Plant Cell Physiol. 45(6): 795–802 (2004)
JSPP © 2004
Short Communication
Sex-Specific Cell Division during Development of Unisexual Flowers in the
Dioecious Plant Silene latifolia
Sachihiro Matsunaga 1, 2, Wakana Uchida and Shigeyuki Kawano
Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, 5-1-5, Kashiwanoha, Kashiwa, Chiba, 277-8562
Japan
;
Keywords: Cell division — Dioecious plant — Flower meristem — Sex chromosome — Sex development — Unisexual
flower.
The nucleotide sequences reported in this paper have been submitted to GenBank, EMBL, and DDBJ under the accession numbers
AB102686 (SlH4) and AB102687 (SlCycA1).
All floral organs are generated by a group of undifferentiated cells, the floral meristem. Like the shoot apical meristem,
the floral meristem is divided into three layers: the epidermal
layer (L1 layer), the subepidermal layer (L2 layer), and the
inner core (L3 layer) (Szymkowiak and Sussex 1996, Traas and
Doonan 2001). Cells of the L1 layer divide anticlinally
throughout development, so that daughter cells remain in the
same layer, whereas cells of the L3 layer divide in all planes
(Vernoux et al. 2000). In contrast, cells of the L2 layer initially
divide anticlinally, but can also divide periclinally during organ
development (Vernoux et al. 2000). The three cell layers are
clonally distinct, and cells of each layer specifically contribute
to the different lineages of the floral organs. The L1 layer contributes to the epidermis, the stigma, part of the transmitting
tract, and the integument of the ovules, while the L2 and L3
layers contribute to the mesophyll and other internal tissues to
different degrees (Jenik and Irish 2000). Cell division patterns
1
2
Present address: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, 565-0871 Osaka,
Japan
Corresponding author: E-mail, [email protected]; Fax, +81-6-6879-7441.
795
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are regulated differentially at different stages of floral development. By differential cell division, floral meristems produce
four different whorls: sepals in the outermost whorl (whorl 1);
petals in whorl 2; stamens in whorl 3; and carpels in the innermost whorl (whorl 4) (Bowman et al. 1991). After floral organ
identity has been established, stamens and carpels develop in
the third and fourth whorls, respectively, of bisexual flowers on
hermaphroditic plants.
Only 6% of angiosperm species are dioecious, with separate individuals producing staminate and pistillate flowers
(Renner and Ricklefs 1995). In dioecious plants, unisexual
flowers develop through the suppression or promotion of each
sex primordia. The sex development patterns of dioecious
plants can be divided into three classes based on the developmental stage at which sex differences morphologically appear
(Matsunaga and Kawano 2001). The first group has flower
buds that rarely form the primordia of the opposite sex. In the
second group, development of opposite-sex primordia is initiated, but then arrested in the early stages. In the third group,
development of the organs of the opposite sex is arrested at
much later stages of development. The dioecious campion
Silene latifolia, in the family Caryophyllaceae, belongs to the
second group. A gynoecium primordium is suppressed in the
male flower and later becomes a rudimentary gynoecium, like
a filamentous rod that lacks an ovary and pistils. In whorl 3
of female flower buds, the growth of stamen primordia is
arrested and the tissues degenerate before each flower opens
(Matsunaga et al. 1996, Uchida et al. 2003). The differences
between male and female flower buds have been detected only
by morphological observations using scanning electron microscopy and paraffin sections (Grant et al. 1994, Farbos et al.
1997). The genetic programs that determine the specific contributions of the different lineages to the male and female floral
organs will be reflected in the sex-specific regulation of the
factors controlling cell division patterns.
Cell proliferation is controlled by highly conserved molecules, including histones and cyclins. Histones are universally
conserved nuclear proteins that can be classified into five
subtypes: H1, H2A, H2B, H3 and H4. The transcription of his-
We analyzed cell division patterns during the differentiation of unisexual flowers of the dioecious plant Silene latifolia using in situ hybridization with histone H4 and cyclin
A1 genes. The gene expression patterns indicated that the
activation of cell divisions in whorls 3 and 4 was reversed in
young male and female flower buds. During maturation of
flower buds, a remarkable reduction in cell division activity occurred in the male gynoecium primordium and female
stamen primordia. Our analyses showed that differential
activation and reduction of cell division strongly correlated
with sex-specific promotion and cessation in the sex differentiation of unisexual flowers.
