Protein localization-GFP-Arabidopsis spp. - Particle Gun
nobody at hgmp.mrc.ac.uk
Thu Jan 16 13:15:18 EST 2003
Gene Expression and Protein Localization in Arabidopsis
To a large degree, the function of a protein can be inferred from its
cellular compartmentalization and its interactions with other
cellular components. Therefore, information on the subcellular
distribution of a protein is crucial to determine its overall role in
the cell. The histological distribution of the mRNA transcript
reflects another important aspect; this aspect, however, has been
covered in the in situ hybridization laboratory and will not be
Several options are available to probe the subcellular localization
of a given protein. In a few cases, it has been possible to design a
specific way to localize a protein directly while inside the cell,
based on a unique functional property or physical characteristic of
the protein (light absorption or fluorescence). In the majority of
cases, however, some form of indirect detection will be necessary.
Methods that should be applicable to almost any protein will be the
focus in this lab.
There are two major strategies for indirect detection:
A. Detection of the native protein: A specific antibody or an
equivalent high affinity probe is used to label the target protein in
situ. The probe is either directly labelled with a fluorescent tag or
detected with secondary antibodies. Final observation is by either
electron microscopy or light microscopy. While electron miscroscopy
has higher resolution, it is also time-consuming and not very
sensitive. In many cases, the light microscope can offer sufficient
spatial resolution (Colasanti et al., 1993; Neuhaus et al., 1993;
Wick, 1993; Matsui et al., 1995).
B. Detection of a recombinant version: Alternatively, a tag can be
incorporated into the target protein and the modified protein
introduced back into the cell by recombinant DNA technology. Thus,
the fusion protein can be localized by monitoring the tag. There are
three types of commonly used tags.
=46irst, epitope tags (8-11 amino acid stretches of well-characterized
foreign proteins such as c-myc or hemagglutinin). These tags are
small, thus minimizing the chance of altering the gross conformation
(or subcellular localization) of the targeted protein. Monitoring the
tag can be carried out according to standard immunological procedures
with commercially available monoclonal antibodies specific for the
chosen tag. The obvious advantage here is elimination of the need to
generate specific antibodies for each new target protein.
Second, =FE-glucuronidase (GUS) acts as a reliable reporter enzyme in
fusion proteins, especially for nuclear versus non-nuclear
localization (Restrepo et al., 1990; Varagona et al., 1991 and 1992;
Abel and Theologis, 1994; Abel et al., 1994; Citovsky et al., 1994;
von Arnim and Deng, 1994). Although the in situ staining for GUS
activity is easier than immunodetection, there are some potential
drawbacks with this method. For example, the in situ enzyme assay can
lead to a loss of spatial resolution.
Third, fluorescent proteins (e.g. GFPs) combine the advantage of high
resolution with detection in vivo (Chalfie et al., 1994, Heim et al.,
1994; Prasher, 1995). This approach has been succesful in yeast and
mammalian cells (e.g. Olson et al., 1995) and in Drosophila (Wang and
Hazelrigg, 1994; Kerrebrock et al., 1995), and has also been
developed for plants.
While a tag can obviously improve the specificity of detection and
avoid the need for generating antibodies for every new protein to
study, it can potentially alter the subcellular localization of the
fused protein due to structural constraints introduced by the tag and
by an aberrant expression level. However, genetic or biochemical
experiments may clarify whether the fusion protein remains
functional. Ideally, conclusions using one method should be
corroborated by an independent approach.
During the two days allocated to this section, we will provide a
theoretical overview and an experimental entryway into methods for
transient gene expression and subcellular localization in
Arabidopsis. The main course of events will be dominated by
Experiment 1, immunofluorescent detection of proteins in protoplasts.
In parallel, we will demonstrate the subcellular detection of
proteins using GUS as a fusion marker (Experiment 2). Experiment 3
will demonstrate the power of GFP as a subcellular marker and the
transient expression of proteins using the particle gun.
Experiment 1: Immunofluorescence detection in Arabidopsis protoplasts
Experiment 2: Subcellular localization of GUS-fusion proteins in
Experiment 3: Transient gene expression of GUS and GFP by particle
Experiment 1 meanwhile...
