERL 1996 ). We examined the subterminal regions of Tag1 and found at the 5' end a motif of AAACCC repeated 12 times in both orientations (Figure 6A). Six of these repeats are perfect, and the other six have one nucleotide change. There is also a 19-bp direct tandem repeat with the putative "TATA" box sequence. Unlike other transposons, the AAACCC motif is not found in the subterminal region at 3' end. Instead, the 3' end region has a TTATT sequence motif repeated 14 times, all in the same orientation (Figure 6B). Eight of the repeats are perfect, and six have one nucleotide change. Two other motifs, TATATA and GACCC, are repeated four times each in direct orientation. This 3' subterminal region is highly AT-rich (80% AT).

View larger version (49K): In this window In a new window Download PPT slide   Figure 6. —Analysis of subterminal regions of Tag1. (A) Sequence at 5' end of Tag1 is shown. Terminal inverted repeat sequence is double underlined. Repeated AAACCC sequences are indicated by thin-lined arrows below. Nine-basepair tandem direct repeats are indicated by arrows with thick line. Putative TATA boxes are in bold. Transcription start site for Tag1 cDNA is indicated by closed circle at nucleotide 262. Transcribed sequences are capitalized. (B) Sequence of the 3' end of Tag1 is shown. Terminal inverted repeat is double underlined. Repeated sequences TTATT are indicated by arrows. TATATA motifs are boxed, and GACCC sequences are indicated by a black bar below. AT-rich sequences are in bold. Transcribed region is capitalized. Stop codon is indicated by an asterisk.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In this article we present data on the expression of Tag1, along with an analysis of the sequence of the putative transposase and its possible binding sites. RNA blot analysis revealed a major 2.3-kb Tag1 transcript ubiquitously present in all major organs of the plant. The transcript encompasses almost the entire length of Tag1 and contains an ORF with two NLS sequences and a region that is highly conserved among transposases of the Ac or hAT family of transposons. This conserved region is required for transposition of hobo elements in Drosophila (CALVI et al. 1991 ). If the major transcript encodes the functional Tag1 transposase, then Tag1 would be most similar to Ac, which requires only one transposase transcript for transposition (POHLMAN et al. 1984 ; COUPLAND et al. 1988 ; KUNZE and STARLINGER 1989 ). If any of the minor transcripts are also required for transposition, then Tag1 would be analogous to Spm, which requires two mRNAs generated by alternative splicing for transposition (MASSON et al. 1991 ). Tag1 transcription does not appear to resemble Mutator, which produces two transcripts by convergent, nonoverlapping transcription (CHOMET et al. 1991 ; HERSHBERGER et al. 1991 , HERSHBERGER et al. 1995 ; JOANIN et al. 1997 ).

Further analysis of the Tag1 transcripts showed that the major mRNA is produced from the removal of four introns, all with the consensus border sequences GT-AG and AT-rich internal sequences (BROWN 1986 ; GOODALL and FILIPOWICZ 1989 ). The first intron is located in the 5' untranslated region, a strategic location for regulation (e.g., see CALLIS et al. 1987 ; FU et al. 1995 ; SIEBURTH and MEYEROWITZ 1997 ). Because we found two minor transcripts along with the major one in leaves of transgenic plants, we wondered if they might be generated by alternative splicing as is the case for Spm (MASSON et al. 1989 ). We obtained several additional cDNA clones by the RT-PCR method described above that were smaller than the major transcript, but none of these clones corresponded in size to the minor transcripts (data not shown). We also hybridized our RNA blots (as seen in Figure 2) with internal Tag1 DNA fragments that were missing from the smaller cDNA clones (probes B and C in Figure 1B) and found that all RNA transcripts, major and minor, were labeled (data not shown). The origin of the smaller transcripts remains unknown.