796
Sex-specific cell division in flower development
tone mRNAs is completely coupled with cell proliferation and
DNA replication. S phase-specific expression of histones
results in a twofold increase in the total amount of histone
(Meshi et al. 2000). Plant cyclins can be grouped into A-, B-,
D- and H-type cyclins, mostly by analogy to their animal
counterparts (Vandepoele et al. 2002). The plant A-type cyclins are classified into three groups, CycA1, CycA2 and CycA3
(Renaudin et al. 1996). Representative members of the three
groups have been found in all angiosperms (Chaubet-Gigot
2000). CycA1 is equally expressed in root and shoot apical
meristems, whereas CycA2 is expressed most strongly in root
apical meristems (Chaubet-Gigot 2000). In synchronized
tobacco BY-2 cells, the CycA1 and CycA2 genes were induced
at mid-S phase, and their transcripts were drastically reduced at
mid-mitosis (Setiady et al. 1995, Reichheld et al. 1996). In soybean, the CycA1 transcript was detected primarily in late S- and
G2-phase cells by in situ hybridization (Kouchi et al. 1995).
Generally, CycA1 is more highly expressed than CycA2 or
CycA3 (Chaubet-Gigot 2000). Therefore, we isolated the histone H4 and CycA1 genes from unisexual flower buds of S. latifolia as S phase and late S to G2 phase-specific markers and
compared developmental cell division patterns between male
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Fig. 1 (A) Alignment of deduced amino acid sequences
of A1-type plant cyclins. Lyces;CycA1;1, Nicta;CycA1;
1, and Glyma;CycA1;1 represent amino acid sequences
of Lyces;CycA1;1 (AJ243451; Joubes et al. 2000), Nicta;
CycA1;1 (X92967; Reichheld et al. 1996) and Glyma;
CycA1;1 (D50870; Kouchi et al. 1995), respectively. The
solid and shaded boxes represent fully and partially identical amino acids, respectively. Asterisks indicate perfect
consensus sequences. The destruction boxes and the
nuclear export signals are underlined and framed, respectively. The cyclin core region is indicated with arrows.
(B) Unrooted phylogenetic tree of the most conserved
part of A-type plant cyclins. The sequences of 197 amino
acids from the cyclin core region, as shown in Fig. 1A,
were aligned, and the tree was constructed using the
DNASpace Ver. 3.5 software (Hitachi Soft Engineering,
Japan). Arath;CycA1;1, Arath;CycA2;1, and Arath;
CycA3;1 from Arabidopsis thaliana (Accession No.
AV556475, Z31589 and AT5g25380; Ferreira et al.
1994; Vandepoele et al. 2002), Glyma;CycA1;1, Glyma;
CycA2;1, and Glyma;CycA3;1 from Glycine max
(D50870, D50969 and D50868; Kouchi et al. 1995),
Lyces;CycA1;1, Lyces;CycA2;1, and Lyces;CycA3;1 from
Lycopersicon esculentum (AJ243451, AJ243452 and
AJ243453; Joubes et al. 2000), Nicta;CycA1;1, Nicta;
CycA2;1, and Nicta;CycA3;1 from Nicotiana tabacum
(X92967, D50736 and X92964; Setiady et al. 1995,
Reichheld et al. 1996).
Sex-specific cell division in flower development
797
and female flowers using in situ hybridization with these
phase-specific markers.
Using RT-PCR and screening of a male flower bud cDNA
library, we obtained two full-length cDNA clones, SlCycA1 and
SlH4, corresponding to A1-type cyclin and histone H4.
SlCycA1 was 1,902 bp in length and contained a 1,461-bp open
reading frame expected to encode a protein of 487 amino acids.
A comparison of the deduced amino acid sequence with
BLASTP showed significant identity with the A1-type mitotic
cyclins in plants, for example, 63% identity with NtcycA30 of
Nicotiana tabacum (Reichheld et al. 1996, Accession No.
