An in vitro transcription system has been developed from maize mitochondria to increase the understanding of transcriptional regulation in this organelle, as similar systems have accomplished for several fungi and for animals cells. Maize has bee chosen as a model system because extensive molecular genetic data relevant to transcription have previously been published, and because mitochondrial mutations in maize can lead to important developmental defects. Data obtained in plants may also shed light on mitochondrial disease mechanisms in animal cells. Maize is one of the most economically important crop species in the U.S. and an important animal feed product. The experimental objectives combine a further analysis of maize mitochondrial promoter structure with an analysis of protein:DNA interactions in the promoter region. Pervious analysis of the atp1 promoter has led to the development of a promoter model that is unique to plants. By further mutagenesis of the atp1 promoter and selective mutational analysis of the cox3, cox2 and atp6 promoters, it will be established whether the atp1 model represents a paradigm for maize and other plant mitochondrial promoters. DNA:protein interactions will be studied by a combination of gel shift and footprinting techniques. A promoter-specific DNA-binding activity that has been identified in preliminary experiments will be purified by chromatography, and its binding site will be used to screen a cDNA expression library. Based on the currently available data and literature, plant mitochondrial genome structure, gene content and transcriptional strategies differ from those of yeast, fungi, protozoa and animals. The findings from the proposed research may also therefore be of interest from an evolutionary perspective.
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| Lupold, D S; Caoile, A G; Stern, D B (1999) The maize mitochondrial cox2 gene has five promoters in two genomic regions, including a complex promoter consisting of seven overlapping units. J Biol Chem 274:3897-903 |
| Lupold, D S; Caoile, A G; Stern, D B (1999) Genomic context influences the activity of maize mitochondrial cox2 promoters. Proc Natl Acad Sci U S A 96:11670-5 |
| Chang, C C; Sheen, J; Bligny, M et al. (1999) Functional analysis of two maize cDNAs encoding T7-like RNA polymerases. Plant Cell 11:911-26 |
| Caoile, A G; Stern, D B (1997) A conserved core element is functionally important for maize mitochondrial promoter activity in vitro. Nucleic Acids Res 25:4055-60 |
