Transactivation of the Bz1 promoter following transfer of the C1 gene into aleurones or embryos: A reporter construct using the firefly luciferase coding region under control of the Bz1 structural gene promoter was used to test for transactivation by the C1 gene product. As an internal control for the efficiency of gene transfer, a plasmid containing the alcohol dehydrogenase-1 promoter driving the chloramphenicol acetyltransferase (CAT) coding region, was included in each DNA sample used for bombardment. The values of C1 transactivation of Bz1-luciferase are therefore expressed as a ratio of luciferase enzyme activity to the CAT enzymatic activity generated from the internal control plasmid. Co-bombardment of the Bz1-luciferase reporter construct with the C1 gene or the expressed C1 cDNA resulted in an 85 to 300-fold induction of the Bz1 promoter relative to tissues receiving the reporter plasmids alone (Table I).

pUC = pUC19.

pMF6 = CaMV 35S Expression Vector without insert.

pBz1Luc = Bz1 promoter driving luciferase.

p35SC1 = C1 cDNA under 35S promoter control.

pC1gen = C1 genomic clone.

pC1GAL10 = Fusion of GAL4 acidic domain at amino acid 258 of C1.

pC1fs2 = Frameshift of C1 at amino acid 258.

pAdhCAT = Adh1 promoter driving Chloamphenicol Acetyltransferase.

Complementation and transactivation with C1-GAL4 fusion proteins: To obtain more definitive evidence that the product of the C1 gene functions as a transcriptional activator, we have replaced the acidic region of C1 with the acidic domain of the yeast transcriptional activator GAL4, and tested for complementation of the anthocyanin pathway in c1 aleurones. Three CaMV 35S-driven fusion protein plasmids were constructed with varying lengths of the C1 protein fused to the C-terminal acidic domain of GAL4. These fusions were made at amino acids 117, 144, and 258 of the intact 273 amino acid C1 protein. Each of these constructs was delivered to c1 R-scm aleurones and resulted in the generation of pigmented cells, however placement of the GAL4 acidic domain immediately adjacent to the putative DNA-binding region (at amino acid 117 of C1) resulted in only weakly pigmented cells. The largest C1-GAL4 fusion (at amino acid 258 of C1) was also tested for its ability to activate the Bz1 promoter. A 115 fold induction of Bz1-luciferase was observed in c1 R-scm aleurones co-bombarded with this C1-GAL4 fusion construct (Table I). A frameshift placed in the C1 acidic region (amino acid 258) renders the altered C1 protein unable to transactivate the Bz1 promoter (Table I). These results demonstrate that the C1 carboxy-terminal acidic region can be replaced by an acidic domain from a known transcriptional activator and result in a functional regulatory protein.

A frameshift mutant of C1 inhibits transactivation of the Bz1 promoter: A frameshift in the carboxy-terminal acidic region of the C1 cDNA coding sequence (amino acid 258) was constructed in vitro in an attempt to mimic the effects of the dominant inhibitor allele of C1 designated C-I. The product of this construct did not complement c1 aleurones nor transactivate the Bz1 promoter. At 1 to 1 ratios of wild-type C1 and this C1 frameshift construct, an 80% reduction of transactivation was observed following gene delivery, and at higher ratios, inhibition of transactivation approached 95%. Thus it is possible to mimic the effects of C-I and generate a dominant inhibitor of C1 with a frameshift in the coding region which disrupts the acidic nature of the C1 carboxyl-terminus.

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