Deliverables
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 database describing the predicted structure of each protein, its expression
pattern and intracellular localization. Also, a Reference Protein Subcellular
Localization Map will be constructed using fluorescently-tagged proteins with
known intracellular targeting.
Deliverable 1: Expression constructs harboring
full-length genomic sequences for each gene under its native promoter and with
the YFP tag flanked by two unique restriction sites engineered inside one of
the exons. The tagged constructs will be supplied as smaller, transient
expression TOPO vectors and as larger binary vectors for stable plant
transformation. In addition to expressing the YFP-tagged genes, these vectors
will be useful for other applications. For example, the YFP tag can be easily
removed using its flanking unique restriction sites and replaced with other
tags, such as T7 or HA, for immunodetection, or tags for fluorescence or
bioluminescence resonance energy transfer (FRET/BRET) protein-protein
interaction studies.
A similar CFP (Cyan fluorescent protein) tag with the
flanking restriction sites will be engineered into a series of selected genes
encoding proteins with known localization patterns for use as a standard
control set for subcellular targeting,. Having confirmed the sequence of the
clones, we will promptly list them on the TAIR website, and they will be
immediately available as DNA stocks from ABRC. All vector DNA stocks will be
supplied together with the description of how they were produced (restriction
map, primers used for PCR, etc.) and instructions for their utilization
(Agrobacterium transformation protocols, etc.).
Deliverable 2: Arabidopsis
T-DNA insertion seed stocks of plant lines expressing the YFP- or CFP- tagged
genes. After the expression of the tagged proteins has been confirmed, these
seed stocks will be listed on the TAIR website and disseminated through ABRC.
Deliverable 3: A website and a searchable database containing information
about the lines and constructs, including the gene sequences highlighted with
the 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. This database will be
supplemented with the Reference Protein Subcellular Localization Map, generated
using fluorescently-tagged proteins of known subcellular localization, and
offered to other researchers to use as controls for studies of protein
targeting. This information will include both the primary data, e.g., images of
the cells and tissues, and our interpretations. It will be disseminated via
unrestricted access to the project website integrated into The Arabidopsis Information Resource (TAIR).
This database will leverage from the database schema and software developed at
TAIR and also serve as (i) a module for integrating protein localization data
and software to the existing data and software at TAIR, and (ii) an essential
training data set for refining bioinformatic algorithms to better predict
intercellular localization signatures for Arabidopsis
and other organisms |