To avoid wasting time and resources, we will
screen our collection only if there is good evidence that
the targeted gene encodes a chloroplast-localized
protein. Any one of the following types of evidence is
sufficient:
a) Predicted chloroplast targeting sequence
If the N-terminal sequence of the gene product is
available, it should be run through two prediction
programs: TargetP (http://www.cbs.dtu.dk/services/TargetP/)
and Predotar (http://www.inra.fr/Internet/Produits/Predotar/)
If both algorithms predict a chloroplast-transit
peptide, this is sufficient evidence for a screen. If
just one algorithm predicts a chloroplast-transit
peptide, we would prefer that some other type of evidence
for chloroplast-localization is provided in addition.
Caution! The ChloroP and PSort algorithms are less
accurate.
Note: A predicted chloroplast targeting sequence on
the predicted ortholog from another species (e.g.
Arabidopsis) is also sufficient. However, the method by
which the orthologous relationship was established must
be provided.
b) Cellular fractionation
Demonstrate that the protein of interest is enriched
in isolated chloroplasts with respect to its
concentration in total leaf extracts. Markers for
cytosol, chloroplasts, and mitochondria must be included
in the analysis.
The data must be provided, preferably as an email
attachment.
c) Chloroplast localization during in vitro import
assays or during transient expression of GFP fusion
proteins.
If a full-length cDNA is available, one can test
whether the protein can be imported into isolated
chloroplasts, or whether a C-terminal fusion with GFP
targets the protein to chloroplasts following transient
expression in cultured cells. The data must be provided,
preferably as an email attachment.
d) Mutant Phenotype
if a mutation in this gene has been obtained
previously that causes one of the phenotypes represented
in our collection (chlorophyll-deficiency or high
chlorophyll fluorescence), this is sufficient evidence
that an allele should be represented in our
collection.
Primers: We need 15-20 nmoles of each
primer (about 110-150 µg for a 20-mer oligo).
Primers can either be lyophilized or diluted in water.
(The exact concentration is not critical, as long as it
is clearly indicated... anywhere from 50-5000 µM.)
Primers can be sent either lyophilized at room
temperature or diluted and sent on WET ice via "overnight
standard" mail. A copy of the information sheet from the
company that synthesized the primers would also be
helpful.
Probes: We will need roughly 200ng of each
working DIG-labelled probe. The probe can either be
lyophilized and sent at room temperature, or frozen in TE
or hybridization buffer and sent on WET ice via
"overnight standard" mail. Please include information on
how the probe was synthesized (template & primers
used). The concentration and/or amount of probe should be
clearly indicated.
Gene Map: A simple cartoon showing the location
of relevant gene features eg; start codon, stop codon and
introns. The map should also indicate primer locations,
and the span of the DIG-labelled probe to be used for
detection of Mu insertions. Ideally we would like this
map in electronic form (PICT, JPEG or TIFF are the
prefered formats)
Mailing address:
Attn: PML Technician
c/o the Barkan Lab
Institute of Molecular Biology
University of Oregon
Eugene, OR 97403