X92966) and 62% identity with cyc3Gm of Glycine max
(Kouchi et al. 1995, Accession No. D50870). Fig. 1a shows an
alignment of the deduced amino acid sequences of Al type
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Fig. 2 In situ hybridization analyses of young flower buds with RNA probes derived from SlCycA1 and SlH4. Fixed flower buds were embedded in paraffin; 8-µm longitudinal sections were allowed to hybridize with a mixture of biotinylated probe derived from SlCycA1 and digoxigeninlabeled RNA probe derived from SlH4. First, the biotinylated antisense probe was detected in the hybridized sections; signals corresponding to
SlCycA1 transcripts appeared brown (A, D, E, F). Next, the digoxigenin-labeled antisense probe was detected; signals corresponding to SlH4
appeared purple (B, G–L). Cells with transcripts of both SlCycA1 and SlH4 appeared dark purple. Sense probes derived from SlCycA1 and SlH4
were not detected above background (C). (A, B, C) male flower buds at stage 4; (D, G) a male flower bud at early stage 5; (E, H) a male flower
bud at late stage 5; (F, I) a male flower bud at stage 6; (J) a female flower bud at early stage 5; (K) a female flower bud at late stage 5; (L) a female
flower bud at stage 6. G, P, Se and St indicate a gynoecium primordium, a petal primordium, a sepal primordium, and a stamen primordium,
respectively. Bars = 50 µm.
798
Sex-specific cell division in flower development
Table 1 Percent of dividing cells in whorls of floral meristems
Stage 5
Male
Whorl 3
Whorl 4
Female
Whorl 3
Whorl 4
Stage 6
Early
Late
L1
L2
L3
L1
L2
L3
42%
38%
32%
11%
8%
17%
11%
13%
18%
30%
38%
26%
46%
53%
46%
17%
24%
31%
L1
L2
L3
L1
L2
L3
18%
19%
33%
40%
32%
35%
45%
38%
30%
18%
13%
47%
35%
27%
37%
44%
27%
43%
into two types (Koning et al. 1991). In one, this occurs during S
phase in the cell cycle. The other undergoes endoreduplication,
in which the nuclear DNA is replicated without mitosis, resulting in polyploidy. Endoreduplication is consistently detected
during flower development (Kudo and Kimura 2001). Cells
with signals from both SlCycA1 and SlH4 appeared dark purple; and were regarded as dividing cells because cyclins were
not expressed during endoreduplication (Kondorosi et al.
2000). The ratio of cells with both signals in the third and
fourth whorls was examined in more than five series of sections from different flower buds at stages 5 and 6, as shown in
Table 1. This percentage of dividing cells based on SlCycA1
expression was underestimated relative to the percentage of
cells in the cell cycle because cells during early S or M phase
were not counted as dividing cells.
At stage 4, the signals corresponding to SlCycA1 completely overlapped those of SlH4 (Fig. 2A, B), suggesting that
SlH4 expression is coupled with the cell cycle. When the
biotin-labeled SlH4 gene and the digoxigenin-labeled SlCycA1
gene were used for in situ hybridization, the result was completely consistent with the result using the above probes. Both
signals appeared to have the same pattern in female and male
flower buds at stage 4 (data not shown).
At stage 5, stamen primordia arose in whorl 3. The dome
of whorl 4 was rounded in female flower buds, but it was
almost flat in the male flower bud (Grant et al. 1994). We
divided stage 5 into two sub-stages, early stage 5 and late stage
5. In both male and female flower buds at late stage 5, the
heights of undifferentiated fourth whorls were greater than
10% and 40% of the widths, respectively. At stage 5, the signal
corresponding to SlH4 completely overlapped with that corresponding to SlCycA1 (Fig. 2D, G, J). At early stage 5 of male
flower buds, patched signals were detected in all layers of
emerging stamen primordia (Fig. 2D, G); only faint signals
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cyclins from four species: Lycopersicon esculentum, N. tabacum, G. max and S. latifolia. This indicates that SlCycA1 has a
cyclin core that generates multiple recognition sites for interactions with other proteins, including cyclin-dependent kinases
(Noble et al. 1997). The N-terminal domain of plant cyclins has
a destruction box that is involved in the degradation of its
cyclin via the ubiquitin pathway (Glotzer et al. 1991). The consensus sequence RXA/PLXNL/IXN (X stands for any amino
acid) for the destruction box typical of A1-type cyclins is perfectly conserved in SlCycA1 (Fig. 1A).