1. Digest tissue 1) 09.00--11.00
2. Wash protoplasts 11.00 --12.00
3. Settle cells onto slides 12.00 --15.00 lunch, instruction set up
particle gun (Expt.3)
4. Fixation etc. of cells 15.00 --17.00 (leave over night)
5. Analyse GUS-stained
seedlings (Expt.2) 17.00--18.30
5. Rehydration and block 1) 09.00--11.30 (TA)
6. Add first antibody 11.30 --13.00 lunch
7. Wash 13.00 --13.30
8. Add second antibody 13.30 --14.30 inspect cells from
(particle gun) 2)
9. Wash 14.30 --15.00
10. Mount slides 15.00 --15.30
11. Analyse results 15.30 --18.30
1) This step will be set up by the TAs
2) Tissue transformed with the particle gun (Day 1) has been mounted
for microscopy by TAs.
Experiment #1: Immunofluorescence detection in Arabidopsis protoplasts
We will concentrate on the direct detection of a protein using
specific antibodies. The major problem is to make the antigen
accessible to the antibody, which is effectively blocked by cell
walls and the cuticle. The effect of cell walls can be overcome by:
- Digestion of cell walls in a whole mount specimen
- Digestion of cell walls under isolation of protoplasts
Several key factors need to be considered when choosing between these option=
=46irst, autofluorescence in plant specimens can be a serious problem.
Sometimes it is possible to work with tissues that show little
autofluorescence, for example roots, which lend themselves to the
whole-mount approach, but this is often impractical. More commonly,
it is necessary to take additional measures to reduce
autofluorescence, for example by washing out the interfering
substances before microscopy. This is more easily achieved in single
cells, such as protoplasts, than in whole mounts. In addition,
careful selection of fluorophores and optical filters is necessary to
reduce the interference of autofluorescence.
Second, the specimen has to be fixed to preserve the subcellular
structures during the antibody incubation and wash steps. The
trade-off: Gentle fixation will preserve antigenicity and keep
autofluorescence low, but may not preserve the subcellular structures
sufficiently. Harsh fixation conditions (especially glutaraldehyde)
will increase autofluorescence.
Third, antigenicity of the target should be preserved. Strong
fixatives will reduce antigenicity and may also unmask fortuitous
epitopes. Embedding of tissue, associated with extreme changes
between aqueous and non-aqueous media and exposure to a wide range of
temperatures, also runs counter the preservation of antigenic sites.
Overall, while detection of a highly abundant antigen may be
straightforward even after rough treatment of the tissue, only the
very optimum of conditions may allow detection of a protein of low
The following strategy has allowed quite consistent detection of a
wide variety of antigens in our hands.
1. Isolation of protoplasts from the tissue of choice
2. Binding of protoplasts to gelatin-coated slides
3. Fixation and permeabilization of the cells
4. Antibody incubations and washes
5. Mounting and inspection
This method has the following advantages:
(1) Access of the antibodies is very rapid.
(2) Fixation is quick and uniform.
(3) Autofluorescence is kept at a low level.
(4) The whole experiment can be carried out in one (long) day.
Disadvantages to be kept in mind are:
(1) Protoplasting may alter certain cellular structures.
(2) Analysis is confined to cell types amenable to protoplasting
(mesophyll cells, etc.).
Part A: Isolation of Arabidopsis seedling protoplasts
1. Harvest one plate of seedlings, grown on solidified agar (GM
medium) for 7 to 10 days.
-> Ecotypes Columbia and No-O work well. Using a small pair of curved
scissors, cut the seedlings just above the agar surface to separate
the roots from the rest of the seedling. Transfer the green organs to
10 ml enzyme solution in a 125 ml Erlenmeyer flask or to 5 ml enzyme
solution in a 6 cm diameter glass petri dish.
2. Apply vacuum for 1 minute in a desiccator/bell jar.
3. Shake slowly (40-70 rpm) for 90 to 120 minutes.
4. Shake for 10 minutes at 80 to 100 rpm to release the protoplasts.
5. Filter the cell suspension through a 75 micrometer nylon mesh into
a 30 ml Corex glass tube.
