2
.
6
. Immunohistochemical staining and fluorescence microscopy
To visualize the expression of AtENT1-His-tag, sf21 cells at 48 h post transfection were collected
by centrifugation 1000 g for 2 min, fixed with 4 paraformaldehyde for 10 min and spread onto
poly-
L
-lysine Sigma treated slides. After 10 min of drying on a warm plate set at 45°C, the slides
were washed with TBS 150 mM NaCl, 100 mM Tris – HCl, pH 7.5 for 5 min, followed by an
incubation in
TBSM TBS
containing 5
skimmed milk powder for 60 min to block non- specific binding sites. After the incubation, the
cells were treated with a mouse monoclonal anti- body that recognized the His-tag Pharmacia,
1:2000 dilution in TBSM for 24 h at 4°C. The antibody reaction was terminated by washing the
cells three times 5 min each in TBS. A goat anti-mouse IgG-alkaline phosphatase conjugate
solution Sigma, 1:2000 dilution in TBS was then applied to the washed cells. After a 1-h reaction,
the cells were washed three times with TBS 5 min for each wash. To detect antibodyconjugate reac-
tion, the cells were treated with a substrate solu- tion containing NBT and BCIP as detailed by
Wang and Maule [31]. To check the specificity of the monoclonal antibody, sf21 cells transfected
with the WT bacmid were stained exactly as de- scribed above. The staining results were pho-
tographed with an Olympus microscope using Kodak color films 400 ASA.
For visualizing the expression of GFP-AtENT1- His-tag by fluorescence microscopy, cells at 48 h
post transfection were collected and spread onto clean slides. The cells were mounted in 10 glyc-
erol in 130 mM NaCl, 7 mM Na
2
HPO
4
, 3 mM NaH
2
PO
4
containing 1 mgml propium iodide Sigma and immediately examined under either a
conventional fluorescence microscope Olympus or a confocal microscope Bio-Rad 680 using
appropriate excitation and emission filters. In con- focal microscopy, selected cells were optically sec-
tioned at a 2 mm interval. On average, about 15 sections
were obtained
for each
of the
examined cells. Fluorescent signals in all sections were carefully examined and recorded. Cells trans-
fected with the P1-GFP bacmid were also exam- ined via confocal microscopy. The GFP fusion
protein specified by this bacmid was used as a positive control for observing GFP fluorescence in
insect cells.
3. Results
3
.
1
. Cloning and amino acid sequence analysis of AtENT
1
cDNA RT-PCR reactions with primers NT1 and NT2
generated a cDNA fragment of approximately 1.3 kb Fig. 1. The size of this fragment corresponded
well with that of the AtENT
1
coding region calcu- lated from the genomic sequence. Using the TA
cloning strategy, the PCR fragment was cloned into the pGEM-T vector. The cDNA sequence of
the insert in two independent clones was exactly the same, and in both sequences a single ORF
coding for a polypeptide of 428 amino acids was identified. Although equal in the number of the
amino acids, the AtENT1 polypeptide specified by the cloned cDNA differed from the one deduced
from the genomic sequence at two amino acid positions owing to two single nucleotide substitu-
tions. In positions 89 and 410 of the polypeptide specified by the cDNA, a proline and a threonine
residue were present, respectively, whereas in the polypeptide deduced from the genomic sequence
the corresponding positions were occupied by a leucine and a methionine residue, respectively. The
amino acid sequence predicted from AtENT
1
cDNA possessed significant similarity to those of several ENTs. Pairwise comparisons revealed that
the putative nucleoside transporter AtENT1 was
Fig. 1. Amplification of AtENT
1
cDNA by RT-PCR. PCR was performed without template 1 or with RT mixture
2 – 5. The presence of a 1.3 kb DNA fragment in lanes 2 – 5 is indicated by an arrow. Lane 6 contains DNA markers.
Fig. 2. Comparison of the deduced amino acid sequences of hENT1, hENT2 and the putative nucleoside transporter AtENT1. The AtENT1 amino acid sequence was derived by conceptual translation of the AtENT
1
cDNA sequence. The three sequences were aligned using programs of the NCBI Network Services. Identical amino acids in the three sequences are shown in white on
a black background. Solid lines above hENT1 and below AtENT1 sequences indicate putative transmembrane TM domains that may be present in the two proteins. The numbers on the left and right of the figure are amino acid positions in each sequence.
24 identical to hENT1, 25 to hENT2, 24 to rENT1 rat equilibrative nucleoside transporter 1,
26 to rENT2, 22 to LdNT1 Leishmania dono- 6
ani nucleoside transporter 1 and 21 to TbNT2 Trypanosoma brucei nucleoside transporter 2. In
addition to above ENTs, two hypothetical Ara- bidopsis proteins AAD25545 and AAF04424
identified by the genome-sequencing project also showed similarity to AtENT1 28 identity was
found between AAD25545 and AtENT1 and 29 between
AAF04424 and
AtENT
1
. Eleven
transmembrane domains were predicted for the putative nucleoside transporter AtENT1. The or-
ganization of the putative transmembrane do- mains in AtENT1 was similar to that in hENT1
Fig. 2.
3
.