With the borders of the major transcript defined, one can locate and analyze the (100–250 bp) nontranscribed regions of Tag1. Typically, sequences adjacent to the inverted repeats (subterminal repeats) of transposons have repeated sequences found at both ends that serve as binding sites for the transposase. For example, Ac transposase binds to the AAACGG and related sequence motifs that existed in both 5' and 3' subterminal regions (KUNZE 1996 ). For Spm, the TNPA component of the transposase binds to 12-bp motifs repeated at both ends (GIERL 1996 ). A model for transposition is that the transposase binds to the subterminal repeats, bringing together both ends of the element for subsequent cleavage next to the terminal inverted repeats. Sequence analysis of the subterminal repeats in Tag1 reveals repeated sequences at each end, but the repeated sequences at the 5' end are different from those at the 3' end. If these sequence motifs serve as transposase binding sites, instead of some other sequence such as the terminal inverted repeat, then it would appear that Tag1 does not fit this model for the mechanism of transposition. Further functional tests, including DNA binding assays, will be needed to determine the mechanism of Tag1 excision.

Tag1 expression is ubiquitous with abundant transcripts in all major organs of Arabidopsis. This finding correlates with the ubiquitous excision activity of Tag1 in all major organs (LIU and CRAWFORD 1998 ). However, the abundance of Tag1 mRNA does not correlate with somatic excision activity, measured as the number of somatic sectors in leaves, in our transgenic lines, nor with germinal reversion rates. These results contrast with those obtained with Ac in dicots where a higher level of transposase expression leads to higher frequency of excision up to a limit (SCOFIELD et al. 1992 , SCOFIELD et al. 1993 ; SWINBURNE et al. 1992 ). For Tag1 it appears that some factor other than Tag1 mRNA abundance as observed on the RNA blots is controlling excision frequency. Perhaps the active transposase levels in these lines are well above the saturating limit in the nucleus. Alternatively, translational or post-translational mechanisms are limiting active transposase levels, regardless of transcript abundance. Last, a component of the transposase not observed on our RNA blots or accounted for by our cDNA clone is limiting or controlling excision frequencies.

Unlike the transgenic lines, untransformed Landsberg plants had no detectable Tag1 transcripts even though they have two endogenous Tag1 elements. Transformation of Landsberg plants with a dTag1 element in the 35S-GUS construct produces plants with high levels of mRNA from the endogenous elements. This apparent activation of Tag1 expression does not occur when Landsberg plants are transformed with DNA containing no dTag1 sequences. It is interesting to compare these results with those of BHATT et al. 1998 , who showed that Tag1 transposition, as measured by Southern blot analysis, is activated by Agrobacterium-mediated transformation with any DNA (i.e., Ac, Ds, and control DNA). A total of 43 new Tag1 insertion events were observed from 241 transgenic lines. No Tag1 transposition was observed in untransformed Landsberg lines (188 lines examined), but two out of 118 Landsberg lines crossed to Columbia (i.e., recombinant inbred lines) showed a new Tag1 insertion. The near absence of transposition in untransformed lines fits with the absence of Tag1 mRNA that is reported here, but the activation of Tag1 transposition with non-Tag1 DNA does not correlate with our finding that transformation with control DNA does not activate Tag1 expression. The reason for this discrepancy is not clear; however, the transformation protocol was different in these two sets of experiments: the work of BHATT et al. used root transformation in tissue culture, whereas the work presented here was done using vacuum infiltration of whole plants. In summary, transformation with dTag1 DNA increases the level of Tag1 mRNA, possibly by activating the Tag1 promoter. We do not know if this activation is dependent on a specific sequence within dTag1, if it requires transcription of the dTag1 element to produce some activating protein product, or if it requires excision of the dTag1. The dTag1 element used in these experiments still retains the 5' upstream regions that include potential binding sites for the transposase and the upstream elements of the promoter. It also can excise from GUS construct (FRANK et al. 1997 ). Further analysis of the activation of Tag1 expression by different dTag1 sequences will be done to distinguish among these possibilities.

