University of Cologne

A reexamination of Ac transcript quantification --Sylvia Schein, Reinhard Kunze and Peter Starlinger In this note we report about recent improvements of the expression analysis of transposable element Ac in maize. The only known Ac-specific transcript is 3.5 kb long and encodes an open reading frame 807 amino acids long. Remarkably, multiple transcription initiation sites are scattered over about 100 bp and no TATA- or CAAT-boxes are found in the upstream sequence. The abundance of the Ac-mRNA was determined to be between 1 and 3x10-7 of the polyA-RNA (Kunze et al., EMBO J. 6:1555, 1987). Recent estimations show a 2-4 times higher amount depending on genotype and tissue. Assuming that maize cells contain similar amounts of messenger RNA per cell as the monocot Tradescantia palludosa (i.e. 0.16 pg, Willing and Mascarenhas, Plant Physiol. 75:865, 1984) we calculated that only one Ac transcript per every 20th to 100th cell is synthesized (Kunze, Ph.D. thesis). This low transcription rate could indicate that the transcription event is the rate limiting step for transposition of Ac. However, at least two observations are in contradiction with this interpretation:

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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