-> Hold the folded mesh with a small plastic funnel or with one hand.
Handle the cells gently with a pasteur pipette. Wash the flask or the
dish with 5 ml of wash solution and pool this fraction with the first.
6. Spin for 2 minutes at room temperature at 150 g (1000 rpm) in a
HB-6 swingout rotor with rubber adapters (up to 500 g is acceptable).
7. Pipette off and discard the 10 ml supernatant, resuspend
protoplasts gently in 10 ml wash solution and incubate for 5 minutes.
8. Spin as above in step 6, remove supernatant, and resuspend the
protoplasts in 0.5 ml wash solution. If necessary, the cells can be
stored on ice.
Note: We thank Dr. Jen Sheen, Massachusetts General Hospital, Boston,
MA, for communicating the basis of this protocol.
SOLUTIONS AND REAGENTS-1A
GM medium (1 liter)
sucrose 10 g
MS salts (Sigma M-5519) 4.3 g
MES buffer 0.5 g
1000x vitamins 1 ml
Adjust pH to 5.7 with KOH, add 8 g Bacto-Agar, autoclave in a 1 l
bottle, cool to about 50=83C, and pour about 30 ml each into deep petri
dishes (Nunc 4026).
100 mg/ml myo-inositol
1 mg/ml thiamine
0.5 mg/ml nicotinic acid
0.5 mg/ml pyridoxine
cellulase R10 1%
macerozyme R10 0.25%
mannitol 0.4 M
MES 10 mM pH 5.7
Heat to 55=83C for 10 minutes to inactivate proteases and to accelerate
enzyme solubilization, then cool to room temperature.
CaCl2 30 mM
=FE-mercaptoethanol 5 mM
=46ilter through 0.45 micron filter. The solution can be stored for
several months at -20=83C without apparent loss of activity.
Cellulase 'Onozuka' R-10 and Macerozyme R-10 are supplied by Yakult
Honsha Inc. Co, Ltd., 1-1-19 Higashi Shinbashi Minato-ku; Tokyo; 105
mannitol 0.5 M
MES, pH 5.7 4 mM
KCl 2 mM
5 mM CaCl2, 2 mM malate, 2 mM ammonium sulfate, 1 mM potassium phosphate.
Part B: Immunofluorescence labeling of Arabidopsis protoplasts
1. Coat slides: Dip 8-well slides (Carlson Scientific, #100806) into
a narrow beaker with coating solution, air dry for 30 minutes by
leaning them against a support; rinse with water, then air dry and
store at 4=83C in a microscope slide holder, wrapped in cling film.
-> Coated slides will be provided.
2. Place slides onto a pair of pasteur pipettes over wet paper towels
in a dish with a tight lid (humid chamber).
3. With a pasteur pipette, apply a drop of protoplasts (ca. 50 =B5l in
wash solution) to each of the 8 wells on the slide, cover the
container and let the cells settle and adhere to the slide for 3
hours at room temperature.
-> Make sure that the cells will stay moist throughout, if not stated
-> Label slides with a pencil.
-> It is convenient to apply only one type of cells per slide and
apply different antisera onto the different wells, rather than the
other way round.
4. Gently remove most of the excess solution with a yellow tip from
the wells of one slide. With a blue tip, immediately add 50 =B5l
fixation solution to each well (need 400 =B5l per slide). Incubate for
5 to 10 minutes at room temperature. Repeat for the other slides.
-> Lots of protoplasts don't stick to the slide and are lost at this step.
-> Work quickly to prevent cells from drying out while they are not
protected by solution. Add and remove droplets of solution from the
edge of the well, avoiding to touch the well itself.
-> Incubation times should be reproducible, but can be optimized for
5. Permeabilize/destain the cells. Gently remove fixative solution
with a yellow tip/Pipetman, again working from the edge of the well.
Immediately add 25 =B5l permeabilization solution to each well and
incubate for 5 to 10 minutes.
-> Keep the slide horizontal to make sure that this solution doesn't
run off the slide.
