Mol. Cells , Vol. 8, No.2, pp. 240-245
Molecules
and
Cells
© Springer-Verlag 1998
Byr4, a Dosage-dependent Regulator of Cytokinesis in S.
pombe, Interacts with a Possible Small GTPase Pathway
Including Spg1 and Cdc16
Miri Jwa and Kiwon Song*
Department of Biochemistry, College of Science, Yons ei University, Seoul 120-749, Korea
(R eceived on January 19, 1998)
Coordination between karyokinesis and cytokinesis in
the cell division cycle is fundamental to a precise
transmission of duplicated genome into dividing
daughter cells. byr4, a previously isolated essential gene,
affects the mitotic cell cycle and cytokinesis in S. pombe.
Phenotypic analyses of the null alleles and the
overexpression of byr4 suggest that byr4 is a dosagedependent coordinator of karyokinesis and cytokinesis
(Song et al., 1996). In this study, the functional
mechanisms of byr4 were investigated using a byr4
mutant that exhibits byr4 overexpression phenotypes in
thiamine deficient media. Genetic suppression analyses
of this byr4 mutant with other cytokinesis regulatory
genes in S. pombe, edc16, ede7, edc15, ede14, and plol,
show that byr4 overexpression phenotypes are
suppressed by the overexpression of edc16 and edc7,
but not by plol, ede14, and edc15. Also, the basal
expression of byr4 and ede7 suppresses the
temperature-sensitive edc16 mutation. However, the
basal expression of either byr4 or edc16 does not
suppress the temperature-sensitive ede7 mutation. The
results of these suppression tests suggest that byr4
genetically interacts with edc16 and ede7: byr4
functions at the same level with or downstream of edc16
and upstream of ede7. In the present study, we also
show that Byr4 interacts with Cdc16 and Spgl in the
yeast two-hybrid assays. Recent reports suggest a
possible small GTPase pathway to regulate the timing
of cytokinesis where Cdc16 function s as a GAP
(GTPase activating protein), Spgl as a GTPase, and
Cdc7 as a downstream effector. Combined genetic and
two-hybrid analyses of this study strongly suggest that
Byr4 directly interacts with this possible small GTPase
* To whom correspondence shou ld be addressed.
Tel : 82-2-36 1-2705 ; Fax: 82-2-362-9897
E-mai l: [email protected]
pathway including Cdcl6, Spgl, and Cdc7 to regulate
cytokinesis in S_ pombe.
Keyword: Byr4; Cytokinesis; GTPase Pathway; S. pombe.
Introduction
Cytokinesis is one of the major events of the cell cycle that
divides the cytoplasm by placing the division apparatus
including the actin contractile ring upon the equator of the
parental cell at the end of karyokinesis. Cytokinesis
normally occurs in late mitosis following the separation of
c hromosomes in anaphase . Premature initiation of
cytoki nesis before the end of karyokinesis could destruct
the mitotic spindle that can lead to an unequal segregation
or a loss of chromosomes. It could also cause cell cycle
arrest in mitosis and sometimes even "cutting" of the
nucleus (Fankhauser and Simanis, 1994a) . Moreover,
fai lure to undergo cytokinesis after karyokinesis can
produce polyploid multinuclear cells that are frequently
observed in tumor cells. Therefore, the proper spatial and
temporal coordination of karyokinesis and cytokinesis is
essential for maintaining the integrity of the genome.
The fission yeast Schizosaccharomyces pombe provides
an excellent eukaryotic system to study the mechanisms
coordi nating karyokinesis and cytokin esi s, since
cytokinesis in S. pombe morphologically resembles that in
mammalian cells (Marks and Hyams, \ 985). In addition, S.
pombe is readily approached with the tools of classical
genetics, molecular genetics, and cell biology (Moreno et
al., 199 1). As in hi gher eukaryotes, the initiation of
cytokinesis in S. pombe depends on the onset of mitosis by
Abbreviations: S. cere visiae, Saccharomyces cerevisiae; S.
pombe, Schizosaccharomyces pombe; X-Gal , 5-Bromo-4-chloro3 -indo l yl-~ -D- gal actopyranoside .
24 1
Mira Jwa & Ki won Song
relocating F-actin to form a medial ring in the middle of
the cell where the di vision septum is fo rmed following
cytokinesis (M arks and Hyam s, 1985).
