The Plant Cell, Vol. 10, 331–341, March 1998 © 1998 American Society of Plant Physiologists

A Novel cis-Acting Element in Promoters of Plant B-Type
Cyclin Genes Activates M Phase–Specific Transcription
Masaki Ito,a,1 Masayuki Iwase,a Hiroaki Kodama,b Philippe Lavisse,a Atsushi Komamine,cRyuichi Nishihama,d Yasunori Machida,d,e and Akira Watanabeaa Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Tokyo 113, Japanb Department of Biology, Faculty of Science, Kyushu University, Fukuoka 812-81, Japanc Department of Chemical and Biological Sciences, Japan Women’s University, Mejirodai, Tokyo 112, Japand Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-01,

Japan
e Department of Plant Science, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake-cho, Sakyo-ku,
Kyoto 606-01, Japan

Plant B-type cyclin genes are expressed late in the G 2 and M phases of the cell cycle. Previously, we showed that thepromoter of a Catharanthus roseus B-type cyclin, CYM, could direct M phase–specific transcription of a b-glucuronidase reporter gene in synchronously dividing BY2 tobacco cells. In this study, we determined the regulatory
elements contained within the CYM promoter by using a luciferase reporter gene. Mutational analysis showed that a 9-bp
element is essential for M phase–specific promoter activity in synchronized BY2 cells. The CYM promoter contains
three other sequences similar to this element. A gain-of-function assay demonstrated that when fused to a heterologous promoter, these elements are sufficient for M phase–specific expression; therefore, we named these elements
M-specific activators (MSAs). We found MSA-like sequences in B-type cyclin promoters from tobacco, soybean, andArabidopsis as well as in the promoters of two M phase–specific genes, NACK1 and NACK2, which encode tobacco kinesin-like proteins. Thus, MSA may be a common cis-acting promoter element that controls M phase–specificexpression of cell cycle–related genes in plants.

INTRODUCTION

During progression through the cell cycle, several sets ofgenes are expressed at specific time points (reviewed in
Müller, 1995). Generally, these genes encode proteins that
control cell division or are involved in metabolism that occurs in a periodic fashion. For example, several mammalian
G1 cyclins and cyclin-dependent kinases are induced sequentially during the G 1 phase. Other genes that encode
cyclin A, Cdc2, DNA synthetic enzymes, and several transcription factors are expressed in the late G 1 and S phases.
Another group of genes, including cyclin B and cdc25C
phosphatase, is expressed in the late S and G 2 phases of
the animal cell cycle.
Mechanisms controlling G 1 and S phase–specific transcription are relatively well studied in animal cells. The E2F/
DP family of transcription factors is thought to play important roles in gene regulation during S phase (reviewed in La
Trangue, 1994). E2F binding sites are found in promoters ofmany genes specifically expressed during the S phase andinclude DNA synthetic enzymes, cyclin A, B- myb, and cdc2
(Müller, 1995). E2F has been shown to form complexes with
1 To whom correspondence should be addressed. E-mail masakito@
biol.s.u-tokyo.ac.jp; fax 81-3-3814-1728.

several proteins, including pRb, p107, Cdk2, and cyclins A
and E, and these complexes both repress and activate thetranscription (Schulze et al., 1995).
In contrast, the control of G2-specific expression is poorly
understood in higher eukaryotes. Recently, two promoter elements, CDE (for cell cycle–dependent element) and CHR(for cell cycle genes homology region), have been identified,
and they are involved in transcriptional repression (Zwicker
et al., 1995). It is believed that a repressor protein binds tothese elements during the G 1 phase and that subsequentdissociation of the repressor in the late S and G2 phases activates transcription. Mutated CDE/CHR elements confer
constitutively high levels of gene expression throughout thecell cycle (Lucibello et al., 1995). CDE/CHR elements arepresent in cyclin A, cdc25C, and cdc2 promoters and may
be common motifs required for the G2-specific gene expression in animal cells (Zwicker et al., 1995). However, a different inductive mechanism may also operate in the G 2 and M
phases. The mammalian cyclin B1 promoter contains abinding site for an upstream stimulatory factor, and thisbinding site is essential for transcriptional activation in theG2 phase (Cogswell et al., 1995). However, no single element in the cyclin B1 promoter has been shown to be