6. Remove the permeabilization solution from one slide by aspiration
or with a yellow tip/Pipetman. Immerse the slide into a glass slide
holder/Coplin jar with acetone/ methanol (1:1 v/v, preferably kept at
-20oC). Repeat for the other slides. Incubate slides for 10 minutes
in the freezer (or on the bench). Using forceps, transfer the slides
to a second Coplin jar with the same solution and incubate for
another 5 minutes.
-> The slide holder is designed to hold up to 10 slides in back-to-back pair=
7. Pull out the slides with forceps and air dry for at least 20 minutes.
-> At this stage all green pigment should be gone.
-> The slides can now be stored in a slide holder, wrapped in
clingfilm, at 4oC over night.
8. Wash the slides once for 30 minutes with PBS (using the slide
holder), then place them into the humid chamber.
-> Keep the samle moist in the humid chamber from now on.
-> It is most convenient and safe to remove solutions from the wells
with a yellow tip at the end of a piece of tubing attached to an
9. Apply 50 =B5l blocking solution per well (PHEM, 2% BSA); incubate
for at least 30 minutes.
10. While the slides are being blocked, prepare the antibody
solutions. About 10 =B5l per well is needed. Make a dilution in
blocking solution in a 0.6 ml eppendorf tube. Spin the antiserum for
10 minutes at 13,000 rpm to remove any insoluble aggregates of
antibody and and transfer the supernatant to a new tube.
-> Leave the two left wells with blocking solution lacking antibody.
These will serve as controls for autofluorescence and for the
secondary antibody alone.
-> To reduce background fluorescence resulting from antibody
aggregates, it helps to store (a 1 in 5 dilution of) the antibody
stock in smalll aliquots at -80oC, after filtering it through a 0.2
-> The concentration of antibody has to be determined empirically. A
concentration of 5 =B5g per ml or a dilution of 1:100 is a point to
begin with. Try 1:30 and 1:300 as well.
11. remove blocking solution by aspiration and apply 8=B5l of antibody
per well. Incubate for 1 hour at room temperature or over night at
-> Make sure that the antibody cannot leak from one well to the next.
-> Overnight incubation tends to increase unspecific binding.
12. Wash. Remove first antibody solution, taking care that the sample
doesn't dry out completely. Add 50=B5l PBS to each well and remove,
then was with PBS containing 0.1% NP-40, then again with PBS (5
-> The second and third wash are done by immersion of the slide into
the slide holder.
13. While the slides are being incubated with the first antibody and
washed, prepare the secondary antibody. Again, make 10 ml per well of
a (suggested) 1:200 or 1:100 dilution in PBS, 0.5% BSA. Spin for 10
minutes at 13,000 rpm and take only the supernatant to remove any
insoluble aggregates. Apply 8 =B5l antibody to each well for 1 hour,
making sure that it doesn't leak from one well to the next.
-> Incubate one of the two wells lacking first antibody with
secondary antibody, and leave the other one with blocking solution
alone (autofluorescence control).
14. Wash slides once with PBS, twice with PBS, 0.1% NP-40, and then
once with PBS again, 5 minutes each.
-> The first wash is done on individual wells, the other washes in
the slide holder.
15. Remove PBS by aspiration and quickly add about 2 to 4 =B5l mount
solution to each well.
-> Spread out the mount solution with a yellow tip and add a
coverslip. Add as little mount solution as possible, but try to avoid
trapping air bubbles. To do this, initially add 2 =B5l; if this is not
enough to cover the well, add aother 2 =B5l along the edge of the well.
Seal the coverslip with nail polish and store slides in the dark.
16. Inspect slides at 1000x magnification (100x objective/lens) with
immersion oil on a microscope with epifluorescence optics.
-> Beforehand, clean any gelatin from the back of the slide with
water and a Kimwipe. Try to limit exposure of the samples to the
microscope light, because this tends to produce a hazy background,
rather than the more desirable black background. It is convenient to
look for nicely fixed cells under the UV/blue channel (DAPI), then
switch to the blue/green (fluorescein) or green/red channels (Texas
Red, rhodamine). If desired, photograph with 400 ASA color slide film.
-> fluorescein bleaches out very easily, even in the anti-fade
solution. Expose only briefly to epifluorescence light source.
