High rates of Ac/Ds germinal transposition in Arabidopsis suitable for gene isolation by insertional mutagenesis.

Abstract

Overexpression of the Activator (Ac) transposase gene in Arabidopsis thaliana resulted in a minimal germinal transposition frequency of 27% in which independent Dissociation (Ds) transposition events were observed. Molecular analysis of 45 F1 generation AciDs plants indicated that high rates of somatic excision had occurred, and independent germinal insertions were identified in F2 generation progeny plants. A tandem cauliflower mosaic virus (CaMV) promoter fused to two different Ac coding sequences signifcantly increased the rate of Ds transposition. The CaMV-Ac fusions activated single and multiple copies of two different Ds elements, DsDHFR and Ds35S-1, and reciprocal crosses resulted in similar transposition frequencies. The improved rate of independent germinal transposition observed makes Arabidopsis an ideal system for insertional mutagenesis.Arabidopsis thaliana is an excellent model plant for classical and molecular genetic studies. Current efforts toward characterizing the genome of Arabidopsis include restriction fragment length polymorphism analysis (1, 2), production and physical mapping of yeast artificial chromosome libraries (3-5), and Agrobacterium tumefaciens T-DNA insertional mutagenesis (6-8). An alternative means of isolating genes, which has been effective in many other model systems, is the use of transposable elements. In this paper, we report the development of a two-element Activator/Dissociation (Ac/ Ds) transposable element system suitable for gene tagging in Arabidopsis.The Ac/Ds transposable element system was originally discovered in maize by McClintock (9). It has since been shown that Ac transposes in Nicotiana tabacum (10) and numerous other dicotyledonous plant species (for review, see ref. 11). Its behavior in heterologous plant species has been most intensively studied in tobacco (10, 12-21), tomato (22-24), and A. thaliana (25-28). Many transposition-related properties associated with Ac activity in maize are also found in heterologous systems. For instance, the 4.6-kilobase (kb) autonomous element forms 8-base-pair duplications upon insertion (10), is capable of mobilizing receptor Ds elements (12,13,25,29), and preferentially transposes to nearby locations (16,28).To develop an efficient transposon tagging system for Arabidopsis and other plants, we have constructed a twoelement Ac/Ds system containing plant selectable markers designed to monitor the presence of Ac, Ds, and Ds-related excision events (25,30). A marked Ds element (DsDHFR) was constructed by inserting a plant selectable marker cassette, including a dihydrofolate reductase (DHFR) gene under the control of a cauliflower mosaic virus (CaMV) 35S promoter, into the central region of a Ds element. We have previously shown that the DsDHFR element transposes in tobacco and Arabidopsis, and the DHFR cassette does not decrease the frequency of Ds transposition (25). Furthermore, it was determined that approximately two-thirds of excised DsDHFR elements reinserted in transgenic tobacco protoplasts while the oth...

Transposition of a Ds element from a plasmid into the plant genome in Nicotiana plumbaginifolia protoplast‐derived cells

“…The assay relied on measuring restoration of GUS activity in the protoplast population. Using the same histochemical test, Becker et al (1992) recently identified more active ORFa constructs: the most active Ac was engineered so that the ORF starts at the 10th ATG. With this construct, the frequency of Ds excision was high even when the ORF was driven by the native Ac promoter.…”

“…One million N. plumbaginifo/ia protoplasts were transformed with 10 I~g target + 20 lig pBAc102 (supplied by B. Masterson). pBAc102 carries the same genomic transposase sequence as the pNT804 effector but under the control of a double CaMV 35S promoter followed by a synthetic oligonucleotide sequence derived from the 5'-leader of tobacco mosaic virus (Grevelding et al, 1992). pBAc102 was used instead of pNT804 because it does not carry the bacterial ampicillin-resistance gene.…”

“…Ac activity has been monitored using artificial Ds elements produced by deletion of the autonomous Ac element; the Ds elements transpose in a heterologous host provided the Ac transposase encoded by ORFa is also present (Altmann et al, 1992;Bancroft et aL, 1992;Grevelding et aL, 1992;Lassner et al, 1989;Swinburne eta/., 1992).…”

Alternative processing of the maize Ac transcript in Arabidopsis

“…A previous study by Grevelding et al (1992) suggested that a significant prorportion of the Ac transcript in Arabidopsis has a size of around 1.1 kb, and it was hypothesized that this may be due to incorrect processing of the mRNA. We have already demonstrated that intron 4 is alternatively spliced in Arabidopsis but none of the gene products was as small as those found by Grevelding.…”

“…The majority of work investigating Ac transposition has utilized the genomic sequence as a transposase source rather than the cDNA, as studies comparing the activity of the two suggest that the cDNA is even less active in tobacco than the genomic Ac element (Schofield et aL, 1993). One explanation for reduced AciDs transposition in Arabidopsis could be that Arabidopsis inefficiently processes Ac pre-mRNA (Grevelding et al, 1992;Keller et al, 1992;Swinburne et al, 1992), as previous research has established that introns derived from monocotyledonous (monocot) genes are often aberrantly spliced in dicot plant species ' (Goodall and Filipowicz, 1991;Keith and Chua, 1986), and possibly aberrantly polyadenyated (Keith and Chua, 1986). In this paper we confirm that the maize Ac transposase transcript is both alternatively spliced and prematurely polyadenyl- Primers 'a' span the first two introns (5' anneals from 1047; 3' to position 2671), and primers "b' span the latter two introns (5' anneals to position 3033; 3' at position 4060).…”