Analysis of functional domains of the C1 encoded protein
--Philipp Franken, Susanne Kartzke, Peter A. Peterson, Heinz Saedler and Udo Wienand
The putative C1 encoded protein contains two major domains: a basic one at the amino terminus with possible DNA binding capacity and an acidic domain for activation of transcription at the carboxy terminus. Functional analysis of these domains has been carried out by the investigation of revertants of transposable element induced mutants. Also in vitro constructs containing fusions of various domains of C1 and C1-related cDNAs have been tested for activity.
The En1 element insertion sites of seven C1 mutants have been determined and revertants of four of these were further investigated. A pale revertant of one such mutant (c1-m55437) was analyzed and showed a deletion of a lysine in the basic domain of the putative C1 encoded protein at amino acid position 30. This indicates that this position in the C1 protein is of functional importance. Colorless, pale and colored revertants of the mutant c1-m11702 have also been investigated. The En1 element in c1-m11702 is integrated in the acidic domain of the putative C1 encoded protein at amino acid position 223. The pale and colorless revertants represent frame shift mutations, whereas the colored revertant contains an additional amino acid. The result of this analysis shows that sequence and charge of the acidic domain of the C1 protein is important for its activator function.
For reconstruction of C1 like proteins, experiments using the C1 cDNA as well as two C1-related cDNAs from maize (Zm1 and Zm38; Marocco et al., MGG 216:1347-1368, 1989) have been used. Particle gun experiments with various constructs carrying different combinations of the putative DNA binding and trans-activating domains indicate flexibility of the acidic carboxy terminus. One of the C1 homologous cDNAs and fusion constructs thereof were also capable of A1 gene activation, but not of activation of the entire pathway.
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