In S. pombe, a number of cell di vision cycle mutants
were identified, whose terminal phenotypes suggest their
participation in the control of cytokinesis (for review, see
Gould and Sirnanis, 1997). T he products of edc3, ede4,
ede8, ede12 , and rng2 are the components of the actin
contractile ring or the requi rements for actin rearrangement
du ring Iilltosis (Balasubramanian et aI., 1992; 1994 ; Chang
et ai. , 1996; McCollum et aI. , 1995; Nurse et ai., 1976).
The phenotypes of ede7, edell, ede14, edc15, edc16, piol ,
and spgl mutations suggest that these genes affect the
timing of cytokinesis (Fankhauser and Simanis, 1993 ,
1994; F ankhauser et ai., 1993 ; 1995 ; Minet et ai., 1979 ;
Nurse et ai. , 1976; Ohkura et aI., 1995 ; Schmidt et ai. ,
1997). In addition, intensive homology search and genetic
analyses of spgl and ede 16 raised a possibility that Spg l
encodes a small GTPase and Cdcl6 encodes a GTPaseactivating protein (GAP) (Neuwald, 1997 ; Schmidt et ai. ,
199 7 ) . Th e refore, a p o ss ibl e s m a ll GTP ase sig n a l
transduction pathway, where Spg l functi ons as a GTPase,
Cdcl6 as a GTPase-activating protein (GAP), and Cdc7 as
a downstream effector of Spg l was suggested for spatial
and timely coordination of cytokinesis to karyokinesis in S.
pombe (Schmidt et ai. , 1997).
We originall y isolated byr4 as a multi copy suppressor of
rasl in S. pombe (Song et ai. , 1996). The overexpression
and knock-out studies of byr4 indicate that it regulates the
mitotic cell cycle and the timing of cytokinesis in a
dosage-dependent manner. In detail, byr4 overexpression
inhibits cytokinesis but cell cycle continues leading to
multinucleate cells. Knock-out of byr4 is lethal, and causes
cell cycle arrest in late mitosis with mutiple cytokinesis
and septation (Song et aI. , 1996). In this study, we have
performed genetic suppression analyses of a byr4 mutant
with other genes affecting the timing of cytokinesis to
in ves tiga te th e fun c ti o n a l m ec h a ni s m of by r4 in
conjunction with other genes. We have also deterIillned the
direct interactions of Byr4 with other gene products that
exhibit positive interactions in genetic suppression tests.
The results of these combined geneti c and two-hybrid
analyses strongly suggest that Byr4 directl y interacts with
a possible small GTPase pathway including Cdcl 6, Spg l ,
and Cdc7 to regulate cytokinesis in S. pombe.
Materials and Methods
Strains and Growth Conditions Table I represents the strains
of S. pambe and S. cerevisiae used in this study.
Standard techniques were used for growth, manipulation, and
transformation of fiss ion yeast S. pambe (Moreno et al., 1991). S.
pambe were grown in yeast extract (YE) or minimal media (MM)
with required supplements at the level of 75 mg/l for adenine,
uracil , leucine, and 0.4 mM thiamine. Permissive temperatures
fo r the temperature-sensitive mutants were 20°C or 25 °C
depending on the strains, and the restricti ve temperature was
35°C. The KFY 18 strain that contains a single integrated copy of
byr4 under the control of nmtl promoter show s normal
proli fera ti on with 0.4 mM thi amin e but di spalys by r4
overexpression phenotypes when cultured without thiamine.
Standard techniques were used for growth of S. cerevisiae
(Sherman, 199 1). S. cerevisiae were grown in YPD or synthetic
complex media (SC) with various necessary additions at the level
of 20 mg/l for adenine, L-tryptophan, and 30mg/1 for L-leucine.
S. cerevisiae were transformed by following LiAc/SS-DNAJPEG
(Gietz and Schiestl, 1996) .
