Germinal Excision Research Articles

Preferential transposition of the maize element Activator to linked chromosomal locations in tobacco.

The autonomous maize transposon Activator (Ac) has been used in maize for gene isolation by tagging and may prove similarly useful in other species. To test the feasibility of gene tagging with heterospecific transposons, we have examined three key genetic properties of a slightly modified Ac in tobacco. First, we show that frequencies of germinal excision of this Ac element from the antibiotic resistance gene streptomycin phosphotransferase can be comparable with or slightly lower than in maize. Second, we show that about half of the progeny carrying a germinal excision product also carry a transposed Ac. Last, we have mapped transposed Ac locations relative to the streptomycin transferase gene excision product and have shown that as in maize Ac in tobacco preferentially transposes to genetically linked sites.

The maize autonomous element Activator (Ac) shows a minimal germinal excision frequency of 0.2%–0.5% in transgenic Arabidopsis thaliana plants

The autonomous mobile element Activator from Zea mays was introduced into Arabidopsis thaliana via Agrobacterium-mediated gene transfer. The use of a chimaeric construct, where the Ac element is located in the leader of the neomycin phosphotransferase (NPT II) gene, enabled the excision of Ac to be monitored by assaying for the reconstitution of NPT II gene activity. Using this approach, the transpositional activity of AC was initially studied in primary transformants. About 50% of the regenerating Ac transformants showed evidence for excision of the element. Reintegration of Ac was confirmed by Southern blot analysis. Transposition events are transmitted to the F1 generation with a minimal frequency of 0.3%. In a few exceptional cases they are detected in a high proportion of the F1 generation. Seedlings from the F2 and F3 generations were assayed for the rate of germinal excisions by scoring for kanamycin resistance. The minimal frequency of germinal excision events amounts to 0.2%–0.5% and hence allows the use of the Ac element for gene tagging purposes in A. thaliana.

Cloning Knotted, the dominant morphological mutant in maize using Ds2 as a transposon tag

The Kn1-2F11 mutation causes protrusions or knots along the lateral veins of the first few leaves of the maize plant. The phenotype is visible when an unlinked gene, presumably Ac, is present in the genome. The mutation is closely linked to a genetically unstable Adh1 mutation that resulted from the insertion of a Ds2 element (Döring et al., 1984; Chen et al., 1986). Using a unique sequence from the Ds2 element as a hybridization probe, a genomic restriction fragment that cosegregated with the knotted phenotype was cloned. It carries the Kn1-2F11 locus by the following criteria. (i) Cosegregation of the fragment is tightly linked to the phenotype. (ii) Somatic and germinal excision produce a fragment which is the expected size of a revertant fragment; progeny containing the revertant size fragment are normal. (iii) The sequences that hybridize to this fragment are significantly altered in the chromosome containing the original knotted mutation, Kn1-O, (iv) The cloned fragment does not hybridize to a chromosome that contains a deletion of Kn1-O.