The RCC1 Protein, a Regulator for the Onset of Chromosome
Condensation Locates in the Nucleus and Binds to DNA
Motoaki Ohtsubo, Hiroshi Okazaki,* and Takeharu Nishimoto
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812, Japan; and * Hoechst
Japan Ltd., Pharma Research Laboratories, Kawagoe, Saitama 350, Japan
Abstract. The RCC1 gene, a regulator for the onset of
chromosome condensation was found to encode a pro-
tein with a molecular mass of 45 kD, determined
using the antibody against the synthetic peptides pre-
pared according to the amino acid sequence of the
putative RCC1 protein. The p45 located in the nuclei
was released from the isolated nuclei, either by DNase
I digestion or by treatment with 0.3 M NaCI. Consis-
tently, p45 bound to the DNA-cellulose column was
eluted with 0.3 M NaCI. After sequential treatment
with DNase I and 2 M NaCI, almost all of the RCC1
protein were released from the nuclei. Thus, RCC1
protein locates on the chromatin and is not a compo-
nent of the nuclear matrix. In mitotic cells, 1M5 is dis-
persed into the cytoplasm. Presumably, RCC1 protein
plays a role in regulating the onset of chromosome
condensation, at the level of transcription or of mRNA
is present in a latent form in the Xenopus oocyte (7, 9) and
is specifically activated at mitosis by the translation of an ac-
tivating agent (for review see reference 10). It is therefore as-
sumed that the interphase cells have a regulatory mechanism
which represses expression of this activator protein. This
view was supported by cell fusion experiments which re-
vealed that mitosis was delayed in G2 phase cells fused with
S phase cells (30), and by isolation of mutants with defects
in negative regulatory factor (s) for MPF activation (23, 26).
In eukaryotic cells, mitosis does not normally occur until
DNA synthesis is completed, but, in these mutants, mitosis
is prematurely induced at interphase under restrictive condi-
In one such temperature-sensitive (ts) mutant, tsBN2, from
the baby hamster kidney (BHK21) cell line (24, 26) prema-
ture chromosome condensation (PCC) occurs at 39.5°C. PCC
is a phenomenon that was first identified in cell fusion exper-
iments (30): after fusion with mitotic cells, interphase cells
undergo rapid transformation resembling that of G2-prophase
transition, leading to the formation of discrete chromosomes.
In the tsBN2 cells, this phenomenon can be induced at the
restrictive temperature without fusing mitotic cells. In addi-
tion to PCC, all mitotic-specific events (such as phosphoryla-
tion of histone H3, appearance of mitotic-specific antigens,
and synthesis of p35 protein) occur in interphase tsBN2 cells
at the nonpermissive temperature (2, 39, 40). These mitotic-
specific events depend on the synthesis of a new protein (24).
Consistently, tsBN2 cells showing PCC possess the ability
HROMOSOME condensation occurs at the transition
from G2 to M phase in the cell cycle, with the aid of
maturation promoting factor (MPF) ~ (20, 36). MPF
1. Abbreviations used in this paper: MPF, maturation promoting factor;
PCC, premature chromosome condensation; ts, temperature sensitive.
to condense the chromatin of interphase cells by cell fusion
(14). Thus, chromosome-condensing factor is newly pro-
duced in tsBN2 cells at the nonpermissive temperature even
in cases of arrest at interphase. As the tsBN2 mutation is
recessive in hybrid cells (22), tsBN2 cells are considered to
have a ts defect in the negative regulator for the onset of chro-
mosome condensation, which inhibits the condensation until
the G2 phase.
We cloned the human RCC1 gene, complementing tsBN2
mutation from HeLa ceils, by DNA-mediated gene transfer
(15). Using the Alu-free DNA fragment of the RCC1 gene,
cDNA clones were isolated from Okayama-Berg's cDNA li-
brary (25). While two of these complement the tsBN2 muta-
tion, the 5' base sequence of these clones differs. Since both
clones share the open reading frame of 1,263 bp, we consid-
ered that this open reading frame may encode the product of
the RCC1 gene, RCC1 protein. According to the base se-
quence of the RCC1 cDNA, the putative RCC1 protein is es-
timated to have a molecular mass of 45 kD and seven homol-
ogous internal repeats of ~60 amino acids.
