University of Cologne
1) A peculiarity among transposable elements is the "negative dosage effect" of Ac: with increasing Ac copies in the genome the reversion frequency drops. In plants homozygous for Ac about twice as much Ac transcript is synthesized as in plants carrying only one Ac (Kunze et al., EMBO J. 6:1555, 1987). If transcription initiation of Ac is a stochastic event, in the homozygotes twice as many cells should contain one Ac mRNA (i.e. one Ac mRNA per 10-50 cells). However, such a higher proportion of cells expressing Ac is inconsistent with a reduced transposition frequency.
2) In a recent paper it was reported that in Ac-free wx-m9::Ds plants the BamHI site at the 5'-end of Ds9 is modified in all cells. In the presence of an active Ac these Ds9 elements are completely cleavable with BamHI, which could be due to a production of the BamHI site by the transposase (Schwartz, Proc. Natl. Acad. Sci. 86:2789, 1989). This interpretation implies that transposase must be present in every single cell, a prediction that is hard to explain with the low abundance of the Ac transcript.
Therefore, we reexamined the transcription
rate of Ac by employing a modification of our quantification procedure:
instead of comparing the transcript band-intensity with that of defined
amounts of denatured Ac-DNA fragments, we used defined amounts of
in vitro transcribed RNA from Ac fragments as concentration markers
on the Northern blots. Unexpectedly, circa 50 times more RNA than DNA had
to be applied to yield hybridization signals of equal intensities. This
phenomenon was observed with four different Ac-probes. A control
experiment with sucrose synthase RNA and DNA had the same result, demonstrating
that the observed difference in hybridization signals between DNA and RNA
is not an Ac-specific phenomenon. Since we believe that the in vitro
transcribed RNA behaves more similar to the Ac-mRNA than DNA fragments,
we have to raise our estimate of the abundance of the Ac-mRNA in
the polyA fraction to ca. 7x10-5 or roughly one transcript per
cell, respectively. In contrast to the former estimate, this value does
not conflict with the above two observations.
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