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The Plant Cellsufficient for G 2-specific activation (Hwang et al., 1995;
Piaggio et al., 1995).
Mechanisms regulating cell cycle–dependent expression
in animals have been well studied; however, much less isknown about these mechanisms in plants. A- and B-type cyclins are the best-characterized cell cycle–dependent proteins in plants (Hirt et al., 1992; Ferreira et al., 1994a; Fobert
et al., 1994). In all plant species studied thus far, A-type cyclins are expressed earlier in the cell cycle than are B-type
cyclins (Kouchi et al., 1995; Setiady et al., 1995; Fuerst etal., 1996; Reichheld et al., 1996; Ito et al., 1997). Based onsequence similarity, A-type cyclins are further subdivided
into CycA1, CycA2, and CycA3, whereas B-type cyclins are
divided into CycB1 and CycB2 classes (Kouchi et al., 1995;
Renaudin et al., 1996). Transcripts of both classes of B-type
cyclins accumulate in G 2 and M phases of the cell cycle(Ferreira et al., 1994a; Fobert et al., 1994; Kouchi et al.,
1995; Setiady et al., 1995; Ito et al., 1997). Recently, it has
been shown that B-type cyclin promoters from Arabidopsis(Shaul et al., 1996) and Catharanthus roseus (Ito et al., 1997)
conferred M phase–specific expression to a b-glucuronidase (GUS) reporter gene in tobacco BY2 cells.
We have chosen to further characterize the M phase–specific activity of the promoter of the CycB1 cyclin CYM fromC. roseus. Reporter activity was measured during the cell
cycle of stably transformed tobacco BY2 cell lines, which
had been synchronized by the aphidicolin method. We haveidentified elements designated as M-specific activators(MSAs) that are necessary and sufficient for activation of the
CYM promoter. The MSA elements placed in a heterologous
context could activate the cauliflower mosaic virus (CaMV)
35S basal promoter specifically during M phase. The MSA
consensus sequence resembles the binding sites of c-Myb
and v-Myb transcription factors. Sequence analysis showed
the presence of MSA-like sequences in promoters of several
other genes expressed during G2 and M phases: these are
the B-type cyclins from tobacco, soybean, and Arabidopsis
and NACK1 and NACK2 that encode plant kinesin-like proteins. Thus, MSA-mediated transcriptional activation may be
a general mechanism operating in the G 2 and M phases ofthe plant cell cycle. In this study, we report on a novel regulatory pathway that activates gene expression specifically in
the G2 and M phases of the cell cycle in higher plants.

RESULTS

Analysis of the 462-bp CYM Promoter
Our previous work showed that a region of the CYM promoter from positions 2462 to 1102 was sufficient to direct
the M phase–specific expression of the GUS reporter gene
(Ito et al., 1997). In this article, the CYM promoter was used
to drive a firefly luciferase gene (LUC). The constructs wereintroduced into tobacco BY2 cells by Agrobacterium-medi-

ated transformation. Stably transformed cell lines were synchronized by the aphidicolin method (Nagata et al., 1992).
Aphidicolin treatment caused complete arrest of the cell division cycle early in S phase. As shown in Figure 1B, the removal of aphidicolin resulted in synchronous progression
through the cell cycle, with a clear peak in the mitotic index(25 to 35%) after 7 or 8 hr.
Cell cycle–dependent changes in the activity of the CYM
promoter were first studied by measuring LUC mRNA levels
by RNA gel blot analysis. As shown in Figures 1A and 1B,
when LUC transcription was mediated by the 462-bp CYM
promoter, LUC transcript levels increased rapidly during M

Figure 1. M Phase–Specific Transcription of the CYM Promoter–
LUC Transgene in BY2 Cells.
(A) Tobacco BY2 cells transformed with the CYM promoter (positions 2462 to 1100)–LUC transgene were arrested for 24 hr in S
phase by aphidicolin. Aphidicolin was removed (point 0), and cells
were harvested at 1-hr intervals. Total RNA was isolated and hybridized, with the LUC coding region as a probe. The ethidium bromide
staining of the rRNA is also shown.
(B) Cell cycle progression was monitored by measuring the mitotic
index (triangles). The relative level of LUC mRNA was determined byquantifying the radioactivity of each signal on the RNA gel blot
(circles).