SOLUTION AND REAGENTS-1B
Polylysine (reusable): 50 mg per liter in 10 mM Tris, pH 8.0; store at 4=83C=
Gelatin: Dissolve 1 g gelatin, 0.1 g chromic potassium sulfate
(Chrome alum, Aldrich) in 100 ml warm water. Make this solution fresh
PHEM (100 ml)
PIPES (MW 302.4) 60 mM 1.81 g
HEPES (MW 238.3) 25 mM 0.60 g
EGTA (MW 380.4) 10 mM 0.38 g
Mg2Cl (MW 203.3) 2 mM 40.6 mg
adjust pH to 6.9 with NaOH.
10x PBS (1 liter)
Na2HPO4 74 mM 10.6 g
NaH2PO4.H2O 14 mM 2.0 g
NaCl 1.5 M 90 g
=46ixation solution: 2% paraformaldehyde in PHEM.
-> To dissolve paraformaldehyde, heat gently to not more than 55=83C
and vortex occasionally. The choice of fixative and the design of the
fixation step are probably the most critical for the success or
failure of the experiment.
Permeabilization solution: 0.5% NP-40 in PHEM
Acetone/methanol 1:1 (v/v)
Blocking solution: 2% BSA in PHEM
Mount solution: Antifade' (Molecular Probes, S-2828), freshly mixed
to contain 1 mg/l DAPI (4',6-diamidino-2-phenylindole).
Experiment #2: Subcellular localization of GUS-fusion proteins in
GUS-fusion proteins have been employed extensively to ask whether a
test protein can drive nuclear localization of the GUS enzyme marker
(Restrepo et al., 1990). The fusion protein genes can be expressed by
particle bombardment (Varagona et al., 1992), transformation of
protoplasts (Abel et al., 1994) or from chromosomal transgenes
(Varagona et al., 1991).
In this experiment, transgenic Arabidopsis seedlings carrying genes
for GUS-COP1, GUS-NIa, or GUS will be provided.
Localization of Arabidopsis proteins with GUS in situ enzyme assay
1. For each sample, add 300 =B5l of stain solution to a well of a
24-well tissue culture dish.
2. Using forceps, harvest seedlings grown on GM for 6 days and
immediately place them into the stain solution.
-> Note: in general, a gentle prefixation of the tissue (2%
formaldehyde in wash solution for 2 minutes), followed by washing,
may preserve the subcellular localization better, but at the expense
of the sensitivity of the detection. We did not notice significant
differences in the relative GUS staining patterns with or without the
3. Incubate seedlings in stain solution at room temperature until the
desired staining intensity is achieved.
-> Monitor progress under the stereomicroscope and/or:
-> Inspect under the light microscope in the presence of 1 =B5g/ml DAPI.
4. If desired, replace stain with wash solution.
5. Replace wash solution with fixation solution, incubate for 30'.
-> Wear gloves when handling fixation solution.
6. Replace fixation solution with 30% ethanol, leave on a rocking
table for 10 minutes, then change to 50% and 70% ethanol. Incubate in
70% ethanol overnight, gradually transfer back to wash solution and
mount the seedlings in wash solution with 1 =B5g/ml DAPI on regular
microscope slides. Seal the slides and store at 4=83C.
7. Observe under light microscope.
SOLUTIONS AND REAGENTS
sodium phosphate, pH 7.0 100 mM
EDTA 1 mM
potassium ferrocyanide 5 mM
potassium ferricyanide 5 mM
Triton-X-100 1 %
X-Gluc 1 mg/ml
-> dissolve X-Gluc in a small volume of dimethylformamide by warming
it for a few seconds at 50=83C.
Wash solution: 100 mM sodium phosphate, 1 mM EDTA, pH 7
=46ixation solution: 3.7% formaldehyde in wash solutionExperiment #3:
Transient expression by particle bombardment
Particle bombardment has been used to introduce test plasmids into
various tissues of Arabidopsis. Potential applications:
- Cell type specificity of promoter fragments
- Strength of promoter fragments in a defined cell type
- Subcellular localization of proteins
1. Prepare DNA:
=46or 5 shots @ 2 microgram plasmid DNA
- Place 50 =B5l of ethanol-washed tungsten particles into a 1.5 ml tube
and vortex heavily.