We prepared antibodies against the peptides of putative
RCC1 protein, in attempts to identify the presence of human
RCC1 protein in HeLa cells. Using these antibodies, human
RCC1 protein was shown to be a new member of the nuclear
DNA-binding protein family. It has a molecular mass of 45
kD, the same as estimated from the base sequence of the
Materials and Methods
Cell Lines and Culture Condition
tsBN2 cells are ts mutants derived from the BHK21 cell line (22), and the
following cell lines are derivatives of tsBN2 cells: BN2-RV, a spontaneous
© The Rockefeller University Press, 0021-9525/89/10/1389/9 $2.00
The Journal of Cell Biology, Volume 109, October 1989 1389-1397 1389
ts + revertant; ST2-7, a secondary ts + transformant transfected by the total
HeLa DNA (22); BN2-peD51, a ts + transformant transfected with the hu-
man RCCI eDNA (pcD51) (25). The BHK21/13 cell line and the HeLa $3
cell line are derived from baby golden hamster kidney and from human cer-
vix carcinoma, respectively, Cell lines were cultured in a humidified at-
mosphere containing 10% CO2 at 37.5"C, except for the tsBN2 cells which
were cultured at 33.5°C.
Preparation of Antipeptide Antibodies
According to the sequence of RCCI eDNA (25), five peptides (peptide 1,
KSKKVKVSHRSHSTE; peptide 2, ENVMERKKPALVSI; peptide 3,
KSRGSRGHVRFQDA; peptide 4, LGLGEGAEEKSIPT; and peptide 5,
HTVLLVKDKEQS) were synthesized by the solid-phase t-Boc procedure,
using the Applied Biosystems, Inc. (Foster City, CA) model 430A automated
synthesizer. Cysteine was added to the carboxy-terminal ends of all peptides
to couple the carrier proteins.
Synthetic peptides were coupled to the carrier protein, keyhole limpet
hemocyanin or BSA using N-(r-maleimidobutyryloxy) succinimide, and
then were used to immunize rabbits (33).
Preparation and Fractionation of Nuclei
To prepare nuclei, cells were pelleted and washed twice in ice cold hypo-
tonic buffer containing 10 mM Hepes, pH 8.0, 5 mM KCI, and 2 mM
MgCl2, then incubated in the hypotonic buffer containing 0.5% NP-40 for
10 rain on ice, and disrupted with a tight-fitting pestle of a Potter
homogenizer until virtually all cells were broken (usually 25 strokes). The
extent of cell breakage was monitored microscopically. Nuclei were then
separated from the cytoplasmic fraction by sedimentation at 1,000 g for 5
rain and washed twice with ice cold hypotonic buffer. The isolated nuclei
were suspended in the ice cold hypotonic buffer and kept on ice. All proce-
dures of cell fractiouation were performed with freshly prepared nuclei, at
a concentration of l0 s nuclei/ml. In the salt extraction experiments, the
hypotonic buffer containing the desired salt concentration was used. Appro-
priate amounts of a solution containing 2 nag of DNase I per milliliter were
added to the nuclei preparation to obtain the desired concentration of
nuclease. All experiments were carried out for 10 rain at 0*C. Nuclease
treatment was terminated by adding EDTA (final concentration, 5 raM), fol-
lowed by an additional incubation for 10 rain on ice. Nuclear supernatant
and residual nuclei were separated by centrifugation. Each fraction was ana-
lyzed by immunobiotting (5, 16).
Cells were lysed in buffer containing 62.5 mM Tris-HCI, pH 6.8, 10 mM
2-mercaptoethanol, 3% (wt/vol) SDS, and 20% glycerol. Cellular proteins
were electrophoresed in a 12.5% SDS-polyacrylamide slab gel (19), and
analyzed by immunobiotting, as described previously (5), using the anti-
body against peptides and a Vectastain ABC kit. Protein concentration was
determined by Bradford's method (4).
Indirect lmmunofluorescence Staining
Cells growing on glass coverslips were fixed in 3 % (wt/vol) paraformalde-
hyde in PBS, permeabilized with 1% NP-40 in PBS containing glycine, and
stained using the anti-peptide 1 and the rhodamine-conjngated goat
anti-rabbit IgG (Tago Inc., Burlingame, CA), as described elsewhere (6).
