Award Abstract:
This pilot project will develop a high-throughput strategy to analyze
native expression patterns and subcellular localization of Arabidopsis
gene products of unknown function. This strategy, Fluorescent Tagging of Full-Length
Proteins (FTFLP), will comprise five major steps: (1) Selection of
functionally unassigned Arabidopsis
genes and prediction of their protein structure and suitable site for
fluorescent tag insertion (2) Amplification of each gene in two parts, with the
junction between the two parts corresponding to our chosen insertion site for
the fluorescent tag(3) Introduction of the fluorescent tag, yellow fluorescent
protein (YFP) using a triple overlap PCR approach (4) Insertion of PCR products
into binary vectors (5) Production of transgenic Arabidopsis lines and
analysis of expression pattern and
intracellular localization for each tagged protein. As a pilot approach, the
project aims to analyze a statistically significant number of genes to support
the applicability to a subsequent wider study. To this end, approximately 800
genes were selected from a total of ca. 8,000 unknown genes. This pilot list was
chosen based on the following sequentially-applied criteria: 1) have matching
full-length cDNA, 2) are annotated as unknown protein or putative protein,
and 3) do not have any Gene Ontology annotations. The selected genes reflect
the diversity of all the unknown Arabidopsis
genes with respect to plant specificity, predicted domain and/or gene family
information, and availability of matching full-length cDNA sequences.
FTFLP as a tool for functional proteomics offers
four significant advantages: it focuses on genes of unknown function, it
produces tagged full length proteins that are more likely to exhibit
faithful intracellular localization, it expresses fusion proteins at native expression levels to minimize artifacts due to over-expression and ectopic expression, and the use of native promoters allows
determination tissue specificity. Three deliverables will be offered to the
research community:
1) Expression vectors harboring full-length
sequences for each gene under its native promoter and tagged with YFP flanked
by unique restriction sites,
2) Arabidopsis
transgenic lines expressing each construct, and
3) A website and a searchable database containing
information about the lines and constructs, including the gene sequences
highlighted with positions of primers and tagging sites, vector construct
information, images and text descriptions of the protein expression pattern and
intracellular localization, and protocols and standard operation procedures in
experimentation, analysis, and interpretation. Also, a Reference Protein
Subcellular Localization Map will be constructed using fluorescently-tagged
proteins with known intracellular targeting.
These resources will be available to the public
through two unrestricted venues: DNA constructs and transgenic seeds will be
distributed through the Arabidopsis
Biological Resource Center (ABRC) whereas gene sequences and expression and
subcellular localization data, including fluorescence microscopy images, will
be disseminated via the project website integrated into The Arabidopsis
Information Resource (TAIR).
Importantly, this sharing of the resources and results of this project through
ABRC and TAIR, respectively, will take place on a continuous basis as the
deliverables become available. Announcements on the availability of new
resources will be made through such electronic media as the Bionet USENET newsgroups
and parallel e-mail lists. This
project significantly advances the overall objectives of the 2010 Project by
characterizing on a large scale the expression and subcellular localization of
unknown Arabidopsis gene products. Our
understanding of Arabidopsis biology
will be significantly incomplete without such knowledge. In addition, this project
has a broader impact on the society and science. Once this pilot project
demonstrates the feasibility of the proposed approach, it will serve a basis
for developing a laboratory curriculum for use in cell biology training of high
school students and teachers as well as beginning investigators at the CSHL DNA
Learning Center and the annual Arabidopsis Molecular Genetics Course,
and at the biannual UCR Plant Cell Biology course. Finally, a teaching outreach
program with community colleges will involve undergraduates in summer research.
Thus, our program will bridge genomic approaches with cell biology in the
laboratory and classroom, and generate important novel information and tools to
characterize the Arabidopsis
proteome.
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