Nucleic acids manipu lations and cloning of other cdc
genes Standard methods were used for DNA manipulations
(Sambrook et ai. , 1989). cdc 16, cdc 7, cdcl4, cdc15, and pial
were cloned by polymerase chain reacti ons (PCR) using the
cDNA library of S. pambe as templates. For PCR and cloning,
oligonucleotides with specific restriction sites were generated
(NdeI fo r cdc7, Sail , and BamHI for cdc16, and NdeI and BamHI
for cdcl4, cdc15, and pial ) and PCR was performed wi th Pfu
polymerase (Stratagene). Each PCR frag ment was cloned into the
pREP41 vector that carries the leu2 marker and inducible nmtl
promoter (Basi et al. , 1993; Maundrell , 1993). Clones were
verified by partial sequencing and by complementation of each
temperature-sensitive mutant allele. cdc7 has an internal NdeI site
and C-terminal 14 amino acids are missing in the cloned cdc7 but
it complements the temperature-sensiti ve cdc7 mutant allele.
Table 1. Yeast strains used in this study.
Genotype
Source
KGY246
h- leul-32 ura4-d18 ade6-2JO
K. Gould
Strai n
S. pambe
KGY320
h- leul-32 ura4-d18 ade6-216 cdc7-24
K. Gould
CAll 7
h- leul-32 ura4-d18 ade6-210 cdc16- 116
C. Albright
KFYl 8
h- leul-32 ura4-d18 ade6-2JO byr4::pbyr4/1K210
C. Albright
MATa gal4 gal80 his3 trp l -901 ade2-101 ura3-52
leu2-3,1l 2 URA3::GAL-7lacZ, bYS2::GAL-7HIS3 cyhr
S. Fields
S. cerevisiae
Yl 90
242
Byr4 Interacts with Spgl and Cdcl6
Two-hybrid assay The cDNAs of cdc7, cdcl6, spgl , and cdcl4
prepared by PCR were respectively cloned into the pAS2 vector
that makes fusion protein with the GAL4 DNA binding domain.
The SpeI/Sall byr4 fragment from pbyr4/REP41 (Song et al. ,
1996), that encodes all but the N-terminal 29 amino acids of byr4,
was cloned into the GAL4 ac ti vation domain fusion vector
pACT!. byr4 in pACT! and each of the cdc 7, cdc16, spgl, and
cdcl4 in pAS2 plasmid were cotransformed into the S. cerevisiae
Y190 strain. Cells cotransformed with pS Ellll (SNF4 fused to
the activation domai n of GAL4 in pACT! ) and pSEll1 2 (SNFI
fused to the DNA-binding domain of GAL4 in pAS I) were used
as positive controls (Fields and Song, 1989). ~ -ga l a to s ida se
assays were performed by the X-Gal filter assay as follows. Fresh
yeast were grown on selective SC-trp, leu plates as patches and
transferred to nitrocellulose filter. The filter was put into liquid
nitrogen to permeabilize the cells for 5-10 s, placed on a 3MM
chromatography paper soaked with Z buffer (60 mM Na2 HP0 4 ,
40 mM NaH 2 P0 4 , 10 mM KCI , 1 mM MgS0 4 , 5 mM ~­
mercaptoethanol) containing I mg/ml X-Gal , and incubated
overni ght at 30°C. For positi ve clones of the X-Gal filter assay,
a quantitative ~- ga lato s ida se acti vity was also measured as
follows. Start culture was prepared from single transformant
colonies, diluted 20-fold into fresh medium , and grown until the
OD 600 reached 0.5 to 1.0. One ml of cells were collected, washed,
and resuspended in 0.15 ml of Z buffer. Cells were broken with
glassbeads and bead beater. Lysate was collected and the protein
concentration was determined by the Lowry method. Assay was
set up by mixing 25 llg of extract with 0.8 ml of Z buffer, adding
0.2 ml of 4 mg/ml ONPG (o- nitrophenyl-~-galactopyra no sid e)
and incubating at 37°C until a yellow color develops within 7-10
min . Reactions were stopped by adding 0.5 ml of I M Na2C0 3
and the OD 42o for each reaction was meas ured. The specific
activity was calculated as the llmole of ONPG hydrolyzed/min!
mg protein. All assays were performed in dupli cate from
independent transformants.