DNA was stained with Hoechst dye 33258 (1 pg/ml) (Hoechst Japan, Ltd.,
D NA-binding Assay
Cells labeled with [35S]methionine were lysed in buffer A (10 mM Tris-
HCI, pH 8.0, 1 mM MgCle, 0.5% NP-40, 0.45 M NaCI, 1 mM PMSF, and
5 pg/ml Pepstatin) (29). After dilution with buffer B (10 mM potassium
phosphate, pH 6.2, 1 mM MgCI2 0.5% NP-40, 1 mM DTT, 10% glycerol),
the lysate was loaded onto the double-stranded calf thymus DNA-cellulose
column. Bound proteins were eluted with buffer containing 10 mM Tris-
HCI, pH 8.0, 1 mM DTT, and increasing concentrations of NaCI. The flow-
through and the eluted fractions were immunoprecipitated and analyzed by
electrophoresis on 12.5 % SDS-polyacrylamide gels. The radioactivity was
detected by fluorography.
Preparation of Antibodies to Peptides Encoded by
Using the cloned human RCC1 genomic DNA as a probe,
two RCC1 cDNAs complementing the tsBN2 mutation were
cloned (25). These clones have a different 5' untranslated re-
gion, but share the open reading frame of 1,263 bp encoding
a protein of 45 kD, which is estimated to be a product of the
human RCC1 gene. According to the amino acid sequence
of this putative RCC1 protein, five peptides were synthe-
sized, as described in Materials and Methods, and the loca-
tion of each is shown in Fig. 1. These peptides were con-
jugated with BSA or keyhole limpet hemocyanin, as a carrier
protein, and then used as antigens to immunize the rabbits.
Identification of the Putative RCC1 Protein in
Of the five synthetic peptide-specific antibodies, the anti-
peptide 1 antibody recognized the protein with a molecular
mass of 45 kD, detected in the lysate of HeLa cells by immu-
noblotting (Fig. 2). Other antibodies which recognized the
synthetic peptides by Ouchterlony analysis did not specif-
ically recognize any protein by immunoblotting (data not
shown). The 45-kD mass is the same as that of the putative
RCC1 protein estimated from the base sequence. This p45
protein was also found in two other cell lines, ST2-7 and
BN2-pcD51, but not in BHK21 and tsBN2 cells (Fig. 2).
Both ST2-7 and BN2-pcD51 cells~contain the active human
RCC1 gene (genomic and its cDNA, respectively) (15, 25).
The presence of p45, therefore, is consistent with the pres-
ence of the active human RCC1 gene.
From these results, we conclude that the IM5 protein de-
tected by the antibody to peptide 1 was the product of the hu-
man RCC1 gene.
RCC1 Protein Is Located in the Nuclei
HeLa cells were swollen in hypotonic solution and the intact
nuclei were collected by gentle centrifugation. Proteins were
extracted from both cytoplasmic and nuclear fractions, and
analyzed by immunoblotting using anti-peptide 1. As shown
in Fig. 3 A, p45 was present only in the nuclear fraction.
The nuclear location of the p45 was further confirmed by
indirect immunofluorescence staining. HeLa cells and two
other cell lines, tsBN2 and ST2-7, grown on glass coverslips
were fixed, permeabilized, and exposed to anti-peptide 1.
The presence of antibody was visualized by rhodamine-
conjugated goat anti-rabbit IgG. The antibody to peptide 1
recognizes only the human RCC1 protein. Therefore, stain-
ing of the cells should be consistent with the presence of ac-
tive human RCC1 protein. As shown in Fig. 4, the nuclei of
both HeLa and ST2-7 were stained but the nuclei of tsBN2
cells were only stained faintly and were barely distinguish-
able from the cytoplasm. Since both HeLa and ST2-7 cells
contain the active human RCC1 gene, these results are con-
sistent with the presence of human RCC1 protein.
Subnuclear Localization of RCCI Protein
To determine whether the RCC1 protein locates on the chro-
The Journal of Cell Biology, Volume 109, 1989 1390
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Ohtsubo et al. RCC1 Protein