-> Tungsten is prepared by suspension of commercially available
tungsten powder at 50 mg per ml ethanol, thorough sonication, and
washing with ethanol for four times. The particles are stored at
-20=83C in 1 ml aliquots.
- Spin in microfuge for a few seconds and wash in 50 =B5l water.
- Repeat wash
- Resuspend in 50 =B5l water
50 =B5l tungsten particles in water
12 =B5l of DNA solution (1 mg/ml of a GUS-expression plasmid in water)
50 =B5l 2.5M CaCl2
20 =B5l 0.1M spermidine, free base
- Incubate 20 minutes on ice with occasional vortexing
- Add 200 =B5l ethanol, spin two minutes, and discard supernatant
- Wash particles 3 times in ethanol
- Resuspend particles in 30 =B5l ethanol, vortex well
- Pipette 5 =B5l of the particles onto each of five macrocarriers
(flying disks (orange)).
-> Make sure the particles are well suspended, while dispensing them.
Discard the last 5 =B5l (too lumpy).
-> Dry particles on macrocarrier on filter paper first in air then in
a petri dish with drierite.
2. Harvest one plate of seedlings (ecotype No-O or Columbia), grown
on solidified agar (GM medium) for 7 to 10 days. Use forceps to
gently pull out the seedlings from the agar and arrange them
(crowded) in the center of a petri dish of solidified MS medium with
the roots in the center and the shoots on the periphery.
3. Bombardment with 1200 psi rupture discs, following specific
instructions for BioRad particle gun.
4. After bombardment, seal the plates with parafilm and incubate over
night under white light at 22=83C.
5. Stain the seedlings in a plastic dish with 3-5 ml of GUS-stain
solution (see Experiment 2).
-> Monitor progress of staining under the stereomicroscope. To stop
the staining, wash the seedlings in 100 mM phosphate, 1 mM EDTA (wash
solution) and fix them in 3.7% formaldehyde in wash solution for 30
minutes, then wash them again before mounting onto microscope slides.
- Open pressurized helium tank, turning grey knob
- adjust pressure setting on helium tank gauge to 200 psi above the
value of the rupture disk to be used.
- Switch on vacuum pump
- Switch on particle gun (left of three red buttons).
1. Insert rupture disk (pressure disk, e.g. BioRad 165-2329): Unscrew
grey disk holder from top of vacuum chamber, insert disk (can be
sterilized in isopropanol beforehand), and screw grey disk holder
-> Make sure disk makes a tight seal with holder and doesn't move
while the holder is screwed back on.
2. Place macrocarrier (orange flying disk) with DNA side up into the
small metal holder. Make edge of disk flush with holder by simply
pressing the disk down with the red 'hat' supplied in the accessories
3. Unscrew lid (ca. 6cm diameter) from white Teflon plate. Insert
stopping screen (BioRad 165-2336, package of 500) into bottom of the
plate, then insert macrocarrier holder (with flying disk and DNA),
DNA-side down, then screw lid back on. Place the whole set into the
first shelf of the vacuum chamber with the tiny screw facing the
door. -> The tiny screw can be loosened with an allen key in the
accessories kit; then the position of the DNA can be adjusted up and
down, if desired.
4. Place sample tissue into second shelf from the bottom. Close chamber door=
5. Pull vacuum (upper position on three-way middle switch of three
-> I use 28 in Hg vacuum (pump will only deliver 27 in right now).
Hold vacuum by quickly pressing three-way switch all the way down.
6. press fire switch (right button) continuously. Pressure will rise
behind pressure disk, disk will burst, etc.
C. Open chamber, repeat procedure for second sample etc.
D. Shut down:
1. Close Helium tank (grey knob) .
2. Pull vacuum (about 15-20in) and hold.
3. Press fire switch to bleed helium line until pressure on gauge is
close to zero.
4. Open pressure adjustment screw until handle runs freely
5. Bleed residual vacuum from chamber.
6. Shut off gun and vacuum pump.
SOLUTIONS AND REAGENTS
MS medium plates
4.3 g MS salts (Sigma), 1 mg thiamine, 10 mg myo-inositol, 180 mg
potassium dihydrogenphosphate, 30 g sucrose, adjust pH to 5.7 with
KOH, 1.2% bacto-agar; autoclave, and pour into shallow petri dishes.