M Phase–Specific Activator Elements

phase, and a peak was observed 1 hr before the peak of themitotic index. Subsequently, the levels of LUC mRNA fell
drastically at the completion of cell division. The observed
pattern of expression of LUC mRNA closely resembled
those of endogenous NtCYM (tobacco homolog of CYM)
mRNA and GUS mRNA driven by the CYM promoter (Ito etal., 1997).
LUC activity was also assayed to monitor the promoter
activity during the cell cycle. As shown in Figure 2A, in cells
transformed with the 462-bp CYM promoter–LUC construct,
LUC activity was low for the first 6 hr after aphidicolin removal. A rapid increase in LUC activity was observed 7 hr
after aphidicolin removal and coincided with the peak of themitotic index. LUC activity reached a maximum at 11 hr andremained at constantly high levels for the rest of the experiment. The differences between changes in LUC mRNA levels (Figure 1B) and LUC activity (Figure 2A) during the cell
cycle suggest that the LUC protein has a low turnover rate in
BY2 cells under the applied conditions. Thus, in our experiments, the rate of change in LUC activity depended mainly
on de novo LUC biosynthesis and reflected CYM promoter
activity.
The rate of change in LUC activity is shown in Figure 2C.
This curve has a single peak occurring at 7 hr after aphidicolin removal. This peak coincides with the peak in the mitotic index. On the other hand, a CaMV 35S promoter– LUC
construct produced constantly high levels of LUC activity
throughout the cell cycle (Figures 2B and 2C). Therefore, theCYM promoter could confer M phase–specific expression to
the LUC gene in BY2 cells.
To define the minimal region sufficient for M phase–specific activation of the LUC gene, 39 and 59 deletions wereprepared from the 462-bp CYM promoter. For each construct, 1000 to 3000 stably transformed BY2 calli werepooled and reintroduced into liquid culture. Cell suspension
cultures of transformants were synchronized as describedabove, and LUC activity was assayed to monitor promoter
activity during the cell cycle.
Promoter fragments deleted from the 5 9 end were fused
directly to the LUC reporter gene. As shown in Figures 3A
and 3B, 59 deletions up to position 2229 (where position 1
corresponds to the transcription start site located 109 bp
upstream of the first ATG codon) did not substantially alter
LUC reporter activity. Deletions to positions 2160 and 280(5D160 and 5D80) severely decreased the levels of maximal
LUC activity approximately five- and 50-fold, respectively.
However, all promoter constructs were still capable of directing the M phase–specific expression.
Promoter fragments deleted from the 39 end were fused to
the CaMV 35S basal promoter–LUC construct to provide atranscription start site. Figures 4A and 4B show that 39 deletions from positions 11 to 240 (3D40) were still able to direct M phase–specific expression and maximal activity
levels similar to those observed for the wild-type promoter(3D1). Deletions to positions 2161 and 2180 (3D161 and
3D180) dramatically reduced the levels of maximal LUC ac-

333

Figure 2. M Phase–Specific Increase of LUC Activity in BY2 Cells
Carrying the CYM Promoter–LUC Transgene.
(A) and (B) Tobacco BY2 cells were transformed with CYM promoter(positions 2462 to 1100)–LUC (A) or CaMV 35S promoter–LUC (B).
Cells were synchronized by the aphidicolin method and harvested at
1-hr intervals for LUC assays (open circles, CaMV 35S promoter–
LUC; solid circles, CYM promoter–LUC). Cell cycle progression wasmonitored by measurement of the mitotic index (triangles).
(C) The rate of change in LUC activity was calculated at each time
point as the difference between activities at two consecutive time
points. Open circles, CaMV 35S promoter–LUC; solid circles, CYM
promoter–LUC.
h, hr; RLU, relative light unit.

tivity. However, these constructs were still expressed specifically during M phase. Only the deletion to position 2203
(3D203) abolished M phase–specific promoter activation.
These results led to the following conclusions. First, theCYM promoter sequence between positions 2462 and2203 does not contain elements required for M phase–

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analyzed further. Mutant promoters containing 10-base substitutions in this 80-bp region are shown in Figure 5A. As
shown in Figure 5B, several mutations (80mut80, 80mut10,
80mut20, 80mut30, and 80mut40) directed lower levels of
LUC activity. However, the expression from these promoters
was still M phase specific. Two mutations, 80mut70 and
80mut60, abolished M phase–specific expression. LUC activity directed by the 80mut60 promoter was very low andconstant. Expression from the 80mut70 promoter was
nearly constant; however, it was higher than that observed
for the 80mut60. These data show that an element necessary for M phase–specific promoter activity is located between positions 270 and 250.

Figure 3. M Phase–Specific Activity of 59 Deletions of the CYM
Promoter.
(A) A series of 59-deleted CYM promoter fragments was transcriptionally fused to the LUC reporter gene. The numbers above theblack bars indicate the distance from the CYM transcription startsite.
(B) BY2 cells transformed with the promoter–LUC constructs weresynchronized by the aphidicolin method and harvested at 1-hr intervals for LUC assays. The inset shows LUC activity of 5D80 with an
expanded scale. Promoter constructs are designated with the symbols shown in (A). RLU, relative light unit.

specific activity. Second, the sequence between positions
2203 and 240 contains several elements that regulate thelevels of promoter activity. Third, promoter elements crucial
for the M phase–specific activity should be present in atleast two regions: from positions 280 to 240 and 2203 to
2182.

