Investigations on genetic mutations that affect the accumulation of zein proteins have suggested the presence of several regulatory mechanisms controlling the expression of specific members of the zein multigene family (Motto et al., Oxford Surv. Plant Mol. Cell. Biol. 6:87-114, 1989).

We are studying one of these mutations, opaque2 (o2). The O2 gene product is a basic leucine-zipper (bZIP) protein (Hartings et al., EMBO J. 8:2795-2801, 1989) that is necessary for transcription of the 22kD family of zein genes and for the b-32 gene (Lohmer et al., EMBO J. 10:617-624, 1991). A computer search for related protein sequences revealed a striking homology with GCN4, which is responsible for the general control of amino acid synthesis in yeast (Hinnebusch, Microbiol. Rev. 52:248-273, 1988). The structural homologies between O2 protein and GCN4 prompted us to test whether they might be functionally related.

The O2 cDNA was cloned in the yeast expression vector pEMBLyex4 under the control of the hybrid inducible UASGAL/CYC1 promoter. The resulting plasmid pIOP2 was used to transform to Ura+ the X4004-3A strain. Transformed yeast cells produced specific O2 mRNA and a specific immunoreactive polypeptide of about 65kD during growth in galactose. The expression of O2 protein does not produce appreciable alterations during batch growth at 30 C. The heterologous protein is properly translocated into the yeast nuclei, as demonstrated by immunofluorescence, indicating that the nuclear targeting sequences of maize are recognized by yeast cells. To test if O2 protein can substitute for GCN4 functions we transformed a gcn4 mutant (L1502 strain) with pIOP2 plasmid. The gcn4 mutants fail to grow in the presence of aminotriazole (AT), and the expression of O2 proteins restores a significant resistance to AT. Since the AT resistance is related to the expression of the HIS3 gene, which is controlled by GCN4, this confirms the hypothesis that O2 protein activates transcription of GCN4 regulated genes.

The interaction at the molecular level between O2 protein and HIS4 promoter was tested by band-shift and DNAse I footprint assays. O2 protein was obtained by expressing the O2 gene as an IPTG-inducible glutathione-S-transferase (GST) fusion protein, GST-02-55 in BL21 (psB161) (Lohmer et al., EMBO J. 10:617-624, 1991).

To test the potential capacity of O2 protein to bind the HIS4 promoter gel retardation assays were performed using purified GST02-55 protein and a fragment of HIS4 promoter containing one of the three GCN4 binding sequences. Incubation of purified O2 protein with labelled HIS4 promoter fragment gave two protein-DNA complexes of reduced electrophoretic mobility compared to free DNA. The formation of the complexes was specific because i) purified GST protein alone was not able to bind the labelled HIS4 promoter region and ii) a 1000 fold molar excess of an unspecific competitor DNA does not prevent binding. The binding could be abolished, however, by a 100-fold molar excess of unlabelled HIS4 promoter fragment. The O2 binding site was mapped by DNAse I footprinting, in which the same fusion protein and end-labelled promoter fragment were used as in the bandshift assay. The sequence protected by O2 corresponds to the sequence TGACTC, which is a GCN4 binding site. These results support the idea that basic mechanisms of transcription control have been highly conserved in eukaryotes. 

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