A hydrophobic nuclear export signal is also found in
SlCycA1 (Fig. 1A). The nuclear export signal can be detected
upstream of the cyclin core in all plant CycA1 cyclins, but is
not found in either CycA2 or CycA3 (Chaubet-Gigot 2000). An
unrooted phylogenetic tree of A-type cyclins constructed using
the cyclin cores clearly showed that SlCycA1 belonged to the
CycA1 family (Fig. 1B). The SlH4 cDNA was 551 bp in length
and contained a 309-bp open reading frame that is expected to
encode a protein of 103 amino acids. The deduced amino acid
sequence of SlH4 matched that of plant histone H4 perfectly.
To examine the distribution of dividing cells in male and
female flower buds, we performed double-labeling in situ
hybridization, in which each tissue section was hybridized with
probes specific for transcripts of SlCycA1 and SlH4. The longitudinal sections of flower buds were hybridized with a mixture
of a biotin-labeled SlCycA1 gene and a digoxigenin-labeled
SlH4 gene. After the detection of positive signals with the
biotin-labeled probe using tyramide amplification with a brown
coloration, the digoxigenin-labeled probe was detected using
an anti-digoxigenin antibody with alkaline phosphatase in combination with a purple color substrate. We allowed many sections to hybridize with both probes.
Early flower development was classified into 12 stages
based on morphological features (Grant et al. 1994). At stage 4,
sepal primordia are established in male and female flower buds.
There are no morphological differences between males and
females at this stage (Grant et al. 1994). Fig. 2 shows an in situ
hybridization analysis of longitudinal sections of the male
flower bud. When antisense probes for SlCycA1 and SlH4 were
used for in situ hybridization analyses, both probes gave signals
significantly above background in the cytoplasm of sepal primordia cells (Fig. 2A, B). These signals were also detected in
some cells of the L1 and L2 layers in the second outermost
region of the flower meristem. This region is the third whorl,
where stamen primordia subsequently appear. When a sense
probe for SlH4 was used, significant signals were not detected
(Fig. 2C). Counterstaining of sections with 4′,6-diamidino-2phenylindole revealed that signals corresponding to neither
SlCycA1 nor SlH4 could be detected in cells with condensed
chromosomes (data not shown). This suggests that these transcripts are absent during M phase. Taken together with the
above phylogenetic analysis (Fig. 1C), the results indicate that
SlCycA1 is expressed predominantly during late S and G2
phases. Plant cells that express histone genes can be classified
Sex-specific cell division in flower development
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were detected in the L1 layer of the fourth whorl of the male
flower bud. At late stage 5 of male flower buds, intense signals
were detected in whorl 4 (Fig. 2E, H). In contrast, female
flower buds at early stage 5 exhibited more patched signals in
whorl 4 than in whorl 3 (Fig. 2J); dividing cells were detected
in all layers of whorl 4. At late stage 5 of female flower buds,
many dividing cells could be detected in the small, young stamen primordia of whorl 3. The numbers of dividing cells in the
L1 and L2 layers were reduced in whorl 4 of female flower
buds, although about half the cells in L3 were dividing (Fig.
2K, Table 1).
In male flower buds at stage 6, a single gynoecium primordium appeared in whorl 4; the ratio of dividing cells
increased to about 50% in all layers of whorl 3 (Fig. 2F, I,
Table 1). Only a few signals were detected in the L1 layer of
whorl 4 (Fig. 2F, I). In female flower buds at stage 6, a gynoecium primordium emerged from whorl 4 (Fig. 2L); the ratio
of dividing cells increased in the L1 and L2 layers of the fourth
whorl (Fig. 2L, Table 1). Although female stamen primordia
were smaller than male stamen primordia, many dividing cells
were found in female stamen primordia. The presence of cell
division in female stamen primordia may reflect the fact that
female stamens differentiate to at least stage 7. At stage 6 of
both male and female flower buds, only 15% of all cells positive for the SlH4 signal lacked the SlCycA1 signal, and these
cells appeared light purple. They were probably cells in early S
phase of the cell cycle or endoreduplicating cells.