Identification of a Regulatory Element Required for M
Phase–Specific Expression
Because 80 bp of the CYM promoter (5D80) was sufficient
to confer M phase–specific expression, this sequence was

Figure 4. M Phase–Specific Activity of 39 Deletions of the CYM
Promoter.
(A) A series of 39-deleted CYM promoter fragments was fused upstream of the CaMV 35S basal promoter (positions 246 to 11). The
numbers above the black bars indicate the distance from the CYM
transcription start site.
(B) BY2 cells transformed with the promoter–LUC constructs weresynchronized by the aphidicolin method and harvested at 1-hr intervals for LUC assays. The inset shows LUC activity of 3D182 and3D203 with an expanded scale. Promoter constructs are designatedwith the symbols shown in (A). RLU, relative light unit.

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335

positions 266 and 258, AGACCGTTG, is a regulatory element essential for M phase–specific promoter activity.

Repeat Sequences in the CYM Promoter Are Sufficientfor M Phase–Specific Promoter Activation
The CYM promoter contains three other regions that sharehigh identity with the element AGACCGTTG identified

Figure 5. M Phase–Specific Activity of the 80-bp CYM Promoter
Mutated with 10-Base Substitutions.
(A) Sequences of the 80-bp CYM promoter and mutant promoters
containing 10-base substitutions. The substituted 10-bp regions are
boxed, and lowercase letters represent the bases that are mutated.
(B) LUC activity from BY2 cells transformed with the constructs
shown in (A). Cell division was synchronized by the aphidicolinmethod, and samples were harvested at 1-hr intervals for LUC assays. The inset shows LUC activity of 80mut30 and 80mut60 with an
expanded scale. Promoter constructs are designated with the symbols shown in (A). RLU, relative light unit.

The location of the element required for M phase–specificactivation was more finely mapped by introducing two-basesubstitutions in the interval from 270 to 250 bp of the 80bp CYM promoter (Figure 6A). Figure 6B shows that any mutation from position 266 to position 258 (80mut58 to
80mut66) abolished M phase–specific activation. Substitutions at positions 268 and 257 greatly reduced the maximal
levels of activity but still activated expression during M
phase. Other mutations did not change the M phase–specific pattern of LUC expression and only altered expression
levels mildly. Thus, we conclude that the sequence between

Figure 6. M Phase–Specific Activity of the 80-bp CYM Promoter
Mutated by Two-Base Substitutions.
(A) Sequences of the 80-bp CYM promoter and mutant promoters
containing two-base substitutions. Mutant bases are shown on a
black background.
(B) LUC activity from BY2 cells transformed with the constructs
shown in (A). Cell division was synchronized by the aphidicolinmethod, and samples were harvested at 1-hr intervals for LUC assays. The inset shows LUC activity with an expanded scale. Promoter constructs are designated with the symbols shown in (A).
RLU, relative light unit.

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LUC reporter gene. Figure 8B shows that the CaMV 35S
basal promoter alone (35S 246) did not produce any detectable expression. In contrast, all four promoters containing
the repeat sequences from the CYM promoter could activate LUC gene expression, specifically in M phase. Both 3 3
RT1 and 3 3 RT1R constructs conferred high levels of reporter gene activity, indicating that the RT1 element canwork in an orientation-independent fashion.
To verify that M phase–specific promoter activation by
these RT sequences also occurs in a noninductive system,
we analyzed the LUC activity in asynchronously growing
BY2 cells transformed with 3 3 RT1. The transgenic BY2
cells were first treated with luciferin for in vivo bioluminescent imaging and then stained with Hoechst 33258. Figures
9A and 9B show that detectable bioluminescence was observed only for cells in the M phase. Our data show that therepeat sequences identified in the CYM promoter are sufficient to confer M phase–specific activity.
Figure 7. Repeat Sequences Similar to the Nonamer Element Identified as Essential for M Phase–Specific Promoter Activation.
(A) Nucleotide sequence of the 462-bp CYM promoter. Four repeat
sequences are boxed, and their orientations are shown with arrows.
Position 11 indicates the transcription start site of CYM.
(B) Alignment of four repeat sequences (RT1, RT2, RT3, and RT4).
The boxed regions are the 9-bp sequences that show high identity
to each other. For RT1, the complementary sequence is used forcomparison.
In (A) and (B), the nonamer element identified by mutational analysis
is shown on a black background.

