COLLEGE STATION, TEXAS

Texas A&M University

The frequency of Ac transposition from a beta-glucuronidase gene in tobacco varies between individual transformants --Brian H. Taylor, Naoma S. Nelson and Chantel F. Scheuring Previous studies of Ac transposition in dicotyledonous hosts such as tobacco and tomato have shown that there is a high degree of variability in the frequency of Ac transposition between individual transformants. For example, in the initial study of Ac transposition in tobacco, Baker et al. (PNAS 83:4844-4848, 1986) found evidence for transposition in 4 out of 9 transformed calli. Transposition was assayed by probing Southern blots of transformant DNA with probes corresponding to either the Ac element or to the donor DNA. Detection of transposed copies of Ac or empty donor sites indicated that transposition was occurring in the transformed tissue. Using a similar approach, Yoder et al. (Mol. Gen. Genet. 213:291-296, 1988) found that 9 out of 12 tomato plants carrying Ac exhibited detectable transposition, with some plants exhibiting evidence of multiple transpositions by the same Ac. Taylor et al. (Plant Mol. Biol. 13:109-118, 1989) examined 22 tobacco transformants containing Ac and found that 9 exhibited relatively high levels of activity, 11 were weakly active and 2 appeared to be completely inactive. While these results clearly indicate that different Ac "alleles" in transgenic plants can have very different transposition frequencies, a major limitation of this approach is that Southern hybridization techniques do not provide information on the distribution of the transposition events within the sampled tissue and are not sufficiently sensitive to detect very low levels of activity.

To circumvent these difficulties, a phenotypic assay for Ac transposition was developed using the beta-glucuronidase (GUS) gene of E. coli as a reporter gene. Recognition sequences for the restriction endonuclease BglII were inserted near each end of the Ac element isolated from the P-vv allele of maize by Peterson and Schwartz (MNL 60:36, 1986). The element was then inserted into a BamHI site located in the untranslated leader of a 35S-GUS expression cassette in the Agrobacterium binary vector Bin19 (pBI121; Jefferson et al., EMBO J. 6:3901-3907, 1987). When the Ac element is present between the 35S promoter and the GUS coding region, the GUS gene is inactive. Excision of the Ac element during transposition, however, reunites the 35S promoter and the GUS coding region and the gene is expressed. This results in the appearance of clonally derived sectors of cells expressing GUS that can be detected using either a sensitive fluorometric assay or by histochemical staining with the indigogenic substrate X-gluc (ibid.). Initial studies with this assay performed in the Peacock laboratory in Australia have been reported (Finnegan et al., Plant Cell 1:757-764, 1989).

To assess the utility of this assay as a means of monitoring Ac transposition frequency, we generated a series of tobacco transformants containing the Ac-GUS gene. Of 26 plants tested, 21 exhibited transposition activity, as determined by the appearance of GUS-expressing blue sectors after staining with X-gluc. Southern analysis of these plants showed that all 21 active plants contained restriction fragments indicative of the complete Ac-GUS gene, whereas none of the remaining inactive plants carried a complete gene. Although it was possible to detect transposition in most tissue types, for convenience our initial assays were performed on hand-cut petiole sections 0.2 to 0.5 mm thick. GUS-positive sectors could be readily detected in the vascular bundle region, cortex and epidermis, however, the transposition frequencies in each of these tissue types were often not correlated. Sector sizes ranged from single cells to complete staining of a particular tissue type. No completely stained sections were obtained and sectors extending from one tissue type into another were rare. Activity levels ranged from extremely low (only one sector observed in over 50 sections) to nearly uniform (very high activity levels and large sectors encompassing entire tissues are difficult to distinguish). The overall distribution of plants into low, medium and high activity classifications is shown in Table 1. In general, the apparent activity levels in sections taken from different leaves on the same shoot or different shoots from the same plant were consistent, although occasional differences were noted. No correlation between the copy number of Ac and the frequency of Ac transposition was observed. Many of the Ac-GUS plants discussed above have been in soil for more than a year. In that time a trend toward loss of sectoring in the vascular bundle and cortex region with continuing transposition in the epidermal tissue has been noted. One transformant exhibited this pattern initially; two plants that earlier showed sectoring in all tissues now show sectoring only in the epidermis and several appear to be in an intermediate stage, exhibiting clear zones encompassing most or all of the vascular bundle region in which no sectoring occurs. The reverse phenotype, sectoring in the vascular bundle and not in the epidermis, has also been observed. The numbers of plants exhibiting these phenotypes are indicated in Table 1. The nature of this apparent tissue specificity and the events leading to inactivation of the Ac-GUS gene in the vascular bundle and cortex are being studied. Initial analyses of epidermal and total leaf DNA from the plant that exhibited the tissue specific pattern originally have uncovered no detectable differences in restriction pattern or methylation state of the resident Ac element.

Table 1. Activity distribution of Ac-GUS tobacco transformants.
 
Activity level Sectors/section
VB+/E+
VB+/E-
VB-/E+
Low <10
3
1
1
Medium 10-30
3
-
-
High >30-uniform
8
1
4

*VB = Vascular bundle region, E = Epidermis. + = GUS-positive sectors, - = no sectors.

The results discussed here indicate that the frequency of Ac transposition varies considerably between individual transformants, is not necessarily uniform throughout each plant and may change with time. The transposition frequency of Ac in dicots is likely to depend on a number of factors, including the level of transposase present, the methylation state of the Ac DNA and, perhaps, factors relating to the specific site of Ac insertion in a given transformant. Further elucidation of the influence of these factors on the frequency, timing and tissue specificity of Ac transposition will increase our understanding of Ac regulation and perhaps lead to ways of optimizing the Ac element for gene tagging. The Ac-GUS phenotypic assay provides a useful means of monitoring Ac element activity in transgenic plants and will also be used to assess the transposition activities of modified Ac elements.


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