A transient assay for Ac excision in parsley (Petroselinum crispum)
--Ralf Lütticke and Reinhard Kunze
A transient excision assay has been developed using Petunia hybrida protoplasts cotransfected with two plasmid DNAs (Houba-Herin et al., MGG 224:17, 1990): one contains a Ds-element (AcÆ) between the octopine TR-DNA 1'-promoter and the E. coli ß-glucuronidase (GUS) gene (pNT 100). The other plasmid carries the Ac-coding sequence (ORFa) or mutants of ORFa behind the 2'-promoter. After expression of a functional Ac protein the Ds element (AcÆ) can be excised, thereby restoring the activity of the ß-glucuronidase gene. GUS activity is detected by histochemical staining (Jefferson et al., EMBO J. 6:3901, 1987) as blue stained protoplasts. The number of blue protoplasts after fixation on a filter is considered to be AcÆ-excision frequency. As only leaves from young sterile plants of a certain age can be used as a protoplast-source, a continuous supply of young plants is required for routine usage.
We tested the suitability of a fast growing parsley suspension culture (obtained from Dierck Scheel and Klaus Hahlbrock, MPI für Züchtungsforschung, Cologne) as a source of protoplasts for the cotransfection experiments. The background frequency of blue spots after transfection with the target plasmid pNT100 (1'Pr. - AcÆ - GUS) alone is slightly higher (2 to 3 blue spots per 105 protoplasts) compared to Petunia protoplasts. Cotransfection of the parsley protoplasts with pNT100 and a plasmid carrying the wildtype Ac element resulted only in a 2-3-fold increase above background. Very similar numbers were obtained when cotransfecting a genomic ORFa fragment or a cDNA ORFa fragment behind the 2'-promoter, respectively.
Li and Starlinger have shown that the 102 N-terminal amino acids of the Ac ORFa protein are dispensable. The truncated ORFa protein mobilized a Ds element with an even higher frequency than the full length ORFa protein (Li and Starlinger, PNAS 87:6044, 1990). This truncated genomic (= intron containing) ORFa placed behind the 2'-promoter (plasmid pNT804) enhanced the AcÆ excision-frequency in Petunia protoplasts even about 15-fold compared to the full length ORFa protein, although the ATG codon at amino acid 103 was preceded by 7 out-of-frame ATGs (Houba-Herin et al., MGG 224:17, 1990). In parsley protoplasts we have results from only 3 independent transfections. Therefore we consider these results to be preliminary, pNT804 increased the AcÆ excision frequency as it does in Petunia, but only about 2-3-fold compared to the wildtype Ac element. Cotransfection with an equally truncated cDNA fragment, however, resulted in a 6-fold increase.
We conclude that parsley protoplasts can be used as an alternative to Petunia protoplasts. The AcÆ excision frequencies are slightly lower than in Petunia, but the qualitative results seem to be identical. The sensitivity of both assay systems is very limited, as only excision frequencies above the level triggered by the wildtype Ac ORFa protein can be detected reliably. The advantages of the parsley cells are their quick and nearly unlimited availability.
We have begun to investigate derivatives of the truncated ORFa protein in the parsley cell system. Initial experiments revealed that removal of the 7 upstream ATGs by placing the 10 in-frame ATG immediately behind the 2' promoter has virtually no influence on the excision frequency. Preliminary experiments indicate that an N-terminally truncated ORFa protein starting with amino acid 137--that is immediately behind a tenfold repeat of the dipeptide Pro-Gln/Glu--abolishes the transposition function. A construct lacking 220 amino acids from the C-terminus of the ORFa protein is transposition deficient, too, or induces such events only with a very much reduced frequency. If these results can be confirmed, we have to conclude that essential functions of the 807 amino acid Ac ORFa protein are located between amino acids 103 and 136, and within the 220 C-terminal amino acids, respectively.
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