above. As shown in Figure 7A, these sequences are locatedbetween positions 2203 and 280 of the CYM promoter. In
addition, these three regions are oriented in the opposite direction from the original sequence identified by mutagenesis. Figure 7B shows an alignment of the repeats and thesurrounding sequences. All repeat sequences (RTs) containthe fully conserved pentamer AACGG. The region of highsimilarity is comprised of 9 bp that overlap seven bases
from the 59 end of the element CAACGGTCT and extend 2
bp upstream. The repeat sequences are referred to as follows: RT1, CCCAACGGT; RT2, TCCAACGGT; RT3, TGCAACGGC; and RT4, TCAAACGGT (Figure 7B).
Three repeat sequences, RT1, RT2, and RT4, were tested
for ability to confer M phase–specific expression. To minimize the potential problem of inadequate spacing between
elements, we prepared each construct from three copies ofa given repeat sequence separated by six adenine residues(3 3 RT1, 3 3 RT3, and 3 3 RT4), as shown in Figure 8A. An
additional construct was made from three complementary
copies of the RT1 sequence (3 3 RT1R). The constructs
were inserted upstream of the CaMV 35S basal promoter–

Figure 8. Repeat Sequences of the CYM Promoter Can Direct the
M Phase–Specific Activation in a Heterologous Context.
(A) The diagram shows constructs containing three copies of the repeat sequences. The repeat sequences (RT1, RT3, and RT4) are
shown on a black background. The directions of the motifs areshown by arrows.
(B) BY2 cells transformed with the promoter–LUC constructs weresynchronized by the aphidicolin method and harvested at 1-hr intervals for LUC assays. Promoter constructs are designated with thesymbols shown in (A). RLU, relative light unit.

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337

consensus sequences of c-Myb and v-Myb binding sites(Howe and Watson, 1991; Grotewold et al., 1994). This observation suggests that MSA elements may bind some planttranscriptional factors related to Myb.

DISCUSSION

Most eukaryotic cyclins accumulate at specific points in thecell cycle. The oscillation of cyclin levels is regulated, atFigure 9. M Phase–Specific Promoter Activation by the Repeat Sequence in Asynchronously Growing BY2 Cells.
Asynchronously growing BY2 cells transformed with 3 3 RT1 werefirst treated with luciferin for bioluminescence imaging and thenstained with Hoechst 33258. Each luminescent image and Hoechst
fluorescent image was captured with the photon-counting videointensified microscope camera and analyzed by the image processor.
Blue was assigned to Hoechst fluorescence, and red was assigned
to luciferase luminescence to produce false-color images.
(A) Hoechst fluorescence. A mitotic cell is indicated by the arrowhead. Bar 5 10 mm.
(B) LUC luminescent image superimposed on the image shown in (A).

MSA Elements in Promoters of Other M Phase–Specific
Genes from Various Plant Species
Several promoters from genes that are expressed specifically in the M phase were examined to determine whether
they contain MSA-like sequences. We cloned promoters of fourB-type cyclins from different plant species: tobacco NtCYM(Ito et al., 1997), soybean cyc4Gm (Kouchi et al., 1995), andArabidopsis cyc1bAt (Day et al., 1996) and cyc2aAt (Ferreira
et al., 1994a). As shown in Figure 10, these promoters contain three to five repeat sequences that are similar to the
MSA identified in the CYM promoter. In addition, promotersof two other genes, NACK1 and NACK2, which encode tobacco kinesin-like proteins, contain two MSA-like sequences. In all analyzed promoters, the MSA-like sequences
are usually located within the first 200 bp upstream of thetranscription start sites.
Comparison of MSA-like promoter motifs has shown that
all of them contain a fully conserved central core pentamer,
AACGG. It is flanked on both sides by less conserved 3-bp
sequences. Figure 10 shows that the MSA consensus sequence (T/C)C(T/C)AACGG(T/C)(T/C)A closely matches the

Figure 10. Alignment of MSA-like Sequences Present in the Promoters from Other Plant Species.
Shown is an alignment of MSA-like sequences found in promoters of
B-type cyclin genes: C. roseus CYM, soybean cyc4Gm, tobacco
NtCYM, Arabidopsis cyc1bAt and cyc2aAt, and tobacco genes forkinesin-like proteins NACK1 and NACK2. The boxed region indicatesthe 11-bp sequences that share high homology with each other. Nucleotides identical to those of the 11-bp consensus sequence areshown on a black background. Numbers indicate the nucleotide positions from the transcription start sites, which were determined byprimer extension. The binding sequences of c-Myb (Howe andWatson, 1991) and v-Myb (Grotewold et al., 1994) are also shown.

