Functional expression of the transcriptional activator Opaque-2 of Zea mays in transformed yeast

Abstract

The aim of this research was to determine whether the structural homology between the O2 gene, a maize transcriptional activator, and the GCN4 gene, a yeast transcriptional factor, is reflected at the level of function. The O2 cDNA was cloned in the yeast expression vector pEMBLyex4 under the control of a hybrid inducible promoter, and used to transform the yeast Saccharomyces cerevisiae. Transformed yeast cells produced O2 mRNA and a polypeptide immunoreactive with anti-O2 antibodies during growth in galactose. The heterologous protein was correctly translocated into the yeast nuclei, as demonstrated by immunofluorescence, indicating that the nuclear targeting sequences of maize are recognized by yeast cells. Further experiments demonstrated the ability of O2 to rescue a gcn4 mutant grown in the presence of aminotriazole, an inhibitor of the HIS3 gene product, suggesting that O2 activates the HIS3 gene, gene normally under control of GCN4. It was shown that the O2 protein is able to trans-activate the HIS4 promoter in yeast cells and binds to it in vitro. The sequence protected by O2, TGACTC, is also the binding site for GCN4. Finally, the expression of O2 protein in yeast did not produce alterations during batch growth at 30 degrees C, while transformants expressing O2 protein showed a conditionally lethal phenotype when grown in galactose at 36 degrees C; this phenotype mimics the behaviour of gcd mutants. The results support the idea that basic mechanisms of transcription control have been highly conserved in eukaryotes.

Chromatin and DNA Modifications in theOpaque2-Mediated Regulation of Gene Transcription during Maize Endosperm Development

“…This observation suggests that O2 can activate transcription of its targets through different mechanisms, which are specific for subsets of genes on the basis of different modalities of interaction with chromatin regulators. A similar scenario was also reported for other transcription activators, including the bZIP class yeast GCN4, which shows several structural and functional homologies with O2 (Mauri et al, 1993;Pirona et al, 2005). It was shown that GCN4 protein is able to recruit multiple coactivators, including yeast ADA2 and GCN5, at its target promoters, but the requirements for these coactivators varies greatly among individual promoters and in many cases is not sufficient to drive transcription (Swanson et al, 2003;Yu et al, 2006).…”

The wheat transcriptional activator SPA: a seed-specific bZIP protein that recognizes the GCN4-like motif in the bifactorial endosperm box of prolamin genes.

“…First, 0 2 can bind to the GLM of the LMW glutenin endosperm box and activate transcription in yeast and plants through this element (Holdsworth et al, 1995). Second, 0 2 expressed in yeast complements a gcn4 mutant by binding and activating the HIS3 gene at its GCN4 binding site (Mauri et al, 1993), indicating that the GCN4 motif, the GLM sequence, and the 0 2 binding sites in maize genes are functionally related. Finally, the recombinant 0 2 protein has been shown to bind in vitro to a GLM sequence of a pea lectin gene that is necessary and sufficient for seed-specific expression in transgenic tobacco plants (de Pater et al, 1993(de Pater et al, , 1994.…”

Wild-Type Opaque2 and Defective opaque2 Polypeptides Form Complexes in Maize Endosperm Cells and Bind the Opaque2-Zein Target Site

“…Moreover, it is known that O2 binding to DNA is, in some way, promiscuous because O2 binds in vitro to different target sequences (Lohmer et al, 1991;Schmidt et al, 1992;Izawa et al, 1993;Yunes et al, 1994). Finally, O2 is able to complement in vivo the bZIP function of the GENERAL-CONTROL-NONDEREPRESSIBLE4 (GCN4) gene product in gcn4 yeast (Saccharomyces cerevisiae) strains (Mauri et al, 1993), as well as to activate in the endosperm different genes bearing variable O2 core-binding sequences in their promoter region (Maddaloni et al, 1996;Kemper et al, 1999).…”