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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032422-12
Application #
2176567
Study Section
Genetics Study Section (GEN)
Project Start
1991-09-01
Project End
1995-08-31
Budget Start
1994-09-01
Budget End
1995-08-31
Support Year
12
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Stanford University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
800771545
City
Stanford
State
CA
Country
United States
Zip Code
94305
Marrs, K A; Walbot, V (1997) Expression and RNA splicing of the maize glutathione S-transferase Bronze2 gene is regulated by cadmium and other stresses. Plant Physiol 113:93-102
Bodeau, J P; Walbot, V (1996) Structure and regulation of the maize Bronze2 promoter. Plant Mol Biol 32:599-609
Luehrsen, K R; Walbot, V (1994) Intron creation and polyadenylation in maize are directed by AU-rich RNA. Genes Dev 8:1117-30
Galway, M E; Masucci, J D; Lloyd, A M et al. (1994) The TTG gene is required to specify epidermal cell fate and cell patterning in the Arabidopsis root. Dev Biol 166:740-54
Carle-Urioste, J C; Ko, C H; Benito, M I et al. (1994) In vivo analysis of intron processing using splicing-dependent reporter gene assays. Plant Mol Biol 26:1785-95
Luehrsen, K R; Walbot, V (1994) Addition of A- and U-rich sequence increases the splicing efficiency of a deleted form of a maize intron. Plant Mol Biol 24:449-63
Lloyd, A M; Schena, M; Walbot, V et al. (1994) Epidermal cell fate determination in Arabidopsis: patterns defined by a steroid-inducible regulator. Science 266:436-9
Bodeau, J P; Walbot, V (1992) Regulated transcription of the maize Bronze-2 promoter in electroporated protoplasts requires the C1 and R gene products. Mol Gen Genet 233:379-87
Luehrsen, K R; de Wet, J R; Walbot, V (1992) Transient expression analysis in plants using firefly luciferase reporter gene. Methods Enzymol 216:397-414
Lloyd, A M; Walbot, V; Davis, R W (1992) Arabidopsis and Nicotiana anthocyanin production activated by maize regulators R and C1. Science 258:1773-5

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