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least partially, at the transcriptional level. To understand themechanism responsible for periodic transcription of cyclin
genes, we have analyzed the promoter of a C. roseus B-type
cyclin, CYM. This cyclin gene has been shown to be expressed strictly during the late G2 and M phases of the cell
cycle in C. roseus and tobacco BY2 cells (Ito et al., 1997). A
462-bp fragment from the CYM promoter can confer M
phase–specific expression to reporter genes (Ito et al., 1997;
this study).
Deletion analysis of the CYM promoter identified a region(2203 to 240) necessary for M phase–specific expression.
This promoter region could direct the levels of LUC expression similar to those observed for the full-length CYM promoter. A smaller region of the CYM promoter (280 to 15)
was sufficient for M phase–specific expression, although theexpression levels were much lower for this construct thanfor those observed with the full-length promoter. A 9-bp
element located between positions 266 and 258 was identified. When mutated, it completely abolished M phase–specific promoter activation. Three additional repeat sequences
sharing high identity with the identified element were foundin the CYM promoter. In a heterologous promoter context,
these repeat sequences could function in an orientationindependent fashion to direct M phase–specific expressionof the LUC reporter gene. Taken together, our data indicatethat the identified MSA elements are necessary and sufficient for M phase–specific expression in cultured tobacco
cells.
Transcription of several B-type cyclins, including a member of the CycB2 class from Arabidopsis, cyc2aAt (Ferreira
et al., 1994a), and two members of the CycB1 class, soybean cyc4Gm (Kouchi et al., 1995) and tobacco NtCYM (Itoet al., 1997), has been shown to be induced during the G 2
and M phases. Phase-specific expression of the Arabidopsis CycB1 cyclin cyc1bAt (Day et al., 1996) has not been examined; however, it has high sequence homology to M
phase–specific cyc1At (Ferreira et al., 1994a). The promoters of these four cyclin genes carry three to five MSA-like elements. Thus, both CycB1 and CycB2 classes of plant
cyclins are likely to be regulated by the same MSA-mediated mechanism.
The genes encoding A-type cyclins from plants have beenshown to be expressed earlier than the B-type cyclin genes(Reichheld et al., 1996; Ito et al., 1997). The promoter of a C.
roseus A-type cyclin, CYS (Ito et al., 1997), does not contain
MSA-like sequences. Also, we could not find MSA-like elements in the published promoter sequences of two plant cyclin-dependent kinases: one is the constitutively expressed
Arabidopsis cdc2aAt gene (Imajuku et al., 1992; Chung andParish, 1995), and the other is cdc2bAt (Imajuku et al.,1992), which is expressed during the S and G 2 phases(Segers et al., 1996). Thus, among various plant cyclins andcyclin-dependent kinases, the MSA-mediated regulatory
pathway seems to be restricted to the B-type cyclins. On
the other hand, we have found MSA-like motifs in the promoters of NACK1 and NACK2 genes. These genes encode