	ACKNOWLEDGMENTS

We wish to thank CHRISTIAN FANKHAUSER and JOANNE CHORY for pCGN transgenic Landsberg plants and MARY FRANK for No-0 plants transformed with 35S-Tag1-GUS. This work was supported by a grant from the National Science Foundation (MCB-9219374).

Manuscript received December 28, 1997; Accepted for publication March 6, 1998.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

BECHTOLD, N., J. ELLIS, and G. PELLETIER, 1993  In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. Mol. Biol. Genet. 316:1194-1199.

BHATT, A. M., C. LISTER, N. CRAWFORD, and C. DEAN, 1998  The transposition frequency of Tag1 elements is increased in transgenic Arabidopsis lines. Plant Cell 10:427-434[Abstract/Free Full Text].

BROWN, J. W. S., 1986  A catalogue of splice junction and putative branch point sequences from plant introns. Nucleic Acids Res. 14:9549-9559[Abstract/Free Full Text].

CALLIS, J., M. FROMM, and V. WALBOT, 1987  Introns increase gene expression in cultured maize cells. Genes Dev. 1:1183-1200[Abstract/Free Full Text].

CALVI, B. R., T. J. HONG, S. D. FINDLEY, and W. M. GELBART, 1991  Evidence for a common evolutionary origin of inverted repeat transposons in Drosophila and plants: hobo, Activator and Tam3. Cell 66:465-471[Medline].

CHOMET, P., D. LISCH, K. J. HARDEMAN, V. L. CHANDLER, and M. FREELING, 1991  Identification of a regulatory transposon that controls the Mutator transposable element system in maize. Genetics 129:261-270[Abstract].

COUPLAND, G., B. BAKER, J. SCHELL, and P. STARINGER, 1988  Characterization of the maize transposable Ac by internal deletions. EMBO J. 7:3653-3659[Medline].

CRAWFORD, N. M., W. H. CAMPBELL, and R. W. DAVIS, 1986  Nitrate reductase from squash: cDNA cloning and nitrate regulation. Proc. Natl. Acad. Sci. USA 83:8073-8076[Abstract/Free Full Text].

ESSERS, L. and R. KUNZE, 1995  Transposable elements Bg (Zea mays) and Tag1 (Arabidopsis thaliana) encode protein sequences with homology to Ac-like transposases. Maize Genetics Cooperation Newsletter 69:39-41.

FRANK, M. J., D. LIU, Y.-F. TSAY, C. USTACH, and N. M. CRAWFORD, 1997  Tag1 is an autonomous transposable element that shows somatic excision in both Arabidopsis and tobacco. Plant Cell 9:1745-1756[Abstract].

FRANK, M. J., D. PREUSS, A. MACK, T. C. KUHLMANN, and N. M. CRAWFORD, 1998  The Arabidopsis transposable element Tag1 is widely distributed among Arabidopsis ecotypes. Mol. Gen. Genet. 257:478-484[Medline].

FROHMAN, F. M., M. K. DUSH, and G. R. MARTIN, 1988  Rapid production of full-length cDNA from rare transcripts: amplication using a single gene specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85:8998-9002[Abstract/Free Full Text].

FU, H., S. Y. KIM, and W. D. PARK, 1995  High-level tuber expression and sucrose inducibility of a potato Sus4 sucrose synthase gene require 5' and 3' flanking sequences and the leader intron. Plant Cell 7:1387-1394[Abstract].

GIERL, A. 1996 The En/Spm transposable element of maize, pp. 145–159 in Transposable Elements, edited by H. SAEDLER and A. GIERL. Springer-Verlag, Berlin.

GOODALL, G. J. and W. FILIPOWICZ, 1989  The AU-rich sequence present in the introns of plant nuclear pre-mRNAs are required for splicing. Cell 58:473-483[Medline].

GRAPPIN, P., C. AUDEON, M.-C. CHUPEAU, and M.-A. GRANDBASTIEN, 1996  Molecular and function characterization of Slide, an Ac-like autonomous transposable element from tobacco. Mol. Gen. Genet. 252:386-397[Medline].

