The shrunken gene on chromosome 9 is regulated during plant development and exhibits positive and negative control mechanisms (Springer et al., Mol. Gen. Genet., in press). It is therefore a good candidate for studying plant gene regulation.
We have started to identify regulatory proteins which interact with the promoter of the shrunken gene. In the first step we had to adapt a nuclei isolation procedure to different tissues of the maize plant including immature kernels (20 days after pollination), primary roots, shoots and mature leaves. Low salt (250-300 mM) protein extracts from the isolated nuclei were prepared and tested in gel retardation experiments for specific protein DNA interactions. With nuclear protein extracts prepared from immature kernels we see several interactions with small radioactively labeled DNA fragments from the promoter region of the shrunken gene. The strongest interactions are found with several overlapping DNA fragments containing the sequences between -234 and -77. Another DNA protein complex is formed with DNA fragments spanning the sequences between -600 and -577. A weak interaction is found between -77 and + 41 with the fragment containing the TATA box and the transcription start. The complexes are stable in the presence of 1000-2000 fold excess of unspecific competitor DNA but formation of visible complexes is abolished in the presence of a low excess of homologous DNA fragments. At the moment we are in the process of footprinting this interaction on the nucleotide level.
Wolfgang Werr, Regina Bellmann, Boris Springer, Hans-Jurgen Joos and
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