plant kinesin-like proteins that are expressed in a fashion
similar to B-type cyclins (R. Nishihama and Y. Machida, unpublished results). We speculate that the MSA-mediatedmechanism may function in a narrow window in late G 2 andM phases to regulate a group of genes specifically expressed at this timing.
MSA-like sequences could not be found in the promotersof mammalian B1 cyclins (Cogswell et al., 1995; Hwang etal., 1995; Piaggio et al., 1995) and cdc25C (Lucibello et al.,1995). At the same time, plant cyclin promoters do not contain motifs similar to animal regulatory elements. Thus,
higher plants may have a unique mechanism of M phase–
specific regulation of gene expression.
It has been suggested that abundance of B-type cyclin
in plants may be a limiting factor that determines the rateof cell division during developmental processes (Ferreira etal., 1994b; Doerner et al., 1996). Ectopic expression of the
Arabidopsis CycB1 cyclin cyc1At stimulates organized growthof roots (Doerner et al., 1996). We have shown that MSA elements are sufficient for specific expression in root tips oftransgenic tobacco plants (M. Ito and H. Kodama, unpublished results). Thus, transcriptional regulation of cyclin
gene expression mediated by MSA elements may play animportant role in controlling growth of established meristems.
The MSA consensus sequence is similar to the consensus
binding sites of c-Myb and v-Myb factors from animals(Howe and Watson, 1991; Grotewold et al., 1994). There issome evidence that Myb-related proteins can participate in
the control of cell division in plants. Recent studies haveshown that an Arabidopsis Myb-related protein, AtCDC5,
may be a specific regulator of gene expression in the G2 andM phases (Hirayama and Shinozaki, 1996). This protein
shares similarity with Schizosaccharomyces pombe Mybrelated protein Cdc5 1, which is essential for progression
through the G2 and M phases (Ohi et al., 1994). However,the DNA sequence recognized by the recombinant AtCDC5protein, CTCAGCG, differs from the consensus MSA element. In our work, gel shift assays performed to showin vitro binding of some nuclear factors to the MSA elements were not successful. We speculate that the putative
MSA binding factor(s) might be present only in cells in the
M phase, when the nuclear envelope is lost. Thus, the factor may be absent in extract prepared from the isolated
nuclei.
In mammalian cells, G 2 phase–specific transcription isregulated by a complex mechanism with elements that arethought to be involved in activation (Ohno et al., 1996) andrepression (Lucibello et al., 1995). Our data indicate that the
MSA-mediated regulatory pathway alone is sufficient to induce the cyclin expression observed during the G 2 and M
phases of the plant cell cycle. We are currently in the process of identifying the protein(s) that interacts with MSA elements. Cloning of the corresponding cDNA will provide themeans to determine the mechanism that controls M phase–
specific gene expression in plants.

M Phase–Specific Activator Elements

METHODS

Plasmid Construction
The two binary vectors pBI-LUC (Nishiuchi et al., 1995) and pBICL46
were used. pBI-LUC contains a cauliflower mosaic virus (CaMV) 35S
promoter–luciferase (LUC) fusion gene originated from pDO432 (Owet al., 1986). For cloning of the modified CYM promoter fragments,
the CaMV 35S promoter was removed by HindIII-BamHI digestion,and the promoter fragments were cloned into the HindIII-BamHI interval of pBI-LUC. A fragment of the CaMV 35S basal promoter (246
to 11) was synthesized by polymerase chain reaction (PCR), using
pDO432 as a template with a set of linker/primers (HindIII-SalI 59 and
BamHI 39). The resulting PCR fragment was then used to replace thefull-length CaMV 35S promoter in pBI-LUC with the 35S basal promoter. Plasmid pBICL46 contains HindIII and SalI sites upstream of
the CaMV 35S basal promoter–LUC fusion. Modified CYM promoter
fragments were cloned into the HindIII-SalI sites.
A series of 59 deletion derivatives of the CYM promoter was obtained by PCR, using the CYM genomic fragment as a template (Itoet al., 1997). Specific linker/primers (28-mers) were used to generate
restriction sites (HindIII 59 and BamHI 39) for directional cloning upstream of the LUC reporter gene in pBI-LUC. 39 end deletions of the
CYM promoter were generated in a similar fashion by using HindIII
59- and SalI 39-specific linker/primers and cloned upstream of the
CaMV 35S basal promoter of pBICL46. Site-directed mutagenesis of
the CYM promoter (280 to 15) was done by PCR, using 85-mer synthetic DNAs with 10- or two-base substitutions as templates. The
mutagenized promoters were cloned as HindIII-BamHI fragments
into pBI-LUC. Constructs containing repeat sequences (RTs) wereprepared by annealing complementary oligonucleotides that carry
HindIII 59 and SalI 39 linkers. Then they were ligated into the HindIIISalI site of pBICL46. All plasmid constructs were sequenced.

Plant Material
Tobacco (Nicotiana tabacum) BY2 cells were maintained at 278C in a
medium that contained Murashige and Skoog salt base (Murashige
and Skoog, 1962), 3% (w/v) sucrose, 0.2 mg/mL KH2PO4, 0.1 mg/mL
myoinositol, 1.0 mg/L thiamine, and 9 3 1027 M 2,4-dichlorophenoxyacetic acid. Plasmid constructs were introduced into Agrobacterium tumefaciens LBA4404 and used to transform tobacco BY2 cells,
essentially as described by An (1987). Approximately 1000 to 3000
kanamycin-resistant colonies were pooled, reintroduced into suspension cultures, and maintained in the same way as BY2 cells. To
synchronize cell division of transgenic tobacco BY2 cells, 10 mL of
7-day-old cells was diluted 10 times with fresh medium containing
aphidicolin (final concentration of 5 mg/mL), cultured for 24 hr, washedwith 1 liter of fresh medium, and finally suspended in 100 mL of medium. The mitotic index was determined by counting cells stained with
Hoechst 33258 (Sigma) in the presence of 0.5% Triton X-100.