HERSHBERGER, R. J., C. A. WARREN, and V. WALBOT, 1991  Mutator activity in maize correlates with the presence and expression of the Mu transposable element Mu9. Proc. Natl. Acad. Sci. USA 88:10198-10202[Abstract/Free Full Text].

HERSHBERGER, R. J., M. I. BENITO, K. J. HARDEMAN, C. A. WARREN, and V. L. CHANDLER et al., 1995  Characterization of the major transcripts encoded by the regulatory MuDR transposable element of maize. Genetics 140:1087-1098[Abstract].

HICKS, G. R., H. M. S. SMITH, and N. V. RAIKHEL, 1995  Three classes of nuclear import signals bind to plant nuclei. Plant Physiol. 107:1055-1058[Abstract].

JOANIN, P., R. J. HERSHBERGER, M.-I. BENITO, and V. WALBOT, 1997  Sense and antisense transcripts of the maize MuDR regulatory transposon localized by in situ hybridization. Plant Mol. Biol. 33:23-36[Medline].

KEMPKEN, F. and U. KUCK, 1996  restless, an active Ac-like transposon from the fungus Tolypocladium inflatum: structure, expression, and alternative RNA splicing. Mol. Cell. Biol. 16:6563-6572[Abstract].

KUNZE, R. 1996 The maize transposable element Activator (Ac), pp. 162–194 in Transposable Elements, edited by H. SAEDLER and A. GIERL. Springer-Verlag, Berlin.

KUNZE, R. and P. STARLINGER, 1989  The putative transposase of transposable element Ac from Zea mays L. interacts with subterminal sequences of Ac.. EMBO J. 8:3177-3185[Medline].

LABRIE, S. T. and N. M. CRAWFORD, 1994  A glycine to aspartic acid change in the MoCo domain of nitrate reductase reduces both activity and phosphorylation levels in Arabidopsis.. J. Biol. Chem. 269:14497-14501[Abstract/Free Full Text].

LAZO, G. R., P. A. STEIN, and R. A. LUDWIG, 1991  A DNA transformation-competent Arabidopsis genomic library in Agrobacterium.. Biotech. 9:963-967.

LIU, D. and N. M. CRAWFORD, 1998  Characterization of the germinal and somatic activity of the Arabidopsis transposable element Tag1.. Genetics 148:445-456[Abstract/Free Full Text].

MASSON, P., G. RUTHERFORD, J. A. BANKS, and N. FEDOROFF, 1989  Essential large transcripts of the maize Spm transposable element are generated by alternative splicing. Cell 58:755-765[Medline].

MASSON, P., M. STREM, and N. FEDOROFF, 1991  The tnpA and tnpD gene products of the Spm element are required for transposition in tobacco. Plant Cell 33:73-75.

MCBRIDE, K. E. and K. R. SUMMERFELT, 1990  Improved binary vectors for Agrobacterum-mediated plant transformation. Plant Mol. Biol. 14:269-276[Medline].

POHLMAN, R. F., N. V. FEDOROFF, and J. MESSING, 1984  The nucleotide sequence of the maize controlling element Activator.. Cell 37:635-643[Medline].

SAEDLER, H., and A. GIERL (Editors), 1996 Transposable Elements. Current Topics in Microbiology and Immunology. Springer-Verlag, Berlin.

SCOFIELD, S. R., K. HARRISON, S. J. NURRISH, and J. D. G. JONES, 1992  Promoter fusions to the Activator transposase gene cause distinct patterns of Dissociation excision in tobacco cotyledons. Plant Cell 4:573-582[Abstract/Free Full Text].

SCOFIELD, S. R., J. J. ENGLISH, and J. D. G. JONES, 1993  High level expression of the Activator transposase gene inhibits the excision of Dissociation in tobacco cotyledons. Cell 75:507-517[Medline].