-> optional: add 2.5 mg amphotericinB and 100 mg carbenicillin per liter.
Other solutions have been described in Experiment 2.
Abel, S. and Theologis, A. (1994). Transient transformation of
Arabidopsis leaf protoplasts: a versatile experimental system to
study gene expression. Plant Journal 5, 421-427.
Abel, S., Oeller, P., and Theologis, A. (1994). Early auxin-induced
genes encode short-lived nuclear proteins. Proc. Natl. Acad. Sci.
(USA) 91, 326-330.
Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W., and Prasher, D.C.
(1994). Green fluorescent protein as a marker for gene expression.
Science 263, 802-805.
-> GFP as a reporter for cell type specific promoter activity
Citovsky, V, Warnick, D, and Zambryski, P. (1994). Nuclear import of
Agrobacterium VirD2 and VirE2 proteins in maize and tobacco. Proc.
Natl. Acad. Sci. (USA) 91, 3210-3214.
Colasanti, J., Cho, S.-O., Wick, S., and Sundaresan, V. (1993).
Localization of the functional p34(cdc2) homolog of maize in root tip
and stomatal complex cells: association with predicted division
sites. Plant Cell 5, 1101-1111.
Doonan, J., Zhang, H., and Traas, J. Indirect immunofluorescence on
Arabidopsis seedlings. Arabidopsis - The Compleat guide.
Heim, R., Prasher, D.C., and Tsien, R.Y. (1994). Wavelength mutations
and posttranslational autoxidation of green fluorescent protein.
Proc. Natl. Acad. Sci. (USA) 91, 12501-12504.
Jefferson, R.A. (1987). Assaying chimeric genes in plants: The GUS
gene fusion system. Plant Mol. Biol. Reporter 5, 387-405.
Kerrebrock, A.W., Moore, D.P., Wu, J.S., and Orr-Weaver, T.L. (1995).
Mei-S332, a Drosophila protein required for sister-chromatid
cohesion, can localize to meiotic centromere regions. Cell 83,
Matsui, M., Stoop, C.D., von Arnim, A.G., Wei, N., and Deng, X.-W.
(1995). Arabidopsis COP1 specifically interacts in vitro with a novel
cytoskeleton associated protein. Proc. Natl. Acad. Sci. (USA) 92,
Neuhaus, G., Bowler, C., Kern, R., and Chua, N.-H. (1993).
Calcium/calmodulin-dependent and -independent phytochrome signal
transduction pathways. Cell 73, 937-952.
-> This report describes a cryosectioning protocol, after stringent
fixation of the tissue with formaldehyde, followed by detection with
fluorescent antibodies. The antigens are abundant plastid proteins.
Olson, K.R., McIntosh, J.R., and Olmsted, J.B. (1995). Analysis of
MAP4 function in living cells using green fluorescent protein (GFP)
chimeras. J. Cell Biol. 130, 639-650.
Prasher, D.C. (1995). Using GFP to see the light. Trends Genet. 11, 320-323.
-> This review is accompanied by a series of others describing the
application of GFP in different species.
Restrepo, M.A., Freed, D.D., and Carrington, J.C. (1990). Nuclear
transport of plant potyviral proteins. Plant Cell 2, 987-998.
-> One of the first papers to describe the use of GUS-fusions for
Varagona, M.J., Schmidt, R.J., and Raikhel, N.V. (1991). Monocot
regulatory protein Opaque-2 is localized in the nucleus of maize
endosperm and transformed tobacco plants. Plant Cell 3, 105-113.
Varagona, M.J., Schmidt, R.J., and Raikhel, N.V. (1992). Nuclear
localization signal(s) required for nuclear targeting of the maize
regulatory protein Opaque-2. Plant Cell 4, 1213-1227.
von Arnim, A.G. and Deng, X.-W. (1994). Light inactivation of
Arabidopsis photomorphogenic repressor COP1 involves a cell-specific
regulation of its nucleocytoplasmic partitioning. Cell 79, 1035-1045.
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