We have tried to study chromatin structure in the 5' upstream region of the sucrose synthase gene. This gene is active in endosperm, inducible by anaerobiosis in roots and shoots, and hardly expressed in leaves (Springer et al., Mol. Gen. Genet., in press). We have tested for the presence of DNaseI hypersensitive sites.
Nuclei were isolated essentially as described by Rowland and Strommer (PNAS 82:2875-2879, 1985) with modifications for tissues other than roots. Though contaminated with cell wall fragments they contained high molecular weight DNA. The chromatin could be degraded by micrococcal nuclease or MPE (Fe2+) to yield the typical nucleosomal ladder of about 180bp repeat length.
The nuclei are active in run-off transcription. By this method it could be shown that anaerobiosis induces sucrose synthase at the transcriptional level. The maximum levels of initiation occur after a few hours submersion of the seedlings in water roughly as for Adh1 (Rowland and Strommer, Mol. Cell. Biol. 6:3368-3372, 1986).
In nuclei isolated from kernels a set of DNaseI-hypersensitive sites could be identified which extends more than 1 kb into the 5' region. The major sites ( + /- 50 bp) are located relative to the transcription start at positions + 50, -25, -180, -280. The same major sites are seen in nuclei from isolated endosperm. No sites were detectable in naked genomic DNA. The same major sites in front of the transcription start are found both in aerobic and anaerobic (6h) roots.
Wolf-Bernd Frommer, Philipp Franken and Peter Starlinger
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