A study will be made of plant nuclear genes encoding the chloroplast proteins ribulose-1, 5-biphosphate carboxylase (RBCS) and the polypeptide components of the light-harvesting chlorophyll a/b (CAB) complex. Cis-acting promoter elements mediating the transcriptional regulation of these photoregulated genes will be characterized in detail. The genes in question will include the rbcs-3A gene from Lycopersicon esculentum (tomato), the rbcS-1A gene from Arabidopsis thaliana, and the cab-E gene from Nicotiana plumbaginifolia (tobacco). These studies will be carried out by constructing chimeric genes containing RBCS and CAB promoter elements fused to reporter genes. These promoter elements will be manipulated in vitro by deletion and site-directed mutation. The expression of the chimeric genes will be examined in transgenic tobacco plants generated by Agrobacterium-mediated transformation. Trans-acting factors that bind to these promoter elements will be further characterized. This will be achieved by band-shift assays, footprinting, and protein purification. A previously-demonstrated role for protein phosphorylation in modulating binding of factors to promoter elements will be characterized. The nature of the DNA sequences and protein factors that mediate diurnal and circadian expression rhythms will be examined. Photoresponse mutants in tomato and Arabidopsis will be examined in order to determine if they lack expression of any individual RBCS or CAB genes. A transient expression assay will be developed in protoplassts so as to facilitate a rapid analysis of cis-acting promoter elements. The described studies will both contribute to a better understanding of the complexities of transcriptional regulation of gene expression and will assist in the development of novel methods by which the nutritional quality of food crops will be improved.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM038409-07
Application #
3294802
Study Section
Molecular Biology Study Section (MBY)
Project Start
1986-09-01
Project End
1994-07-31
Budget Start
1992-08-01
Budget End
1993-07-31
Support Year
7
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Type
Schools of Arts and Sciences
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Menkens, A E; Cashmore, A R (1994) Isolation and characterization of a fourth Arabidopsis thaliana G-box-binding factor, which has similarities to Fos oncoprotein. Proc Natl Acad Sci U S A 91:2522-6
Schindler, U; Beckmann, H; Cashmore, A R (1993) HAT3.1, a novel Arabidopsis homeodomain protein containing a conserved cysteine-rich region. Plant J 4:137-50
Klimczak, L J; Cashmore, A R (1993) Purification and characterization of casein kinase I from broccoli. Biochem J 293 ( Pt 1):283-8
Schindler, U; Beckmann, H; Cashmore, A R (1992) TGA1 and G-box binding factors: two distinct classes of Arabidopsis leucine zipper proteins compete for the G-box-like element TGACGTGG. Plant Cell 4:1309-19
Schindler, U; Terzaghi, W; Beckmann, H et al. (1992) DNA binding site preferences and transcriptional activation properties of the Arabidopsis transcription factor GBF1. EMBO J 11:1275-89
Granell, A; Pereto, J G; Schindler, U et al. (1992) Nuclear factors binding to the extensin promoter exhibit differential activity in carrot protoplasts and cells. Plant Mol Biol 18:739-48
Schindler, U; Menkens, A E; Beckmann, H et al. (1992) Heterodimerization between light-regulated and ubiquitously expressed Arabidopsis GBF bZIP proteins. EMBO J 11:1261-73
Bar-Zvi, D; Shagan, T; Schindler, U et al. (1992) RNP-T, a ribonucleoprotein from Arabidopsis thaliana, contains two RNP-80 motifs and a novel acidic repeat arranged in an alpha-helix conformation. Plant Mol Biol 20:833-8
Klimczak, L J; Schindler, U; Cashmore, A R (1992) DNA binding activity of the Arabidopsis G-box binding factor GBF1 is stimulated by phosphorylation by casein kinase II from broccoli. Plant Cell 4:87-98
Schindler, U; Ecker, J R; Cashmore, A R (1991) An Arabidopsis thaliana G-box-binding protein similar to the wheat leucine zipper protein identified as HBP-1. Symp Soc Exp Biol 45:211-8

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