Results and Discussion
byr4 overexpression phenotype is suppressed by
overexpression of cdc16 and cdc7 In order to
understand the posssible mechanisms by which by r4
functions in cytokinesis, we needed to perform genetic
complementation or s uppres si on analyses of byr4
conditional mutants with other known genes that affect the
timing of cytokinesis in S. pambe. We used a conditional
byr4 overexpression mutant (KFYI8) that exhibits byr4
overexpression phenotypes in thiamine deficient media,
since byr4 in the genome is under the control of the
heterologou s nmtl promoter that is repressed in the
presence of thiamine but induced without thiamine (Basi et
aI., 1993 ; Maundrell, 1993). KFY18 grew normally on
thiamine plate but could not form colonies without
thiamine, as shown in Fig. 1. When each of the cloned
cdcJ6, cdc 7, cdcJ 5, cdcJ4, and pial under nmtl promoter
was transformed into KFY18 , byr4 the overexpression was
suppressed by the overexpression of cdcJ 6 and cdc7 (Fig.
I). However, it was not suppressed by the overexpression
of pial, cdc J4 , and cdcJ 5 (Fig. 1, data not shown). To
+T
-T
cdc7 cdc16
vector
cdc15 only
Fig. 1. Suppress ion of byr4 overexpression phenotypes by
overexpression of cdcl6 and cdc 7. The KFY 18 strain grows
normally in the presence of 0.4 mM thiamine but can not form
co lonies in thiamine deficient medi a by exhibiting byr4
overexpression phenotypes. KFY 18 was respectively transformed
with cdcl6 in pREP41 , cdc7 in pREP41 , cdcl5 in pREP41 , and
only pREP4l as shown, and cultured on O.4mM thiamine plates
(+T) or on thiamine deficient plates (-T).
exclude the possibility that co-overexpressions of byr4 and
cdcJ6 or byr4 and cdc7 were toxic to cells and made them
revert to form colonies, KFY18 cells freshly transformed
with cdcJ6 or cdc7 were induced in thiamine deficient
liquid media and observed during a time course. No signs
of genetic reversion were observed (data not shown). This
result suggests that byr4 overexpression phenotypes are
specifically suppressed by overexpression of cdcJ 6 and
cdc 7, indicating genetic interactions of byr4 with cdcl 6
and cdc7.
cdc16 mutation is suppressed by byr4 and cdc7 but cdc7
mutation is not suppressed by byr4 and cdc16 The
temperature-sensitive cdcJ6 mutant, cdcJ6-116, can grow
normally at 25°C but not at 35°C. When byr4 or cdc7 in
pREP41 was tran sformed into the cdcJ 6-116 mutant
(CA 117), CA 117 cells formed colonies at restrictive
temperature (35 °C) in the presence of thiamine, as shown
in Fig. 2A. Other cytokinesis regulatory genes did not
suppress cdcJ6-1J6 (data not shown). byr4 and cdc7
s uppressed cdcJ6-1J6 in the presence of thiamine ,
suggesting that the basal expression of byr4 and cdc7 was
enough to suppress the temperature- sensitive cdcJ 6
mutation . In the presence of thiamine, basal expression of
byr4 and cdc7 in a wild-type cell strain (KGY246) did not
show any overexpression phenotypes (data not shown).
Basi et al. reported that transcription activity of nmtl
promoter in pREP41 increases more than 200 fold without
thiamine (Basi et al., 1993). In the absence of thiamine,
transformed byr4 and cd c7 caused overexpression
phenotypes and cytokinesis defects of byr4 and cdc 7.
However, when byr4 or cdcJ6 in pREP41 was transformed
into the cdc7 temperature-sensitive mutant (KGY320) that
grows normally at 20°C but do not form colonies at 35°C,
the cdc7 mutation was not suppressed by basal expression
9 f either by r4 or cdcJ6 (Fig. 2B). These genetic
suppression results suggest that byr4 and cdcJ6 function
Mira Jwa & Kiwon Song
A.
p
R
byr4 cdc7
vector
only
B.
p
R
byr4 cdc16
vector
only
Fig. 2. A. Complementation of the cdc16 temperature-sensitive
mutant by byr4 and cdc7. CA 11 7, a temperature-sensitive cdcl6
mutant, grows normall y at 25 °C, the permissive temperature, but
can not form colonies at 35°C, the restrictive temperature. CA 117
transformed with byr4 in pREP41 , cdc7 in pREP41 , and pREP41
onl y as shown . B. No complementation of cdc7 temperaturesensitive mutant by byr4 and cdcl6. KGY320 , a temperaturesensitive cdc7 mutant grows normall y at 20°C, the permissive
tempera tur e, but ca n not grow at 35 °C , th e re strictive
temperature. KGY320 was transformed with byr4 in pREP41 ,
cdcl6 in pREP41 , and pREP41 only as shown . P stands for cell s
grown at the permissive temperature and R for cells cultured at
the restrictive temperature. P and R are applied to both (A) and
(B ).
upstream of cdc7. In essence, byr4 genetically interacts
with cdc16 and cdc7 to regulate the timing of cytokinesis:
byr4 functions at the same level with or downstream of
cdc16, and cdc7 functions downstream of byr4 and cdcl6.