339

reading frame of the LUC gene (BamHI DNA fragment of pBI-LUC)
was labeled with 32P, using the Megaprime DNA Labelling System(Amersham). Hybridization and washings were performed as described previously (Ito et al., 1997). Signals were detected using the
Fuji Imaging Analyzer (model BAS2000; Fuji Photofilm, Tokyo, Japan).

LUC Assay
Protein extracts were obtained by homogenizing BY2 cells in LUC extraction buffer (50 mM sodium phosphate, 2 mM DTT, and 0.1% Triton
X-100). LUC activity assays were performed using a PicaGene assay
kit (Toyo Co. Inc., Tokyo, Japan), according to the manufacturer’s instructions. Measurements of LUC activity were performed with a luminometer (model LB9501; Berthold, Bad Wildbad, Germany).
For imaging of in vivo LUC bioluminescence, 2 mL of 3-day-old
BY2 cell suspension was treated with 5 mM luciferin (Sigma) for 30
min to diminish the activity of accumulated LUC. BY2 cells were thenwashed with 100 mL of fresh medium, resuspended in 2 mL of themedium, and cultured for 8 to 10 hr. Cells were placed on glass
slides coated with poly-L-lysine (Sigma). They were first treated with
5 mM luciferin to detect luminescence and then stained with Hoechst
33258 (Sigma). Each luminescent image and Hoechst fluorescent image was captured with the photon-counting video-intensified microscope camera connected to the microscope and analyzed by theimage processor ARGUS-20 (Hamamatsu Phototonics, Hamamatsu,
Japan). When producing the false-color images, blue was assigned
to the Hoechst fluorescence, and red was assigned to LUC luminescence.

Isolation of Promoter Sequences by Inverse PCR
Total genomic DNA of Arabidopsis thaliana ecotype Columbia andtobacco BY2 cells was isolated by the method of Murray andThompson (1980). Soybean (Glycine max cv Akisengoku) genomic
DNA was kindly supplied by H. Kouchi (National Institute of Agrobiological Resources, Tsukuba, Japan). Genomic DNA was cut withseveral restriction enzymes, ligated with a DNA ligation kit (Takara,
Kyoto, Japan), and used as a template for PCR with specific primer
sets (25-mers). Nucleotide sequences of the primers were designed
using published cDNA sequences. Amplified fragments were directly
cloned into pGEM-T vector (Promega) and sequenced. Fidelity of thedetermined sequences was confirmed by sequencing DNA fragments obtained by independently performed PCR reactions.
Transcription start sites of NtCYM, cyc4Gm, cyc1bAt, cyc2aAt,
NACK1, and NACK2 were determined by primer extension, using
poly(A)1 RNA extracted from soybean (cv Akisengoku), tobacco BY2,and Arabidopsis T87 cell lines (Axelos et al., 1992), as described previously (Ito et al., 1997).

ACKNOWLEDGMENTS
RNA Extraction and Gel Blot Hybridization
Extraction of total RNA was performed as previously described (Ito etal., 1997). RNA was subjected to electrophoresis on 1% agarosegels that contained 2% formaldehyde by a modified version of theprocedure of Lehrach et al. (1977). RNA was then blotted onto nylon
membranes (Biodyne A; Pall, Port Washington, NY). The entire open

We thank Atsuhiko Aoyama and Dr. Tamotsu Kawazu at the University of Tokyo for technical advice. We are grateful to Drs. Maria Smithand Shannon Frances for critical review of the manuscript. The soybean genomic DNA and soybean seeds were kindly supplied by Dr.
Hiroshi Kouchi at the National Institute of Agrobiological Resources.
The Arabidopsis cell line T87 was obtained from the RIKEN Plant Cell

340

The Plant Cell

Bank (Tsukuba, Japan). This research was supported by a Grant-inAid for Scientific Research (No. 09740589) from the Ministry of Education, Science, and Culture, Japan.

Hwang, A., Maity, A., McKenna, W.G., and Muschel, R.J. (1995).
Cell cycle–dependent regulation of the cyclin B1 promoter. J. Biol.
Chem. 270, 28419–28424.

Received October 17, 1997; accepted January 19, 1998.

Imajuku, Y., Hirayama, T., Endoh, H., and Oka, A. (1992). Exonintron organization of the Arabidopsis thaliana protein kinase
genes CDC2a and CDC2b. FEBS Lett. 304, 73–77.

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