At stage 7 of male flower buds, stamen primordia began to
differentiate into anthers and filaments. Dividing cells were
detected in the sepal, petal, and stamen primordia, and dividing cells drastically decreased in the suppressed gynoecium primordium (Fig. 3A). At stage 8, stamens became lobed, and a
gynoecium primordium in whorl 4 began to elongate into a filamentous structure. Many dividing cells appeared in developing
anthers (Fig. 3B). In contrast, only a few signals corresponding
to both transcripts were found in the undifferentiated gynoecium. At stage 9, the anthers developed pollen mother cells
within the epidermis; all pollen mother cells and epidermal
cells accumulated both transcripts (Fig. 3C). No dividing cells
were detected in the undifferentiated gynoecium. At stage 10,
meiotic tetrads and mature tapetal cells were visible in the
anthers. Dividing cells were found in the epidermis and tapetum (Fig. 3D). At mature stages after stage 10 of the male
flower bud, the undifferentiated gynoecium elongated in only
an upward direction and did not expand in width. A few dividing cells and endoreduplicating cells were identified in the
undifferentiated gynoecium.
In the female flower bud at stage 10, the styles grew from
carpel tips, the ovary walls became thicker, and the ovules
developed within the ovary. Many dividing cells were found in
the developing primordia of carpels and petals, especially in the
ovules and primordia of styles (Fig. 3E). In contrast, in suppressed stamen primordia, the number of cells with signals
799
Fig. 3 In situ hybridization analyses of mature flower buds with antisense RNA probes derived from SlCycA1 and SlH4; 8-µm longitudinal
sections were allowed to hybridize with a mixture of biotinylated
probe derived from SlCycA1 and digoxigenin-labeled probe derived
from SlH4. Cells in which only SlH4 accumulated appeared purple;
cells in which both SlCycA1 and SlH4 transcripts accumulated
appeared dark purple. (A) A male flower bud at stage 7; (B) a male
flower bud at stage 8; (C) a male flower bud at stage 9; (D) a male
flower bud at stage 10; (E) a female flower bud at stage 10; (F) a suppressed stamen and a developing petal in the female flower bud at
stage 10. G, P, and St indicate a suppressed gynoecium, a developing
petal, and a suppressed stamen, respectively. Bars = 150 µm.
800
Sex-specific cell division in flower development
decreased as the female flower buds developed. At stage 10,
undifferentiated stamen primordia stopped elongation and
expansion. No dividing cells or endoreduplicating cells were
seen in the upper round region of the suppressed stamen primordia, but a few dividing and endoreduplicating cells were
detected in the lower region (Fig. 3F).
Although morphological differences in the development of
unisexual flowers have been reported, this is the first report of
sex differences based on the expression of cell cycle-specific
genes. Our analyses demonstrated that the activation of cell
division in whorls 3 and 4 was reversed in male and female
flower buds (Fig. 4). The differences in the timing of mitotic
activation were particularly remarkable in the L1 layer, which
divided only anticlinally. The stamen primordia of male flower
buds were initiated at early stage 5 by cell divisions in all layers of whorl 3 (Fig. 4, Table 1); cell division in whorl 4 was
extensively suppressed at early stage 5. This gives rise to the
reduced size of whorl 4 in male flower buds. At late stage 5, a
gynoecium primordium was initiated by cell divisions in all
layers of whorl 4. In contrast, at early stage 5 of female flower
buds, active cell division occurred in all layers of whorl 4,
especially in the L1 layer (Fig. 4, Table 1). Moreover, the ratio
of dividing cells in the L1 and L2 layers of the stamen primordia of female flower buds decreased at early stage 5, although
cells in the L3 layer were actively dividing (Table 1). Interestingly, the activation of cell division in whorls 3 and 4 was
reversed at stage 5 of male and female flower buds (Fig. 4).
There are two possible explanations for this phenomenon. First,
whorl-specific activation makes it possible to concentrate materials for mitotic division in the specific whorls. Second, the
temporal suppression of cell division clarifies the boundary
between whorls 3 and 4. After all, preferential activation of cell
division at early stage 5 leads to subsequent differentiation of
primordia. The whorl-specific activation unambiguously causes
the initial morphological sex difference in which the size of
male whorl 4 is smaller than that of female whorl 4 (Grant et
al. 1994). Our analyses strongly suggest that specific activation
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Fig. 4 Model of differentiation in unisexual flowers based on cell
division. Shaded ellipses represent flower bud meristems. Crosshatched and hatched ellipses represent the stamen primordia (whorl 3)
and gynoecium primordia (whorl 4) in which cells divide actively,
respectively.
of cell division in whorls 3 or 4 determines the distinct sex in
dioecious plants.