Byr4 physically interacts with Cdc16 and Spgl The
genetic suppression tests reported above suggest that byr4
genetically interacts with cdc16 and cdc7. While we were
conducting the genetic suppression tests , spg J was
identified as a multicopy suppressor of a dominantnegative cdc7 mutant and Spgl was reported to physically
interact with Cdc7 kinase (Schmidt et a!., 1997). Also, the
intense homology search as well as genetic
complementation test of cdcl6 suggested the possibility
that Cdcl6 functions as a GAP (GTPase activating protein)
for a possible small GTPase, Spgl (Neuwald, 1997 ;
Schmidt et a!. , 1997). On the basis of genetic interactions
of byr4 with the cdc16 and cdc7 pathways , direct
interactions of Byr4 with Cdcl6, Spgl, and Cdc7 were
investigated by yeast two-hybrid assays as described in
Materials and Methods. Byr4 was driven to make the
243
GAL4 activation domain fusion protein and Cdc16, Spg1 ,
and Cdc7 were driven to make the GAL4 DNA-binding
domain fu sion protein s. In the X-Gal filter ass ay,
individual introduction of either byr4 , cdc16, or spgJ into
S. cerevisiae Y 190, that contains a LacZ gene driven by a
GAL4 promoter, did not stimulate ~-galacto s ida s e
expression, whereas the co-presences of byr4 and cdc16 or
byr4 and spgJ stimulated high ~-galactosidase expression
and produced strong blue colors (data not shown) . No ~­
galactosidase activity was detected in cells cotransformed
with byr4 and cdc7 on X-gal media (data not shown). For
cells of positive two-hybrid interactions on X-GAL assays,
the quantitative ~-galactosidase activities were measured
and the re sults are summarized in Table 2 . Cell s
cotransformed with pSEllll (SNF4 fused to the activation
domain of GAL4 in pACTl) and pSEl1l2 (SNFI fu sed to
the DNA-binding domain of GAL4 in pAS1) were used as
positive controls (Fields and Song, 1989). Since byr4 did
not show any interaction with cdc14 in genetic
complementation tests, the two-hybrid interaction of Byr4
and Cdc14 was measured as a negative control to show the
specificity of the interactions of Byr4 with Cdc16 and
Spgl. When ~-galactosidase activity by the interaction of
a positive control, SNFI and SNF4, was assumed at 100%,
the relative activities by interaction s of Byr4 fu sion
proteins with Cdcl6 or with Spgl fu sion proteins were
56.8% and 94.6%, respectively (Table 2). These results
indicate that Byr4 physically interacts with Cdc16 and
Spgl . Direct interactions of Byr4 with Cdc 16 and the lack
of direct interactions between Byr4 and Cdc7 are
consistent with the genetic complementation data that byr4
function s at the same level with or downstream of cdc16
and upstream of cdc7 (data not shown, Table 2) .
A possible mechanism for Byr4 function As recently
suggested, if Cdc16 functions as a GAP (GTPa se
activating protein) and Spgl function s as a small GTPase
(Neuwald, 1997; Schmidt et a!. , 1997), Cdcl6 is expected
to regulate Spg1 activity negatively. While knock-out
mutants of cdc16 are arrested with multiple cytokinesis,
knock-out cells of spgJ and cd c 7 do not proceed
cytokinesis to lead to elongated multinuclear cell s
(Fankhauser and Simanis, 1994; Schmidt et a!. , 1997 ).
These opposite phenotypes of the knock-out mutants of
cdcl6 and spg J or cdc7 also suggest that Cdcl6 negatively
regulates Spg1 (Fankhauser et at., 1993; Schmidt et at. ,
1997).