2
. Detection of AtENT
1
related DNA sequence in Arabidopsis genome
Genomic DNA was extracted from Arabidopsis plants and was digested with several restriction
enzymes. The gel blots were hybridized with
32
P- labelled probes. Post-hybridization washes used
both high and low stringency washing conditions. Fig. 3 showed a typical example of the autoradio-
graphies obtained. Two hybridizing bands were present in EcoRI digested DNA Fig. 3. Three
hybridizing bands were found for HindIII and DraI digested DNA, but the two smallest bands
Fig. 3, arrowed were not sufficiently large to represent intact genes on their own. These results
showed that, in the Arabidopsis genome, there was
only one additional sequence gene that was re- lated to AtENT
1
gene.
3
.
3
. Expression of AtENT
1
mRNA in different organs and in plants at different de6elopmental
stages Two sets of Northern blot hybridization experi-
ments were performed to investigate the expres- sion of AtENT
1
in Arabidopsis. The intensity of the hybridization signals generated from total
RNA samples extracted from leaf, root, rachis and flower of fully-grown Arabidopsis plants was of a
similar level Fig. 4A. When total RNA samples derived from plants at eight different developmen-
tal stages were hybridized, there was also little difference in the hybridization signal in between
the eight samples Fig. 4B.
3
.
4
. In 6itro expression of AtENT
1
fusion genes in insect cells
For future functional study, it is important to test the expression of AtENT
1
in an eukaryotic expression system and to obtain information on
intracellular localization of the putative nucleoside transporter AtENT1 in eukaryotic cells. For this
purpose, we expressed the AtENT1-His-tag fusion gene in insect cells. By immunocytochemical stain-
ing using a monoclonal antibody specific for the His-tag, positive expression of the AtENT1-His-
tag fusion gene in sf21 cells was demonstrated Fig. 5A and B. Although the signal produced by
immunocytochemical staining was sufficient for checking fusion gene expression, it was not condu-
cive for examining intracellular localization of the expressed product. To overcome this problem, a
second fusion gene, prepared by adding the GFP coding region to the 5 end of AtENT1-His-tag,
was expressed in sf21 cells. When observed under a conventional fluorescence microscope, 30 – 50
of the cells exhibited GFP fluorescence and the GFP fluorescence was associated with the plasma
membrane Fig. 5C. No GFP fluorescence was detected in the cells expressing the WT type
genome of the baculovirus Fig. 5D. Expressing cells were further examined under a confocal mi-
croscope, because we wished to investigate if there was also some GFP fluorescence present in intra-
cellular membrane systems such as endoplasmic reticulum and nuclear membrane. Optical section-
ing permitted a more detailed, and hence more precise, localization of GFP fluorescence within
the entire contents of the examined cells. The results showed that, in the cells expressing GFP-
AtENT1-His-tag, GFP fluorescence was localized
Fig. 3. Detection of AtENT
1
related DNA sequence in Ara- bidopsis genome by Southern blot hybridization. Aliquots 5
m g each of genomic DNA were digested with EcoRI lane E,
HindIII lane H or DraI lane D, the digested DNA samples were fractionated on 1 agarose gels followed by transfer
onto Hybond N
+
membrane. The resulted blots were hy- bridized with
32
P-labelled probes prepared with AtENT
1
cDNA. The size of DNA markers is indicated in kilobase kb pairs. The two bands indicated by arrow are too small to
represent intact genes.
Fig. 4. Northern hybridization analysis of AtENT
1
mRNA in different organs of fully-grown Arabidopsis plants A and in
Arabidopsis plants at different developmental stages B. Total RNA samples 20 mg each from root lane RO, rachis lane
RA, leaf lane LE, flower lane FL, plants at four vegeta- tive stages lanes 1 – 4 and plants at four reproductive stages
lane 5 – 8 were fractionated on agarose gels prior to transfer to Nylon membrane. The resultant blots were hybridized
firstly with
32
P-labelled probes prepared with AtENT
1
cDNA to detect AtENT
1
mRNA. After stripping, the blots were further hybridized with
32
P-labelled probes specific for 18S rRNA to check the loading of the RNA samples.
Fig. 5. Expression of two AtENT
1
fusion genes in insect cells. AtENT1-His-tag expressing sf 21 cells A and the cells expressing wild type baculovirus genome B were immuno-stained with an anti-His-tag monoclonal antibody. The cells shown in A were
positively stained. In C, a sf21 cell expressing GFP-AtENT1-His-tag was examined and photographed with a conventional fluorescent microscope, GFP fluorescence green color was associated with the cell periphery. No GFP fluorescence was observed
in the sf21 cell expressing wild type baculovirus genome D. In E, a confocal image of a sf21 cell expressing GFP-AtENT1-His- tag is shown. GFP fluorescence was associated specifically with the plasma membrane. In contrast, in the cell expressing a
predominantly cytosolic GFP fusion protein F, GFP fluorescence was distributed mainly in the cytoplasm F. In C – F, cells were counter-stained with a PI solution to reveal the nuclei arrowed. Bars represent 60 mm in A and B, 15 mm in C and D
and 10 mm in E and F.
to the plasma membrane Fig. 5E and no GFP fluorescence was detected to be associated with
other cellular constituents. In contrast, in the cells expressing the cytosolic P1-GFP fusion protein,
GFP fluorescence resided predominately in the cytoplasm Fig. 5F.
4. Discussions