SIEBURTH, L. E. and E. M. MEYEROWITZ, 1997  Molecular dissection of the AGAMOUS control region shows that cis elements for spatial regulation are located intragenically. Plant Cell 9:355-365[Abstract].

SWINBURNE, J., L. BALCELLS, S. R. SCOFIELD, J. D. G. JONES, and G. COUPLAND, 1992  Elevated levels of Activator transposase mRNA are associated with high frequencies of Dissociation excision in Arabidopsis. Plant Cell 4:583-595[Abstract/Free Full Text].

TSAY, Y.-F., M. J. FRANK, T. PAGE, C. DEAN, and N. M. CRAWFORD, 1993  Identification of a mobile endogenous transposon in Arabidopsis thaliana.. Science 260:342-344[Abstract/Free Full Text].

WARREN, W. D., P. W. ATKINSON, and D. A. O'BROCHTA, 1994  The Hermes transposable element from the house fly, Musca domestica, is a short inverted repeat-type element of the hobo, Ac and Tam3 (hAT) element family. Genet. Res. 64:87-97[Medline].

This article has been cited by other articles:

				G. J. Grundy, J. E. Hesse, and M. Gellert Requirements for DNA hairpin formation by RAG1/2 PNAS, February 27, 2007; 104(9): 3078 - 3083. [Abstract] [Full Text] [PDF]

				Z. Xu and H. K. Dooner Mx-rMx, a Family of Interacting Transposons in the Growing hAT Superfamily of Maize PLANT CELL, February 1, 2005; 17(2): 375 - 388. [Abstract] [Full Text] [PDF]

				M. Galli, A. Theriault, D. Liu, and N. M. Crawford Expression of the Arabidopsis Transposable Element Tag1 Is Targeted to Developing Gametophytes Genetics, December 1, 2003; 165(4): 2093 - 2105. [Abstract] [Full Text] [PDF]

				A. M. Mack and N. M. Crawford The Arabidopsis TAG1 Transposase Has an N-Terminal Zinc Finger DNA Binding Domain That Recognizes Distinct Subterminal Motifs PLANT CELL, October 1, 2001; 13(10): 2319 - 2331. [Abstract] [Full Text] [PDF]

				D. Liu, R. Wang, M. Galli, and N. M. Crawford Somatic and Germinal Excision Activities of the Arabidopsis Transposon Tag1 Are Controlled by Distinct Regulatory Sequences within Tag1 PLANT CELL, August 1, 2001; 13(8): 1851 - 1863. [Abstract] [Full Text] [PDF]

				D. Liu, M. Galli, and N. M. Crawford Engineering Variegated Floral Patterns in Tobacco Plants Using the Arabidopsis Transposable Element Tag1 Plant Cell Physiol., April 1, 2001; 42(4): 419 - 423. [Abstract] [Full Text] [PDF]

				D. Liu, A. Mack, R. Wang, M. Galli, J. Belk, N. I. Ketpura, and N. M. Crawford Functional Dissection of the cis-Acting Sequences of the Arabidopsis Transposable Element Tag1 Reveals Dissimilar Subterminal Sequence and Minimal Spacing Requirements for Transposition Genetics, February 1, 2001; 157(2): 817 - 830. [Abstract] [Full Text]

				R. Wang, K. Guegler, S. T. LaBrie, and N. M. Crawford Genomic Analysis of a Nutrient Response in Arabidopsis Reveals Diverse Expression Patterns and Novel Metabolic and Potential Regulatory Genes Induced by Nitrate PLANT CELL, August 1, 2000; 12(8): 1491 - 1510. [Abstract] [Full Text]

				A. D. Henk, R. F. Warren, and R. W. Innes A New Ac-Like Transposon of Arabidopsis Is Associated With a Deletion of the RPS5 Disease Resistance Gene Genetics, April 1, 1999; 151(4): 1581 - 1589. [Abstract] [Full Text]