Functional expression of the transcriptional activator Opaque-2 of Zea mays in transformed yeast.
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.
Mauri
I
I
Instituto Biochimica Comparata, Facoltà di Scienze, Università di Milano, Italy.
Maddaloni
M
M
Lohmer
S
S
Motto
M
M
Salamini
F
F
Thompson
R
R
Martegani
E
E
D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D015151:Immunoblotting; D010940:Plant Proteins; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation; D014170:Transformation, Genetic; D003313:Zea mays / Q000235:genetics*
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C067402:opaque-2 protein, Zea mays; D011494:Protein Kinases