The genes that regulate the initial patterns of cell division
in the floral meristems are of interest because genes that regulate cell division genes may give rise to the spatial and temporal differences in cell division patterns between stamen and
gynoecium primordia. Specific floral organ identity genes are
involved in the establishment of reproductive organ identity.
The AGAMOUS orthologue SLM1, the PISTILLATA orthologue SLM2, and the two APETALA orthologues SLM3 and
SlAP3 have been isolated and characterized (Hardenack et al.
1994, Matsunaga et al. 2003). SLM1 is expressed with the same
pattern in both early male and female flower buds; SlAP3 is
preferentially expressed in mature flower buds. SLM2 and
SLM3 are expressed more closely to the center of the male
whorl 4 than the female whorl 4 at stage 3, suggesting that
these two genes correlate with a reduction in the size of whorl
4. However, clonal analyses using Arabidopsis mutants of
APETALA3 or AGAMOUS suggested that the initial specification of whorl-specific cell division patterns does not depend on
the expression of floral identity genes (Jenik and Irish 2000).
Another possible regulator gene is SUPERMAN. In SUPERMAN loss-of-function mutants, the number of third-whorl stamens is increased, and the number of fourth whorl carpels is
decreased (Bowman et al. 1992). SUPERMAN controls the balance of cell division activity in the third and fourth whorls
(Sakai et al. 2000). Therefore, regulators involved in the cell
proliferation activity of the third and fourth whorls, like
SUPERMAN, would be candidates for sex determination genes
in S. latifolia.
Our in situ hybridization analyses clearly show that the
formation of reproductive organs relies on the coordination of
patterns of cell division. At mature stages of flower buds, the
expressions of both SlH4 and SlCycA1 drastically decrease in
the suppressed gynoecium of the male flower bud and in the
stamens of the female flower bud, whereas developing stamens
in male flower buds and pistils in female flower buds express
high levels of these cell division-specific genes. The suppressed
stamens stop elongating in the early mature stage. By contrast,
the rudimentary gynoecium continues to elongate until it is the
same length as the filaments of mature stamens. The reduction
in the expression of cell cycle-specific genes in the rudimentary gynoecium suggests that the elongation is due mainly to
polar expansion of the cells. Similar expression patterns have
also been observed in a snapdragon staminode, which is an
undifferentiated stamen (Gaudin et al. 2000). The staminode
expressed extremely low levels of histone H4 and low or no
detectable transcripts of cyclins B and D. Our data clearly demonstrate that the reduction of cell division activity in the suppressed organs is correlated with the sex-specific cessation of
cell proliferation. Moreover, the full development of reproductive organs requires the expression of cell cycle-specific genes.
Further analyses of genes that regulate involved sex-specific
Sex-specific cell division in flower development
salt/2-bromo-4-chloro-3-indolyl phosphatase toluidinium salt
(Roche).
Acknowledgments
This work was supported by Grants-in-Aid of Scientific Research
to S.K. (No. 15013215) and S.M. (No. 13740469) from the Ministry of
Education, Science, Culture, Sports, Science and Technology, Japan,
and by the Research for the Future program of the Japan Society for
the Promotion of Science.
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Shuzo) in a total volume of 50 µl. After an initial denaturation
step at 94°C for 3 min, the samples were amplified for five
cycles (94°C for 25 s, 37°C for 2 min, and 72°C for 1 min) and
30 cycles (94°C for 20 s, 45°C for 1 min, and 72°C for 1 min).
Amplified fragments were subcloned into the TOPO-TA vector
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16 h. After hybridization and washing, the biotinylated
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a DAKO GenPoint System (Dako Cytomation, Glostrup,
Denmark). Digoxigenin-labeled probes were detected next with
the anti-digoxigenin alkaline phosphatase conjugate (Roche,
Basel, Switzerland) in combination with nitro blue tetrazolium
801
802
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(Received October 21, 2003; Accepted March 1, 2004)
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