The influence of promoter strength on Ds excision frequency in a transient excision assay
--Detlef Becker, Ralf Lütticke and Peter Starlinger
We have developed a transient petunia protoplast assay to test the biological activity of different Ac constructs (Houba-Herin et al., MGG 224:17-23, 1990). This assay allows the measurement of the relative transposase activity of Ac and derivatives of Ac.
The protoplasts have been cotransfected with two plasmid DNA's. One plasmid carries the b-glucuronidase (GUS) reporter gene under the control of the 1'-promoter. An insertion of a nonautonomous Ds-like element between the promoter and the coding sequence in this target plasmid abolishes the expression of the GUS gene. The second plasmid provides the transposase function. The excision of Ds leads to restoration of the reporter gene. After fixation of protoplasts on nitrocellulose filters and histochemical staining it is possible to detect b-glucuronidase activity as blue spots on the filters. The number of blue stained protoplasts is a measure of the excision frequency.
To our surprise we discovered in experiments with this transient expression system that a truncated piece of the ORFa coding sequence of Ac under the control of the 2'-promoter leads to a high excision frequency of the Ds element out of the target plasmid. As we did not do any transcription measurement, we cannot say for sure whether transcription of the Ac-coding sequence has been enhanced by the 2'-promoter or if the stability of the transcript has been changed by the truncation.
An analysis of transcriptional fusions using the complete ORFa coding sequence (with all introns) under the control of CaMV35S, 1'- and 2'-, Nos- and Ac-promoter leads to a low number of blue stained cells. The 2'-promoter enhanced the excision frequency 4-fold and the Nos-promoter 2-fold compared to the Ac-promoter. It seems that the number of blue stained cells reflects the strength of the different promoters.
If a truncated ORFa coding sequence with 3 out of frame ATG's upstream of ATG10 (codon 103 in the full length ORFa protein) is under the control of CaMV35S-, 1'- and 2'-promoter a significantly higher number of blue spots can be observed compared to the complete ORFa sequence. A lower rate of excision is obtained if the same truncated ORFa coding sequence is under the control of the Ac- or Nos-promoter. The removal of all out of frame ATG's upstream of ATG10 and expression under the control of the 2'-, Nos- or Ac-promoter leads to high excision frequency of the Ds regardless of promoter strength.
These results suggest that the maximal number of spots in a particular experiment can already be reached by the truncated ORFa coding sequence under the control of the weak Ac-promoter. But if the translation efficiency of the truncated ORFa coding sequence is reduced by upstream out of frame ATG's or the complete ORFa coding sequence has been used the excision frequency becomes dependent on transcription rate/promoter strength. The results suggest also that the number of excision events in a particular experiment reaches a saturation level.
The same results can be obtained using a cDNA of ORFa under the control of the 2'-promoter. The complete ORFa cDNA leads to a low number of blue spots whereas the truncated cDNA shows comparable results to the truncated genomic ORFa sequence. Further deletion of the ORFa coding sequence beyond the 10.ATG starting at amino acid 137, that is immediately behind a ten-fold repeat of the dipeptide Pro-Gln/Glu, abolishes transposase function.
The data with the complete ORFa sequence (genomic or cDNA) suggest that the N-terminal part of the ORFa protein exerts a regulatory function on transposition frequency. It is possible that the frequency of the transposition/excision rate is regulated on the protein level. This idea is supported by the following observation: a mixture of full length coding sequence and truncated coding sequence, both under the control of a 2'-promoter, leads to a number of blue stained protoplasts, which is lower than the number obtained with the truncated ORFa sequence alone. It seems that the presence of full length ORFa protein has a negative influence on the high activity of the truncated protein.
A surprising result has been observed by using different target plasmids. The presence of the luciferase gene in the vicinity of the Ds-like element leads to a 6-10-fold higher number of blue spots although the Ds elements in both plasmids are identical. We do not know yet if this result reflects a higher excision rate of Ds out of the luciferase containing target plasmid or if this result is due to a higher expression level of GUS-enzyme after excision of Ds.
Preliminary experiments indicate that the removal of the DNA sequence downstream of the BamHI site (bp 181) in the Ds element leads to a dramatic decrease in excision rate in the presence of complete or truncated ORFa coding sequence. This result, which is different from the results obtained in a callus assay in tobacco by Li and Starlinger (PNAS 87:60446048, 1990), suggests that in Petunia hybrida the DNA sequence located 3' to the BamHI site is important with regard to the cis-acting sequence requirements for excision. This result also shows that the quality of transposase action has not been changed by the removal of the first 102 amino acids of the ORFa protein. The tobacco results may then be due to a saturation effect of Ac excision under the conditions used in the callus assay.
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