Knock-out mutants of byr4 are arrested in late mitosis
with repeated cytokinesis and byr4 overexpressed cells fail
to undergo cytokinesis to lead to elongated multinuclear
cells (Fankhauser et a!., 1993 ; Song et aI. , 1996). Since the
knock-out and overexpression mutants of byr4 show the
exact opposite phenotypes of spgJ and cdc7 mutants, Byr4
is also suggested to negatively regulate Spg 1. Therefore,
the genetic interactions of byr4 with cdcl6-cdc7 pathway,
244
Byr4 Interacts with Spgl and Cdcl6
Table 2. Interactions of Byr4 with Cdcl6 and Spgl in two-hybrid assays .
Activation
Domain Fusion
DNA-Binding
Domain Fusion
~-galacto s idase
activity (units)
SNF4
Relative
activity (%)
~-galactosidase
1.6
0.4
1.3
0.3
l.5
0.3
Spgl
1.5
0.3
Cdcl6
1.5
0.3
SNFI
Byr4
Cdc14
1.7
0.4
SNFI
444.7
100.0
Byr4
Spgl
420.5
94.6
Byr4
Cdcl6
252.8
56.8
Byr4
Cdcl4
2.2
0.5
SNF4
Units represent f,Lmoles of o-nitrophenyl -~- galactopyranoside (ONPG) hydrolysed per min per mg protein. Interactions between
SNF4 fu sed to the GAL4 activation domain and SNFI fu sed to the GAL4 DNA-binding were used as positive controls (Fields and
Song, 1989). The relative ~-galactosidase activity was calculated by assuming the interaction of SNFI and SNF4 as 100%.
the two-hybrid interactions of Byr4 with Cdc 16 or Spg 1, as
well as the opposite phenotypes of byr4 and spgJ or cdc7
strongly indicated that Byr4 directly interacts with Cdc16
and Spgl to negatively regulate the activity of Spgl. A
possible model for Byr4 function is presented in Fig. 3.
The quantitative interaction between Byr4 and Cdc16
could also be proposed, because byr4 overexpression was
suppressed by overexpression of cdcJ 6. The analysis of
one of our byr4 deletion mutant supports this possibility.
We isolated the byr4 deletion mutant that produces the
knock-out phenotypes of byr4 and cdc16 when
overexpressed in wild-type cells, suggesting that this
deletion mutant might block normal cytokinesis by binding
and saturating-out the functional Cdcl6.
In this study, we showed, using combined genetic and
two-hybrid analyses, that Byr4 interacts with the suggested
components of a possible small GTPase pathway, Cdcl6
and Spg I , for a spatial and timely regulation of cytokinesis
in S. pombe . For further analyses of the functional
mechanism of Byr4, the biochemical activities of Cdcl6 as
a GAP, Spgl as a GTPase, and the effect of Byr4 on these
activities should be investigated.
Acknowledgements
We would like to thank Dr. C. Albright,
Dr. K. Gould, and K. Forge (Vanderbilt University, USA) for
generous gifts of plasmids and strains, Dr. J. Fikes for the cDNA
library, and Dr. M. Balasubramanian (Institute of Molecular
Agrobiology, Singapore) for the spgJ clone. This work was
supported by a research grant from the Genetic Engineering
Program, Ministry of Education, Republic of Korea (1997) and
Yonsei University Research Fund of 1997 given to K. Song.
?
Spgl-GDP
t
GAP ~ .
(Cdc16?)'"
GEF I
+
Spgl-GTP
t
~B
karyokinesis
?/ spindle defect
/'
Cdc7
cytokinesis
Fig. 3. A possible mechanism of Byr4 functioning to regulate
cytokinesis in S. pombe. GAP stands for the GTPase-activating
protein and GEF stands for the guanine nucleotide exchange
factor. If Spgl functions as a GTPase, it is only active when
bound with GTP. When the spindle defect is monitored by a
presently unknown mechanism and transmittied to Cdcl6, Spg 1GTP activity is negatively regulated by Cdcl6, a possible GAP
(Schmidt et al., 1997). Since byr4 mutants show the defects both
in karyokinesis and cytokinesis, and Byr4 protein genetically and
physically interacts with Cdcl6 and Spgl , Byr4 possibly also
negatively regulates Spgl-GTP in concert with Cdcl6 to block
cytokinesis in the presence of spindle defects.
Mira Jwa & Kiwon Song
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