In vitro analysis of protein - DNA - interactions between the sucrose synthase gene promoter and nuclear factors

The shrunken gene on chromosome 9, coding for the enzyme sucrose synthase, is regulated during plant development. The transcription rate is specific for different organs of the plant and the gene is inducible by anaerobic stress (Springer et al., 1986). We suppose that nuclear factors should bind to cis-acting sequences in the vicinity or within the gene to regulate the level of transcription.

In gel retardation experiments, using crude nuclear extracts of immature kernels and different promoter fragments, spanning the region between -1592 and + 393 (with regard to the transcription start), we have observed multiple protein-DNA interactions. Two major binding activities (called MNP 1 and MNP 2 for maize nuclear protein) seem to recognize multiple binding sites distributed over the 1.5 kb upstream region of the shrunken gene, which can be concluded from competition experiments with heterologous unlabelled promoter fragments and is supported by DNase I footprint experiments or sequence homologies. At the moment we are trying to purify these factors by affinity chromatography.

Jorg Schurmann, Boris Springer, Thomas Lugert, Regina Bellmann and Wolfgang Werr

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