The proposed research concerns the co-ordinate expression of genes in the anthocyanin pigment pathway of maize. The range of mutants in this pathway and the easy assay for gene function provides an excellent model for unraveling the complex transcriptional and post-transcriptional events required to modulate eukaryotic genes. Although the mRNA levels from the B zeta 2 gene are low, the promoter of this gene is highly active in transient assays, exceeding by >30X the activity of other promoters in the same pathway. To resolve this paradox, the transcriptional and post- transcriptional regulation of this gene will be analyzed by making mutations in the promoter, defining the binding sites of two known regulatory genes (R and C1), and analyzing splicing, translation and mRNA turnover characteristics of elements of the B zeta 2 mRNA. These results will be compared to parallel studies of B zeta 1 to elucidate the differences in regulation at each step in mRNA production. Preliminary results suggest that the B zeta 2 intron is inefficiently spliced and that this contributes to the low functional mRNA levels. Other preliminary studies have defined a number of unusual features of intron splicing maize, including an apparent requirement that internal intron sequences be recognized for efficient splicing. To explore these phenomena, new vectors have been designed so that reporter gene expression is contingent on splicing failure or alternative on splicing success. RNase protection assays will be used to verify pre-mRNA fates. The motifs important for internal intron recognition will be defined by mutagenesis and the protein factor(s) binding to such sites will be cloned for further analysis.
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